Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Accepted manuscripts
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Cell Scientists to Watch
    • First Person
    • Sign up for alerts
  • About us
    • About JCS
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Fast-track manuscripts
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • JCS Prize
    • Manuscript transfer network
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contact JCS
    • Subscriptions
    • Advertising
    • Feedback
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

User menu

  • Log in

Search

  • Advanced search
Journal of Cell Science
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

supporting biologistsinspiring biology

Journal of Cell Science

  • Log in
Advanced search

RSS   Twitter  Facebook   YouTube  

  • Home
  • Articles
    • Accepted manuscripts
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Cell Scientists to Watch
    • First Person
    • Sign up for alerts
  • About us
    • About JCS
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Fast-track manuscripts
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • JCS Prize
    • Manuscript transfer network
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contact JCS
    • Subscriptions
    • Advertising
    • Feedback
Review
Reconstruction of destruction – in vitro reconstitution methods in autophagy research
Satish Babu Moparthi, Thomas Wollert
Journal of Cell Science 2019 132: jcs223792 doi: 10.1242/jcs.223792 Published 31 October 2018
Satish Babu Moparthi
Membrane Biochemistry and Transport, Institute Pasteur, 28 rue du Dr Roux, 75015 Paris, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Satish Babu Moparthi
Thomas Wollert
Membrane Biochemistry and Transport, Institute Pasteur, 28 rue du Dr Roux, 75015 Paris, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Thomas Wollert
  • For correspondence: thomas.wollert@pasteur.fr
  • Article
  • Figures & tables
  • Info & metrics
  • PDF
Loading

ABSTRACT

Autophagy is one of the most elaborative membrane remodeling systems in eukaryotic cells. Its major function is to recycle cytoplasmic material by delivering it to lysosomes for degradation. To achieve this, a membrane cisterna is formed that gradually captures cargo such as organelles or protein aggregates. The diversity of cargo requires autophagy to be highly versatile to adapt the shape of the phagophore to its substrate. Upon closure of the phagophore, a double-membrane-surrounded autophagosome is formed that eventually fuses with lysosomes. In response to environmental cues such as cytotoxicity or starvation, bulk cytoplasm can be captured and delivered to lysosomes. Autophagy thus supports cellular survival under adverse conditions. During the past decades, groundbreaking genetic and cell biological studies have identified the core machinery involved in the process. In this Review, we are focusing on in vitro reconstitution approaches to decipher the details and spatiotemporal control of autophagy, and how such studies contributed to our current understanding of the pathways in yeast and mammals. We highlight studies that revealed the function of the autophagy machinery at a molecular level with respect to its capacity to remodel membranes.

Introduction

Eukaryotic cells have evolved a highly conserved and sophisticated degradation and recycling system for damaged or superfluous organelles, termed macroautophagy (hereafter referred to as autophagy) (Dikic and Elazar, 2018). This system requires cargo selection in a spatiotemporally defined and regulated fashion (Khaminets et al., 2016), and the degradation of cargo with very different sizes and shapes, demanding versatility. The complexity of these requirements is reflected in the complexity of autophagy, which involves the coordinated action of more than 40 dedicated autophagy-related (Atg) proteins (Mercer et al., 2018) that collaborate and are intertwined with a large number of cellular signaling, trafficking, and stress response pathways (Davis et al., 2017).

Autophagy was first characterized at a morphological and ultrastructural level as a pathway that delivers cytoplasm to lysosomes (Ashford and Porter, 1962; De Duve, 1963), and seminal work identified a set of Atg proteins through a genetic screen in yeast (Tsukada and Ohsumi, 1993), followed by related screens that expanded the repertoire of Atg proteins (Harding et al., 1995; Thumm et al., 1994). The identification of human homologs of yeast Atg proteins established that autophagy is conserved in higher eukaryotes (Kabeya et al., 2000; Mizushima et al., 1998). Furthermore, autophagy is involved in the onset of various human diseases, such as cancer and neurodegeneration, which has revealed the importance of the pathway in maintaining cellular homeostasis under normal conditions and in response to cytotoxic stress (Liang et al., 1999; Ravikumar et al., 2002).

Although yeast and human autophagy pathways are conserved, there are significant mechanistic differences, and the human autophagic machinery is much more elaborate compared to its yeast counterpart. In yeast, autophagy is initiated at the phagophore assembly site (PAS) at the vacuole, and involves activation, assembly and recruitment of the Atg1 kinase complex (Fig. 1A) (Cheong et al., 2005; Kabeya et al., 2005, 2009). Following its activation, the Atg1 kinase complex recruits vesicles that contain the membrane protein Atg9 (hereafter Atg9 vesicles) to the PAS (Fig. 1A) (Mari et al., 2010; Rao et al., 2016; Sekito et al., 2009). These vesicles fuse to give rise to a precursor membrane, termed a phagophore (Nair et al., 2011; Yamamoto et al., 2012). The expansion of these membrane cisternae allows cytoplasm to be sequestered and enclosed. The high demand of lipids that are needed for phagophore expansion requires a constant supply with membranes of various origins including the ER, the Golgi, the ER–Golgi intermediate compartment and mitochondria (Graef et al., 2013; Meiling-Wesse et al., 2005; Ohashi and Munro, 2010; Reggiori et al., 2005). Moreover, the early phagophore is enriched in phosphatidylinositol 3-phosphate [PtdIns(3)P] through the action of an autophagy-specific class III phosphatidylinositol 3-kinase (PI3K) complex (Fig. 1B). The recruitment of this lipid kinase to the PAS is mediated by an interaction of Atg13 with the PI3K subunit Atg14 (Jao et al., 2013; Suzuki et al., 2007).

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Schematic representation of autophagy in yeast. (A) The nucleation of phagophores occurs at the phagophore assembly site and requires activation and assembly of the Atg1 kinase complex as well as recruitment and tethering of Atg9 vesicles. (B) The PI3K complex generates PtdIns(3)P at the early phagophore (red section of membrane), which is required to recruit the Atg8 conjugation machinery (shown here is the E2 enzyme Atg3). Before Atg8 is conjugated to the phagophore, its C-terminus is cleaved by Atg4 (priming). (C) The concave (inner membrane) pool of Atg8 tethers cargo to the phagophore, whereas the convex pool forms, together with Atg12–Atg5–Atg16, a two-dimensional protein network that stabilizes the membrane. Expansion of the phagophore requires incorporation of membranes, probably mediated by fusion of vesicles of different origins with the rim of the phagophore. (D) Sealing of the phagophore gives rise to the double-membrane-surrounded autophagosome. The kinase activity of Atg1 promotes release of most Atg proteins on the cytoplasmic face of autophagosomes into the cytoplasm. Recycling of Atg8 requires the proteolytic activity of Atg4. (E) The fusion of autophagosomes with the yeast vacuole is coordinated by the HOPS complex, which tethers both compartments to each other, and by SNARE proteins. Zippering of the lysosomal SNARE with autophagic SNAREs drive membrane fusion. Proteolytic enzymes within the lysosome degrade the inner autophagosome membrane, cargo and Atg proteins (e.g. Atg8).

Enrichment of the phagophore with PtdIns(3)P allows PtdIns(3)P-binding proteins, such as the yeast β-propellers that bind phosphoinositides (PROPPIN)-repeat-containing Atg18 and Atg21, to be recruited (Nascimbeni et al., 2017). Atg21 initiates the transition from phagophore initiation to phagophore expansion by recruiting Atg16 (Juris et al., 2015), whereas Atg18, in conjugation with its interaction partner Atg2, stimulates phagophore expansion by establishing ER–phagophore contact sites (Gómez-Sánchez et al., 2018). The expansion of the phagophore membrane is regulated by a ubiquitin (Ub)-like conjugation machinery that catalyzes the covalent attachment of the Ub-like protein Atg8 to phosphatidylethanolamine (PE) within the phagophore (Fig. 1B) (Ichimura et al., 2000). This reaction is initiated by an Atg4-dependent priming of Atg8 through proteolytic cleavage of its C-terminal residue (Kirisako et al., 2000). The conjugation of Atg8 itself involves the sequential activity of the E1-like Atg7 and the E2-like Atg3 enzymes as well as the E3-like ligase complex Atg12–Atg5–Atg16 (Geng and Klionsky, 2008). Apart from its function during phagophore expansion, Atg8 also coordinates selection of cargo through its interaction with cargo receptors such as Atg19 or Atg32 (Fig. 1C). Moreover, Atg8 promotes sealing of phagophores and fusion with the yeast vacuole (Fig. 1D,E) (Abeliovich et al., 2000; Kaufmann et al., 2014; Kondo-Okamoto et al., 2012; Nakatogawa et al., 2007; Noda et al., 2010; Suzuki et al., 2010; Xie et al., 2008). Shortly before fusion of the two organelles occurs, the cytosolic accessible pool of Atg8 is cleaved from the membrane by Atg4 (Fig. 1D) (Abreu et al., 2017; Kirisako et al., 2000; Yu et al., 2012). This ‘uncoating reaction’ is followed by tethering of autophagosomes to the vacuole through the homotypic fusion and vacuole protein sorting (HOPS) complex and SNARE-mediated fusion of the outer autophagosomal and the vacuolar membranes (Fig. 1E) (Nair et al., 2011; Rieder and Emr, 1997). Although autophagy is well understood at a molecular level in yeast, mechanistic insights into the process as well as its spatiotemporal regulation are still limited. The core autophagy machinery is highly conserved and close homologs of the yeast proteins are expressed in human cells (Yang et al., 2017) (see Box 1). Significant mechanistic differences between the pathways in these species reflect regulatory requirements that are needed for cooperation of cells in multicellular organisms (Boya et al., 2018) as well as autophagy-unrelated functions of ATG proteins in higher eukaryotes (Schaaf et al., 2016).

Box 1. Autophagy in human cells

In contrast to autophagy initiation in yeast that occurs at a single PAS (Cheong et al., 2008; Suzuki et al., 2001), many autophagosomes are formed simultaneously in human cells. Moreover, the endoplasmic reticulum (ER) has been shown to serve as a platform for nucleation of phagophores whereas the yeast PAS is in close proximity to the vacuole with the phagophore edges contacting the ER (Axe et al., 2008; Biazik et al., 2015; Hayashi-Nishino et al., 2009). The ER-domain at which autophagosomes are nucleated, termed omegasome, adopts a characteristic omega-like shape and is enriched in PtdIns(3)P (Hayashi-Nishino et al., 2009). Similar to yeast, the recruitment of the human ULK1 kinase complex (Atg1 kinase complex homolog) is the earliest event in autophagy and requires ATG13 to be dephosphorylated (Hosokawa et al., 2009; Kim et al., 2011; Puente et al., 2016). After translocation to the ER, the ULK1 complex recruits the PtdIns(3)-kinase complex III (Park et al., 2016, 2018), which consists of the enzyme VPS34 that converts PtdIns into PtdIns(3)P, VPS15, its autophagy-specific subunit ATG14 and the adaptor protein Beclin 1. The enrichment of the omegasome in PtdIns(3)P leads to the recruitment of double FYVE-containing protein 1 (DFCP1) and the WD repeat domain phosphoinositide-interacting (WIPI) proteins (Proikas-Cezanne et al., 2004). WIPI1 is involved in the nucleation of the phagophore membrane at the omegasome (Axe et al., 2008; Proikas-Cezanne et al., 2015). The expansion of the phagophore is regulated by WIPI2, which recruits ATG16L1 to initiate the conjugation of ATG8 homologs to the phagophore (Dooley et al., 2014). Although the Ub-like conjugation system in yeast and humans is highly conserved, human cells express six ATG8 homologs with non-redundant functions (Weidberg et al., 2010). According to their sequence homology, two ATG8 subfamilies with the members light chain 3A (LC3A), LC3B and LC3C as well as gamma-aminobutyric acid receptor-associated protein (GABARAP), GABARAPL1 and GABARAPL2/Golgi-associated ATPase enhancer of 16 kDa (GATE-16) can be distinguished (Schaaf et al., 2016). GABARAPs have been shown to play essential functions during both phagophore initiation and closure (Joachim et al., 2017; Wang et al., 2015; Weidberg et al., 2011). Deletion of LC3 homologs leads to the formation of smaller autophagosomes, suggesting that LC3s coordinate the expansion of autophagosomes (Nguyen et al., 2016; Weidberg et al., 2010), a process that also requires cooperation with the ATG9 compartment (Karanasios et al., 2016). Furthermore, all ATG8 homologs target specific cargo to the phagophore based on their interaction with a linear peptide motif, LC3-interacting region (LIR), that is present in most autophagy receptors (Rogov et al., 2014). After cargo has been confined by the expanded phagophore, the membrane is sealed giving rise to the double-membrane-surrounded autophagosome that fuses with lysosomes (Itakura et al., 2012; Jiang et al., 2014).

Many insights into the nucleation of autophagosomes, the selection of cargo and its tethering to autophagic membranes as well as autophagosome completion and fusion with lysosomes have been revealed by in vitro reconstitution methods. The advantages of in vitro approaches in characterizing complex biological pathways were first recognized by Günter Blobel, who developed a cell-free system to recapitulate the translocation of proteins across the ER membrane (Blobel and Sabatini, 1971). A similar approach was used in seminal studies by Randy Schekman, which led to the identification of components of the secretory pathway (Rexach and Schekman, 1991). Advances in membrane physics and recombinant protein production made it possible to reconstitute entire biological pathways from purified components in the test tube. Here, we highlight such reconstitution approaches and summarize how they contributed to our current understanding of autophagy in yeast and humans.

Out of nowhere – nucleation of the phagophore in yeast and humans

The coordinated activity of the Atg1 (Ulk1 in mammals) kinase complex, the PI3K complex and the Atg9 compartment is required to initiate autophagy in yeast and humans. The two subunits Atg17 and Atg11 are major scaffolds for the assembly of two distinct Atg1 kinase complexes that function in selective (Atg11) or non-selective (Atg17) autophagy in yeast (Kabeya et al., 2005, 2009; Kamber et al., 2015; Yorimitsu and Klionsky, 2005). Moreover, both proteins recruit the transmembrane protein Atg9 to the PAS, linking autophagy initiation to nucleation of the phagophore (He et al., 2006; Sekito et al., 2009). First insights into membrane-binding properties of the Atg1 kinase complex came from in vitro reconstitutions of a minimal complex, comprising Atg17, Atg31 and Atg29, as well as fragments of Atg1 and Atg13 (Ragusa et al., 2012). In experiments using Folch liposomes with different diameters as membrane substrate (see Box 2), the C-terminal domain of Atg1 was shown to bind to membranes in a curvature-dependent manner, preferring liposomes with high curvature that corresponds to vesicle diameters of ∼30 nm (Ragusa et al., 2012). Atg9 vesicles that are recruited by the Atg1 kinase complex in vivo possess similar diameters, suggesting that curvature sensing by Atg1 contributes to the selective recruitment of these vesicles.

Box 2. Membrane model systems in synthetic biology

Model membranes are composed of synthetic phospholipids with defined compositions or lipid extracts with complex lipid mixtures. A widely applied phospholipid extract is derived from brain tissues based on the Folch extraction method (Folch et al., 1957). The lipids are, depending on their hydrophobicity, dissolved in chloroform or chloroform:methanole(:water) mixtures. Liposomes of various sizes can be generated by producing a thin film of dried lipids on glass surfaces. Multilamellar vesicles of various sizes are spontaneously formed upon hydration of lipid films with buffer solutions. Unilamellar vesicles can be generated by sonication (small unilamellear vesicles, SUVs) or extrusion through filters with defined pore sizes (large unilamellar vesicles, LUVs) (Mui and Hope, 2009). Giant unilamellar vesicles (GUVs) can be formed by electroformation (Angelova and Dimitrov, 1986). SUVs and LUVs are widely used membrane structures to investigate protein-lipid interactions or to incorporate transmembrane proteins. GUVs are often used to investigate protein binding and protein mobility based on fluorescence microscopy using fluorescent-labeled, recombinant proteins (Wollert, 2012). The dynamics of proteins or lipids within membranes or at the surface of membranes can also be investigated using fluorescence correlation spectroscopic techniques (Ries and Schwille, 2008). In contrast to liposomes, which are ‘free-standing membranes’, supported lipid bilayers (SLBs) are formed on a solid support (for example glass or mica). SLBs are produced from SUVs that are spontaneously fusing upon contact with the surface of the support (Hardy et al., 2013). The advantage of SLBs is that high-resolution techniques such as atomic force microscopy or total internal reflection microscopy can be applied. However, the diffusion rates of lipids and membrane proteins are significantly lower in SLBs compared to free-standing membranes.

Furthermore, mutational analysis and immunoprecipitation suggested that both Atg17 and Atg13 bind Atg9 (Sekito et al., 2009; Suzuki et al., 2015), although which of the two proteins directly interacted with Atg9 was unclear. This was addressed by in vitro reconstitutions using artificial Atg9 vesicles and purified subunits of the Atg1 kinase complex: Atg9 vesicles were generated by incorporating recombinant and purified Atg9core, which contains the highly conserved transmembrane region of Atg9 but lacks the unstructured N- and C-termini, into small unilamellar vesicles (SUVs) (see Box 2). Floatation experiments of these vesicles with purified Atg1 kinase complex subunits (see Box 3) revealed that Atg17 physically interacts with Atg9, suggesting that the binding of Atg9 by the Atg1 kinase complex mainly depends on Atg17 (Rao et al., 2016). Furthermore, the crystal structure of the Atg17–Atg31–Atg29 subcomplex revealed two interesting properties: first, that Atg17 is a highly elongated crescent-shaped molecule that forms tail-to-tail dimers; second, that Atg31 directly binds Atg17 and both Atg31 and Atg29 occupy the central region of the Atg17 crescent (Ragusa et al., 2012). A plausible explanation for this unusual architecture was, again, revealed by reconstitution approaches (Rao et al., 2016). The Atg17 dimer was found to bind two Atg9 vesicles independently and electron microscopy revealed that these vesicles were tightly tethered to each other (Fig. 1A). The Atg17–Atg31–Atg29 subcomplex was, however, not able to tether such vesicles, although the subcomplex retained a limited capacity to bind Atg9. This suggests that the prominent localization of Atg31 and Atg29 that has been observed in the crystal structure inhibits binding of Atg17 to Atg9.

Box 3. Experimental approaches involving artificial membranes

Vesicles are particularly useful to study protein–lipid interactions as well as interactions of membrane proteins with soluble factors. Experiments are based on the physical separation of membrane vesicles from unbound proteins in order to quantify bound and unbound protein fractions. This can be achieved by sedimentation assays, in which both fractions are separated by ultracentrifugation, which sediments vesicles much faster than soluble proteins (Julkowska et al., 2013). The disadvantage of this approach is that protein aggregates co-sediment, leading to contamination of the vesicle fraction with unbound proteins. This problem can be circumvented by applying floatation assays, which are based on centrifugation of the sample in a density gradient. Membranes have a much lower density than proteins, allowing them to ‘float’ to the lightest top fraction of a density gradient. Bound proteins can ‘co-float’ together with membrane vesicles, whereas unbound proteins remain in the dense fraction and protein aggregate sediment. This allows all species to be separated (Busse et al., 2016). The drawback of the physical separation of unbound and bound fractions is the perturbation of the biophysical equilibrium and the dissociation of weak interactions. In order to study protein–lipid interactions under equilibrated conditions, fluorescence-based methods can be applied. One such method utilizes GUVs with diameters of 10 to 100 µm and the incorporation of fluorescent lipids. In combination with fluorescent-labeled proteins, binding can be observed in real time by imaging GUVs upon addition of proteins (Sezgin and Schwille, 2012). Moreover, fluorescent recovery after photobleaching (FRAP) of the membrane-bound protein fraction reveals insights into the mobility of proteins on the membrane as well as dynamics of the exchange of bound and unbound proteins (Rayan et al., 2010). Fluorescence correlation and cross-correlation spectroscopy can be used to determine the two-dimensional diffusion coefficients of membrane-bound proteins and fluorescent lipids independently from each other (Bacia et al., 2014). The interaction of fluorescent proteins with membranes can be studied at a single-molecule level by combining reconstitution on supported lipid bilayers with total internal reflection (TIRF) microscopy (Kiessling et al., 2017). Furthermore, new, ultrafast atomic force microscopes allow assembly of macromolecular machines as well as their dynamics to be structurally characterized with unmatched spatial resolution of height profiles in real time (Chiaruttini et al., 2015). Electron microscopy, electron tomography and subtomogram averaging are, in turn, powerful methods to reveal static high-resolution structures of proteins on membranes. Structural characterization of large membrane proteins by EM is achieved through nano-discs, which are lipid bilayer islands that are surrounded by amphipathic proteins, such as apolioprotein A1 (Thonghin et al., 2018).

Embedded Image

Upon induction of autophagy, the pentameric Atg1 kinase complex assembles at the PAS in vivo (Kabeya et al., 2005). In agreement with this observation, interaction studies with purified components revealed that this complex binds Atg9 and tethers Atg9 vesicles in vitro (Fig. 1A) (Rao et al., 2016). The combination of in vivo experiments, crystal structures and in vitro reconstitution of the Atg1 kinase complex thus provided a detailed model for its function during autophagy initiation. However, the high demand of lipids for nucleation and expansion of autophagosomes cannot be fulfilled by just two Atg9 vesicles, suggesting that the early-stage PAS contains more than one Atg1 kinase complex and multiple tethered Atg9 vesicles. A combinatorial in vitro and in vivo approach revealed that Atg13 has two Atg17-binding sites. One of these sites activates Atg17–Atg31–Atg29 within the same complex whereas the other binding site links two Atg1 kinase complexes together by binding Atg17 of another complex (Yamamoto et al., 2016). This crosslinking allows the formation of larger super-molecular assemblies and thus provides the means to recruit and tether multiple Atg9 vesicles in order to nucleate the phagophore. This model is supported by fluorescence-based quantifications in vivo, which demonstrated that 20 to 60 Atg1 kinase complexes are recruited to the PAS upon induction of autophagy (Geng and Klionsky, 2008; Köfinger et al., 2015; Sweeney and Holzbaur, 2018).

The inhibition of Atg17 by Atg31–Atg29 limits its function to conditions under which autophagy is induced. However, selective cargo, such as the vacuolar aminopeptidase 1 (Ape1), is delivered to the vacuole even under non-induced conditions and independently of Atg17 (Shintani et al., 2002). The initiation complex in selective autophagy consists of Atg1, Atg13 and the scaffolding protein Atg11 (Kim et al., 2001). In vitro reconstitution experiments using Atg11 purified from yeast cell lysates indicated that the major function of Atg11 involves activation of Atg1. By using an engineered Atg1 kinase that metabolized an ATPγS analog instead of conventional ATP, that study revealed that the activation of Atg1 depends not only on Atg11, but requires the formation of a complex of Atg1 with cargo, cargo receptor and Atg11 (Kamber et al., 2015). However, the kinase activity of Atg1 had previously been found to be important for autophagy expansion, but dispensible for the nucleation of autophagosomes (Cheong and Klionsky, 2008). This suggests that the Atg1–Atg11 complex has two functions in autophagy: promoting autophagy initiation and regulating phagophore expansion. The function of Atg11 during nucleation of selective autophagosomes might depend on properties that are similar to those of Atg17, including the recruitment of Atg9 vesicles to the PAS (Backues and Klionsky, 2012).

A lipid diet for autophagy – expansion of the phagophore membrane

A key step during the phagophore initiation to expansion transition is the acquisition of Atg8 (LC3 proteins; see Box 1), a process that involves recruitment of the conjugation machinery to the phagophore. Key factors in this are the two PtdIns(3)P-binding proteins WIPI2 (mammals) and Atg21 (yeast), which recruit ATG16L1 (mammals) and Atg16 (yeast) to the phagophore (Fig. 2A). Both WIPI2 and Atg21 thus promote expansion by coupling Atg8/LC3 lipidation to PtdIns(3)P-containing phagophores (Dooley et al., 2014; Juris et al., 2015). In order to identify the membrane source that becomes the substrate for ATG8 conjugation, a cell-free reconstitution approach combined cytosol fractions of wild-type cells with membrane fractionation from ATG5-knockout cell lysates (Ge et al., 2013). Owing to the absence of ATG5, these membranes were devoid of LC3-conjugated PE. Combining them with wild-type cytosol complemented ATG5 and initiated lipidation of LC3. The fraction that was enriched in membranes from the ER–Golgi intermediate compartment was found to be the best substrate for LC3 lipidation, implicating that this compartment donates membrane for autophagosome biogenesis (Ge et al., 2013). By applying a similar approach, coat protein complex II (COPII) has been found to mediate the budding of vesicles from the ER–Golgi intermediate compartment upon autophagy induction, suggesting that such vesicles contribute to the formation of autophagosomes (Ge et al., 2014). Furthermore, several other studies have reported that COPII is required for initiation and expansion of autophagosomes (Fig. 2B) (Ge et al., 2017; Graef et al., 2013; Stadel et al., 2015). Whether COPII vesicles transport membranes from the ER to autophagosomes remains, however, to be investigated.

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

Expansion of autophagosomes requires spatiotemporal coordination of many processes. (A) The human WIPI2 protein binds PtdIns(3)P at the outer face of the phagophore and recruits the ATG12–ATG5–ATG16L1 complex, which facilitates the transfer of ATG8 proteins from ATG3 to PE of the phagophore membrane. (B) The yeast Atg1 kinase complex strictly localizes to the phagophore rim, whereas Atg1 is also recruited to the phagophore membrane (by binding Atg8). The Atg1 kinase complex might be involved in coordinating fusion of vesicles with the phagophore rim. Expansion of the membrane requires transport of membranes from different sources by vesicular carriers, such as Atg9 or COPII vesicles. (C) The amphipathic helix of ATG14 targets the human PI3K complex to the phagophore rim where it converts PtdIns into PtdIns(3)P. It is unknown how the asymmetric distribution of PI3P, which is only found in the outer membrane of the phagophore, is achieved and maintained. (D) Human ATG3 possesses an ALPS motif, which might target a subpopulation of ATG3 to the phagophore rim. This localization allows ATG8 to reach the convex (outer) and concave (inner) face of the phagophore after conjugation. Insertion of the ALPS motif of ATG3 or the amphipathic helix of ATG14 into the membrane is facilitated by lipid-packaging defects that are caused by strong bending of the membrane at the rim of phagophores.

The most likely region of the phagophore to which COPII and other donor vesicles might fuse is its highly bent membrane at the phagophore rim (Nguyen et al., 2017). This suggests that factors that are involved in this process are targeted to this membrane region by sensing membrane curvature (Fig. 2B,D). The localization of Atg proteins at the phagophore in yeast was analyzed by inducing the assembly of a giant ApeI substrate through overexpression of ApeI. Under these conditions, a large spherical ApeI structure is formed in the cytoplasm at which the phagophore membrane could be visualized. It was shown that subunits of the Atg1 kinase and PI3K complexes, as well as Atg9, colocalize with the edges of the phagophore, whereas Atg1 itself and the Ub-like conjugation system cover the entire membrane (Suzuki et al., 2013). Thus, autophagy initiation complexes are found exclusively at the edges of phagophores, whereas the phagophore expansion machinery is more or less evenly distributed on yeast phagophores. The spatially restricted localization of Atg17 and Atg9 implies that Atg17 tethers Atg9 vesicles not only to each other upon autophagy induction, but also to the phagophore rim during expansion (Fig. 2B).

The localization of the PI3K complex to the rim of phagophores can be explained by curvature-sensing motifs in VPS34 (also known as PIK3C3) and ATG14 that have been identified in vitro using large unilamellar vesicles (LUVs) with different sizes (Fan et al., 2011; Miller et al., 2010). The curvature-sensing property of ATG14 was found to be counteracted by increasing concentrations of PtdInsPs. This suggests that during autophagy initiation, a positive-feedback loop enriches ATG14 at the PtdIns(3)P-containing omegasome, whereas during phagophore expansion, incoming PtdIns from donor vesicles are phosphorylated in a concerted manner at the phagophore rim (Fan et al., 2011) (Fig. 2C).

Interestingly, membrane curvature-sensing is not only restricted to components of the initiation machinery: an in vitro reconstitution of the human Ub-conjugation machinery identified ATG3 as a curvature sensor. As shown for ATG14, ATG3 contains an amphipathic lipid-packing sensor (ALPS) motif, which forms, upon contact with lipid bilayers, an amphipathic helix that partially inserts into membranes (Nath et al., 2014). The penetration of this helix is promoted by lipid-packaging defects in the bilayer of highly curved membranes, explaining why such motifs are curvature sensors (Antonny, 2011; Bigay et al., 2005) (Fig. 2D). The localization of ATG3 at the phagophore rim spatially restricts conjugation of ATG8, which allows incoming membranes during expansion to be decorated with ATG8 in a spatiotemporally concerted manner. Moreover, ATG8 is found on both membranes of the phagophore, but the high curvature of its rim forms an effective diffusion barrier for lipids and thus for lipidated ATG8s as well (Domanov et al., 2011). Targeting ATG3 to the rim permits the ATG8 proteins to reach both faces of the phagophore after conjugation (Fig. 2D).

In contrast to these observations in vitro, yeast Atg3 has been found to be evenly distributed at the entire area of the phagophore in vivo (Ngu et al., 2015). This indicates that curvature sensing by ATG3 can be overruled by other factors such as the ATG12–ATG5–ATG16L1 complex, which is known to recruit ATG3 in vivo (Hanada et al., 2007). ATG16L1 also covers the surface of phagophores, but its localization is confined to the outer phagophore membrane (Mizushima et al., 2003). The restriction of ATG16L1 to one face of the phagophore might be a consequence of the asymmetric distribution of PtdIns(3)P in human cells (Fig. 2C). PtdIns(3)P is enriched in the outer membrane and bound by WIPI2 (Cheng et al., 2014), which also physically interacts with ATG16L1 (Dooley et al., 2014). How the asymmetric distribution of PtdIns(3)P is achieved and maintained remains to be shown. Moreover, the functional consequence of the asymmetry is currently unclear since in yeast, PtdIns(3)P is enriched at the inner and not at the outer membrane (Cheng et al., 2014).

The components and activity of the autophagy-specific conjugation system were revealed in landmark in vitro reconstitution studies from the laboratory of Yoshinori Ohsumi, which used purified proteins to recapitulate the enzymatic conjugation of Atg12 to Atg5 by Atg7 and Atg10 (Mizushima et al., 1998). Subsequently, a second conjugation system that targets Atg8 to membranes through its conjugation to PE was identified (Kirisako et al., 2000). Using LUVs as model membranes, this conjugation could be recapitulated with purified Atg8, Atg7 and Atg3 (Ichimura et al., 2004), and a functional connection of both conjugation reactions was revealed by the observation that Atg12–Atg5 promotes lipidation of Atg8 in vivo. Furthermore, reconstitution experiments of Atg8 lipidation in the presence of Atg12–Atg5 showed that the latter functions as an E3 ligase and promotes the transfer of Atg8 from the E2-like enzyme Atg3 to PE (Hanada et al., 2007).

The observation that, at least in human cells, the ATG12–ATG5–ATG16L1 complex localizes at the outer membrane of the expanding phagophore raises the question as to why the E3 ligase complex is retained on the phagophore after conjugation of ATG8 has taken place. This was addressed by a reconstitution of the entire yeast conjugation machinery on model membranes (Kaufmann et al., 2014). Atomic force microscopy of supported lipid bilayers (SLBs) (see Box 2) to which Atg8 was conjugated in the absence of Atg16 revealed that Atg8 associates with the Atg12–Atg5 conjugate in uniform particles composed of at least two subunits each. In the presence of Atg16, however, Atg8–Atg12–Atg5 became immobile and, instead of single particles, a continuous 12 nm thin membrane coat was detected on SLBs. The meshwork-like appearance of the coat was caused by the 16 nm-long coiled-coil domain of Atg16 that spatially separated two Atg8–Atg12–Atg5 complexes (Fig. 1C). An Atg16 mutant that could not self-assemble into tail-to-tail dimers caused a remarkable autophagy defect: cells that expressed this mutant produced large phagophore structures with an unusually long persistence in the cytoplasm. Moreover, these phagophores did not transport autophagic cargo to the vacuole, implying that the formed membrane structures correspond to immature and non-productive autophagosomes (Kaufmann et al., 2014). In vivo data from human cells supported this model by showing that deletion of all human ATG8 homologs led to a maturation and closure defect of autophagosomes, although autophagy progressed at a reduced level (Nguyen et al., 2016; Tsuboyama et al., 2016).

Taken together, the combination of in vitro reconstitution experiments with in vivo experiments has revealed that the Atg8 conjugation system is an important regulator of phagophore expansion, but is not essential for autophagy. Which membranes promote expansion in response to different environmental conditions, how their transport is coordinated and how incoming membranes merge with the phagophore are questions that remain to be answered.

Fusion or fission? Maturation and sealing of the phagophore

Electron tomography of autophagic structures in human cells showed that the fully expanded phagophore adopts a spherical shape (Biazik et al., 2017). Sealing of the structure requires that the membrane rims of the remaining spherical pore come into very close proximity to eventually merge. How this process is coordinated and which proteins promote sealing of the phagophore remain unknown. In principle, the process should be similar to the fusion of two small vesicles of high membrane curvature or to the scission of a membrane neck that projects away from the cytoplasm into the lumen of organelles or the extracellular space. Research over the past decades has tried to clarify which of the two diametrically opposed mechanisms serve this purpose.

Initial efforts focused on the fusion model in yeast and human cells. Many fusion reactions in cells are carried out by SNARE proteins, which merge membranes of vesicles with those of organelles or the plasma membrane. To overcome the high energy barrier for membrane fusion, SNAREs from two opposite membranes form a four-strand helical bundle that brings the opposite membranes in such close proximity that fusion takes place (Poirier et al., 1998). However, a recent study reported that, although priming of SNARE proteins is required for their maturation, they are not involved in the formation of autophagosomes (Abada et al., 2017; Reggiori et al., 2004).

The lack of experimental evidence for a SNARE-mediated sealing mechanism has shifted the focus towards Atg proteins with potentially fusogenic properties. First indications for the involvement of yeast Atg8 in this process came from reconstitution experiments of the lipidation reaction on LUVs. Conjugation of Atg8 to LUVs resulted in massive clustering of the vesicles, which was macroscopically visible by an increase in the turbidity of the suspension (Nakatogawa et al., 2007). Further characterization of this process revealed that Atg8 molecules on opposite membranes interact with each other to promote tethering. Atg8-mediated fusion of theses membranes was, however, only observed if membranes with unphysiologically high levels of unsaturated phospholipids were used (Nair et al., 2011). This does not necessarily exclude the participation of Atg8 during phagophore closure since packaging defects of lipids lead to a destabilization of the membrane at the phagophore rim. However, additional experiments are needed to confirm the contribution of Atg8 in sealing of phagophores in vivo (Nair et al., 2011). Reconstitution reactions of human ATG8 homologs on model membranes revealed another potential mechanism by which ATG8 can promote fusion. The two ATG8 family members, light chain 3B (LC3B, also known as MAP1LC3B) and γ-aminobutyric acid receptor-associated protein L2 (GABARAPL2) possess highly basic N-terminal helices that are required and sufficient to tether and fuse membranes in vitro by binding negative charges of opposing phospholipid bilayers (Weidberg et al., 2011). Since lipidation of ATG8 might be restricted to the phagophore rim (Nath et al., 2014), these ATG8 proteins would be positioned at the right place to promote fusion (Fig. 2D). However, their small size would allow them to only bridge distances that are smaller than ∼5 nm. Spontaneous membrane fusion occurs will occur if membranes are as close as 2 nm (Kozlovsky and Kozlov, 2003), suggesting that ATG8 proteins can only catalyze the very last step of sealing at which the remaining phagophore pore is sufficiently constricted. Furthermore, other proteins, such as Atg16, have been found to tether membranes in in vitro reconstituted systems. As for the ATG8 proteins, tethering of model membranes was based on interactions of positively charged amino acids with acidic phospholipids (Romanov et al., 2012). Further studies thus need to clarify how these different tethering events contribute to autophagosome formation.

Recent progress in identifying the biophysical principles of membrane scission reactions indicates that sealing of the autophagosome does not necessarily require fusion, but rather scission of membranes (Knorr et al., 2015; Renard et al., 2018). Although intuitively less obvious, the topology of the phagophore pore is very similar to that of a membrane neck in membrane abscission processes during multivesicular body biogenesis or viral budding. Both scission reactions are carried out by the endosomal sorting complex required for transport (ESCRT) complex III (Wenzel et al., 2018; Wollert et al., 2009). In vitro reconstitutions of this process on giant unilamellar vesicles (GUVs) (see Box 2) using purified ESCRT components have demonstrated that the correct membrane topology is required and sufficient for cutting of the membrane by ESCRT-III (Wollert and Hurley, 2010). In vivo, ESCRT-III appears to cooperate with the AAA-ATPase VPS4 in order to sever not only membrane necks in multivesicular body biogenesis, but also during nuclear envelope assembly and midbody abscission during cell division, as well as for repair of damaged plasma or lysosomal membranes (Adell et al., 2017; Christ et al., 2017; Mierzwa et al., 2017). Owing to the diversity of cellular processes that rely on ESCRT-mediated membrane scission and that share a similar topology of the membrane neck that needs to be cut, a potential role of ESCRTs in autophagy was proposed. However, convincing experimental evidence was missing until a recent study reported that the ESCRT-III subunit-charged multivesicular body protein 2A (CHMP2A) is required for sealing of the phagophore membrane (Takahashi et al., 2018). In related reactions, ESCRT-III cooperates with upstream factors to generate membrane necks that are narrow enough to be cleaved by ESCRT-III. This suggests that – as discussed for a potential ATG8-mediated fusion reaction – the phagophore rim needs to be sufficiently constricted to become a substrate for ESCRT-III. Whether phagophore expansion inherently leads to a sufficiently constricted pore remains a challenging question. Furthermore, a collaboration of Atg8-mediated fusion and ESCRT-III-mediated scission mechanisms remain a possibility. Innovative experiments using physiologically relevant templates such as cup-shaped membranes, might be able to answer this question in the future.

Born to die – fusion of autophagosomes with lysosomes

The final step in autophagy requires the outer membrane of the autophagosome to fuse with the limiting membrane of the lysosome. Before this, autophagosomes need to be transported to lysosomes and both compartments need to be tethered to each other. Yeast autophagosomes form in the immediate vicinity of the vacuole, allowing their completion to be coordinated with fusion independently of microtubule-based transport mechanisms (Kirisako et al., 1999). In human cells, however, autophagosomes are formed at various locations (Jahreiss et al., 2008) and therefore need to be transported to the perinuclear region where lysosomes are located (Korolchuk et al., 2011). This process depends on microtubules, the minus-end-directed motor dynein, which mediates retrograde transport of cargo to lysosomes, but also the plus-end-directed kinesin motor KIF5B, which is important for autophagosome positioning and fusion with lysosomes (Cardoso et al., 2009; Kimura et al., 2008; Köchl et al., 2006). Advanced in vitro approaches have been based on fusion of single dynein or kinesin motors to fluorescent beads and their incubation with microtubule-coated coverslips (Svoboda and Block, 1994). Optical traps allowed movement of single motor proteins on microtubules to be recorded and forces in the pico-Newton range to be measured (Simmons, 1996): Dynein, which drives the anterograde transport of autophagosomes, moves with a speed of ∼0.7 µm/s and can exert a force up to 5 pN (King and Schroer, 2000; Toba et al., 2006). Neurons are particularly well suited for the study of transport processes in vivo. Most autophagosomes are produced at the distal axon of neurons and thus need to be transported to the soma, where lysosomes are located, over distances of 500–1000 µm (Maday and Holzbaur, 2014). Based on the measured velocity of dynein, it takes 12–24 min for an autophagosome to be transported from the distal axon to the soma. Compared to myosin-based transport on actin filaments, dynein-mediated trafficking is rather slow (Schott et al., 2002).

Having reached perinuclear lysosomes, autophagosomes need to be recognized as fusion partners. This requires selective markers on their surface to provide identity and one such marker, the SNARE protein syntaxin 17 (STX17), is recruited to autophagosomes after their completion (Itakura et al., 2012; Tsuboyama et al., 2016).

The activity of SNARE proteins is tightly regulated; before fusion occurs, organelles need to be brought in close vicinity. This tethering is mediated by the lysosomal HOPS complex, which binds autophagosomes and lysosomes through its interaction with the small GTPases such as ADP-ribosylation factor-like protein 8 (ARL8) and the Ras-related protein RAB2 in humans and Ypt7 (a RAB7 homolog) in yeast (Reggiori and Ungermann, 2017). The molecular architecture of the yeast HOPS complex was revealed by single-particle electron microscopy (Bröcker et al., 2012). The complex spans 30 nm, possesses Rab-binding sites (in Vsp41 and Vps39) on both ends and its headpiece contains the SNARE-interacting subunit Vps33. In vitro reconstitution experiments with LUVs revealed that the HOPS complex tethers membranes based on its interaction with Rab GTPases (Ho and Stroupe, 2015), but also through a curvature-dependent direct interaction with membrane vesicles (Ho and Stroupe, 2016). Moreover, the cooperation of HOPS, SNAREs, Ypt7 and the SNARE chaperones Sec17 and Sec18 is required to drive robust membrane fusion (Stroupe et al., 2009). The HOPS complex thus not only tethers autophagosomes to lysosomes, it also promotes SNARE assembly (Fig. 3A). In autophagy, the HOPS complex promotes fusion by cooperating with STX17 (Jiang et al., 2014) as well as with SNAP29 and VAMP8 (Fig. 3A) (Itakura et al., 2012). Mechanistic details on how SNAREs drive membrane fusion have been revealed using SNARE-containing LUVs. Examples include the specificity of SNARE proteins (Paumet et al., 2004) and the process of zippering into a four-strand helical bundle (Jakhanwal et al., 2017; Stein et al., 2009), as well as the process of membrane hemi-fusion and fusion (Hernandez et al., 2012).

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

Autophagosome–lysosome fusion and cargo capture by Atg8. (A) Fusion of autophagosomes and lysosomes is driven by the membrane-tethering HOPS complex and SNAREs. RAB7 (which has RAB7A and RAB7B forms) is a small GTPase that is recruited to autophagosomes and lysosomes. Direct or RAB effector-mediated interactions with RAB7 allow the HOPS complex to bind and tether both membranes. The two cytoplasmic SNAREs STX17 and SNAP29 are recruited to the phagophore after phagophore completion. ATG14 facilitates assembly and priming of the autophagic SNAREs. The HOPS complex promotes zippering of lysosomal and autophagic SNAREs into a four-strand helical bundle that drives membrane fusion. (B) The multiple functions of Atg8 on phagophores. The concave pool of Atg8 binds autophagy receptors to capture cargo. Depicted here is the interaction of the Ape1 complex (a cargo in the yeast cytoplasm-to-vacuole targeting pathway) with its receptor Atg19 and Atg8. Atg19 binds Atg16 in vitro, which might promote recruitment of the Atg12–Atg5–Atg16 complex to the phagophore and position it close to the phagophore rim, where it could stabilize Atg3. The amphipathic helix of Atg3 contributes to the rim localization of Atg3. As a result, conjugation of Atg8 to lipids at the rim of the phagophore is promoted, which would allow Atg8 to reach the inner (concave) and outer (convex) face of the phagophore. At the convex membrane, a scaffold composed of the Atg12–Atg5–Atg16 complex and Atg8 might promote and guide phagophore expansion.

Even in the presence of tethering factors, the spontaneous assembly of SNAREs is inefficient and controlled by Sec1/Munc18-like (SM) proteins (Gerber et al., 2008). Insights into the mechanism by which SM proteins promote assembly of SNAREs came from in vitro reconstitutions and structural characterizations: the SM protein Vps33 helps to assemble the t-SNARE motifs of SNAREs within the target membrane such that they are in register for interaction with the v-SNARE within the vesicle membrane (Baker et al., 2015). A similar assisted assembly of SNAREs was found in autophagy. Unexpectedly, however, ATG14, the component of PI3K complex that initiates autophagy, promotes this step (Fig. 3A). The structure of the SNARE–ATG14 complex revealed that the coiled-coil domain of ATG14 stabilizes the interaction of STX17 and SNAP29 to prime both SNAREs for zippering with the corresponding partner VAMP8 (Baker et al., 2015). Consequently, reconstitutions on LUVs showed that SNARE-mediated membrane fusion was strongly enhanced in the presence of ATG14 (Diao et al., 2015). Taken together, ATG14 appears to have different functions in autophagy: as a component of the PI3K complex, it promotes induction of autophagy at the ER, whereas its function in autophagosome–lysosome fusion is independent of the other PI3K complex subunits (Fig. 3A).

Before fusion of autophagosomes and lysosomes occurs, Atg proteins – including the cytoplasmic accessible pool of Atg8–PE or its human homologs – are released from the autophagosome. Recycling of Atg8 is achieved through proteolytic cleavage of the amide bond between the C-terminal glycine of Atg8 and the amine function of PE by Atg4 (Kirisako et al., 2000). The recruitment and activity of Atg4 needs to be tightly regulated to prevent premature cleavage of Atg8 (Abreu et al., 2017). This is achieved by Atg1/ULK1-mediated phosphorylation of Atg4 or its human homologs at a conserved serine residue near its catalytic site (Pengo et al., 2017; Sánchez-Wandelmer et al., 2017). The importance of this serine residue was confirmed by reconstituting the deconjugation reactions using SUVs with enzymatically conjugated Atg8 and incubating these with Atg4 or related serine mutants (Sánchez-Wandelmer et al., 2017). The observation that Atg1/ULK1 is present on phagophore membranes during their expansion and released shortly after autophagosome completion strongly implies that deconjugation of Atg8 proteins and fusion of autophagosomes with lysosomes are interdependent and precisely regulated processes (Fig. 1D,E) (Cebollero et al., 2012; Yu et al., 2012). Spatiotemporal insights into Atg4-mediated deconjugation were obtained using fluorescently labeled Atg8 conjugated to GUVs (Kaufmann et al., 2014). Atg4 efficiently recycles Atg8 from model membranes in vitro, even if Atg8 is engaged in a membrane scaffold with Atg12–Atg5–Atg16. Consequently, the scaffold formation, which is predicted to regulate phagophore expansion, and recruitment of Atg4 by Atg8 are mutually exclusive (Fig. 1D) (Kaufmann et al., 2014).

The deconjugation of human ATG8 proteins is more complex, given that six ATG8 homologs are conjugated to autophagosomes and four ATG4 homologs catalyze the priming and recycling reactions (Kabeya et al., 2004). The human ATG4B protein is the most potent isoform involved in priming human ATG8 proteins and efficiently cuts the LC3 family proteins GABARAP and GABARAPL2 (Li et al., 2011; Sou et al., 2006). Although the other ATG4 homologs are considerably slower in priming of human ATG8 proteins, they possess similar catalytic activities to ATG4B for deconjugating ATG8 proteins in vitro (Kauffman et al., 2018). The precise timing of the recycling reaction, as well as the physiological functions of the individual ATG4 homologs in human cells, remain to be characterized. Interestingly, depletion of Atg4 in yeast or ATG4 homologs in humans leads to the formation of considerably smaller autophagosomes and impact on the sealing of the phagophore (Fujita et al., 2008; Hirata et al., 2017; Nair et al., 2012). This suggests that deconjugation of Atg8 or its human homologs not only takes place shortly before, but also during phagophore expansion and sealing.

After autophagosomes and lysosomes have fused and autophagic cargo has been degraded, lysosomes need to be regenerated. The involved process has been termed autophagic lysosome reformation (ALR) and involves the formation of membrane extrusions from autolysosomes. Proto-lysosomes bud from theses tubes and their maturation restores the population of functional lysosomes (Chen and Yu, 2017).

Insights into the inside – cargo selection in autophagy

The fusion of the outer membrane of autophagosomes with lysosomes delivers autophagic cargo to the lysosomal lumen where it is degraded. Much attention has thus been devoted to reveal how specific cargo is selected and targeted to phagophores. This involves a family of conserved autophagy receptors, which bind cargo on the one hand and the phagophore on the other (Khaminets et al., 2016). Tethering of cargo to the phagophore depends on a LC3-interacting region (LIR)- or Atg8-family interacting motif (AIM)-mediated interaction of autophagy receptors with Atg8 family proteins (Lamark et al., 2017) (Fig. 3B). The classical LIR motif is characterized by two bulky hydrophobic residues that are separated by two random residues and flanked by acidic amino acids. More recently, a number of motifs that distinguish between different ATG8 family members have been identified, suggesting that binding of cargo receptors is more complex in higher eukaryotes (von Muhlinen et al., 2012; Rogov et al., 2017; Stolz et al., 2017). The interaction of purified yeast and human Atg8 proteins with peptides that contain the various LIR/AIM motifs has been investigated and the underlying structural, and to a certain extend regulatory, principles are well understood (Kim et al., 2016; Wild et al., 2013). Despite the important contributions of these in vitro binding studies, they have some limitations. The LIR motif of tectonin β-propeller repeat-containing protein 2 (TECPR2), for example, binds all human ATG8 proteins with similar affinity in vitro, but TECPR2 selectively interacts with LC3C (MAP1LC3C) in vivo (Stadel et al., 2015). Furthermore, increasing evidence suggests that lipidated and non-lipidated Atg8 proteins adapt distinct structural conformations that impact on their affinity to LIR motifs (Coyle et al., 2002; Ichimura et al., 2004; Kumeta et al., 2010; Nakatogawa et al., 2007), which should be systematically investigated in the future.

Another interesting insight into selectivity of autophagy was the finding that multiple Atg8-binding sites on Atg19 are critical for a tight tethering of the cargo ApeI to membranes in order to avoid uptake of other material (Sawa-Makarska et al., 2014). A related mechanism to achieve selective and exclusive uptake of distinct cargo was revealed by recapitulation of aggrephagy: the receptor p62 (also known as SQSTM1) oligomerizes upon binding to protein aggregates, which facilitates efficient tethering of such aggregates to LC3-conjugated membranes in vitro (Wurzer et al., 2015). However, cargo such as yeast ApeI does not only activate its corresponding cargo receptor Atg19, it also appears to initiate lipidation of Atg8 by recruiting the E3 ligase complex (Fig. 3B). This couples receptor-mediated tethering of cargo to Atg8 conjugation, providing a possible mechanism to spatiotemporally coordinate cargo tethering with expansion of the isolation membrane in a selective and exclusive manner (Fracchiolla et al., 2016).

Taken together, the combination of in vitro and in vivo studies greatly advanced our understanding of how cargo is tethered to membranes during selective autophagy. One of the most important questions that remain to be investigated concerns the two faces of autophagy – how can the cell transform a highly selective pathway that degrades defined cargo in an exclusive manner into a non-selective pathway that degrades bulk cytoplasm?

Conclusions and perspectives

In vitro reconstitutions of biological processes have proven to be powerful tools to reveal unprecedented insights into fundamental mechanisms in biology. The combination of different model membranes, such as vesicles and supported lipid bilayers (see Box 2), with structural, fluorescent-based, biophysical and biochemical methods (see Box 3) have allowed the dissection of molecular principles that govern autophagy induction, and phagophore expansion and maturation, as well as phagophore fusion with lysosomes. The advantage of in vitro systems to provide a chemically well-defined environment is, however, at the same time its strongest limitation. Often, biophysical properties of isolated components in vitro simply cannot be correlated one-to-one with their functions in vivo. This might reflect fundamental differences of artificial membranes, which are composed of pure phospholipids (see Box 2), and biological membranes, which are much more complex in terms of their lipid composition. Biological membranes contain also a substantial amount of membrane proteins that strongly impact on physical properties, spatial organization and shape of these membranes. Moreover, many experiments with reconstituted systems are ‘out of equilibrium’, relying on physical separation of components to analyze or quantify them independently (see Box 3). Examples include a vast number of binding studies with liposomes and recombinant proteins. In vivo systems are, however, mostly in chemical equilibrium and fine-tuned by variations in expression levels of proteins or physical compartmentalization of membranes into microdomains. A successful strategy to overcome these limitations is a combinatorial approach in which in vitro experiments are complemented by in vivo approaches. Quite often, these combinations create positive-feedback loops in which one method complements the limitations of the other. The resulting synergy will be well suited to tackle the most challenging remaining questions in autophagy in the future. Combined with new biophysical approaches, even more complicated aspects of autophagy can hopefully be reconstituted in vitro. The final goal in ‘reconstructing destruction’ remains to build an autophagosome from purified components in the test tube.

Acknowledgements

We thank Lisa Dempfle for comments on the manuscript.

Footnotes

  • Competing interests

    The authors declare no competing or financial interests.

  • Funding

    Research in the laboratory Membrane Biochemistry and Transport was supported by the European Research Council grant Autophagy in vitro (638603) and the Deutsche Forschungsgemeinschaft (WO1770/2-1).

  • © 2018. Published by The Company of Biologists Ltd

References

  1. ↵
    1. Abada, A.,
    2. Levin-Zaidman, S.,
    3. Porat, Z.,
    4. Dadosh, T. and
    5. Elazar, Z.
    (2017). SNARE priming is essential for maturation of autophagosomes but not for their formation. Proc. Natl. Acad. Sci. USA 114, 12749-12754. doi:10.1073/pnas.1705572114
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Abeliovich, H.,
    2. Dunn, W. A.,
    3. Kim, J. and
    4. Klionsky, D. J.
    (2000). Dissection of autophagosome biogenesis into distinct nucleation and expansion steps. J. Cell Biol. 151, 1025-1034. doi:10.1083/jcb.151.5.1025
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Abreu, S.,
    2. Kriegenburg, F.,
    3. Gómez-Sánchez, R.,
    4. Mari, M.,
    5. Sánchez-Wandelmer, J.,
    6. Skytte Rasmussen, M.,
    7. Soares Guimarães, R.,
    8. Zens, B.,
    9. Schuschnig, M.,
    10. Hardenberg, R. et al.
    (2017). Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation. EMBO Rep. 18, 765-780. doi:10.15252/embr.201643146
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Adell, M. A. Y.,
    2. Migliano, S. M.,
    3. Upadhyayula, S.,
    4. Bykov, Y. S.,
    5. Sprenger, S.,
    6. Pakdel, M.,
    7. Vogel, G. F.,
    8. Jih, G.,
    9. Skillern, W.,
    10. Behrouzi, R. et al.
    (2017). Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding. eLife 6, e31652. doi:10.7554/eLife.31652
    OpenUrlCrossRef
  5. ↵
    1. Angelova, M. I. and
    2. Dimitrov, D. S.
    (1986). Liposome electroformation. Faraday Discuss. Chem. Soc. 81, 303-311. doi:10.1039/dc9868100303
    OpenUrlCrossRefWeb of Science
  6. ↵
    1. Antonny, B.
    (2011). Mechanisms of membrane curvature sensing. Annu. Rev. Biochem. 80, 101-123. doi:10.1146/annurev-biochem-052809-155121
    OpenUrlCrossRefPubMedWeb of Science
  7. ↵
    1. Ashford, T. P. and
    2. Porter, K. R.
    (1962). Cytoplasmic components in hepatic cell lysosomes. J. Cell Biol. 12, 198-202. doi:10.1083/jcb.12.1.198
    OpenUrlFREE Full Text
  8. ↵
    1. Axe, E. L.,
    2. Walker, S. A.,
    3. Manifava, M.,
    4. Chandra, P.,
    5. Roderick, H. L.,
    6. Habermann, A.,
    7. Griffiths, G. and
    8. Ktistakis, N. T.
    (2008). Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182, 685-701. doi:10.1083/jcb.200803137
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Bacia, K.,
    2. Haustein, E. and
    3. Schwille, P.
    (2014). Fluorescence correlation spectroscopy: principles and applications. Cold Spring Harb. Protoc. 2014, 709-725. doi:10.1101/pdb.top081802
    OpenUrlCrossRef
  10. ↵
    1. Backues, S. K. and
    2. Klionsky, D. J.
    (2012). Atg11: a Rab-dependent, coiled-coil membrane protein that acts as a tether for autophagy. Autophagy 8, 1275-1278. doi:10.4161/auto.21153
    OpenUrlCrossRefPubMed
  11. ↵
    1. Baker, R. W.,
    2. Jeffrey, P. D.,
    3. Zick, M.,
    4. Phillips, B. P.,
    5. Wickner, W. T. and
    6. Hughson, F. M.
    (2015). A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly. Science 349, 1111-1114. doi:10.1126/science.aac7906
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Biazik, J.,
    2. Ylä-Anttila, P.,
    3. Vihinen, H.,
    4. Jokitalo, E. and
    5. Eskelinen, E.-L.
    (2015). Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy 11, 439-451. doi:10.1080/15548627.2015.1017178
    OpenUrlCrossRefPubMed
  13. ↵
    1. Biazik, J.,
    2. Vihinen, H.,
    3. Jokitalo, E. and
    4. Eskelinen, E.-L.
    (2017). Ultrastructural characterization of phagophores using electron tomography on cryoimmobilized and freeze substituted samples. Methods Enzymol. 587, 331-349. doi:10.1016/bs.mie.2016.09.063
    OpenUrlCrossRef
  14. ↵
    1. Bigay, J.,
    2. Casella, J.-F.,
    3. Drin, G.,
    4. Mesmin, B. and
    5. Antonny, B.
    (2005). ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. EMBO J. 24, 2244-2253. doi:10.1038/sj.emboj.7600714
    OpenUrlAbstract
  15. ↵
    1. Blobel, G. and
    2. Sabatini, D. D.
    (1971). Ribosome-membrane interaction in eukaryotic cells. In Biomembranes, pp. 193-195 (ed: L. A. Manson). Boston, MA, Springer US.
  16. ↵
    1. Boya, P.,
    2. Codogno, P. and
    3. Rodriguez-Muela, N.
    (2018). Autophagy in stem cells: repair, remodelling and metabolic reprogramming. Development 145, dev146506. doi:10.1242/dev.146506
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Bröcker, C.,
    2. Kuhlee, A.,
    3. Gatsogiannis, C.,
    4. Balderhaar, H. J. K.,
    5. Hönscher, C.,
    6. Engelbrecht-Vandre, S.,
    7. Ungermann, C. and
    8. Raunser, S.
    (2012). Molecular architecture of the multisubunit homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Proc. Natl. Acad. Sci. USA 109, 1991-1996. doi:10.1073/pnas.1117797109
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Busse, R. A.,
    2. Scacioc, A.,
    3. Schalk, A. M.,
    4. Krick, R.,
    5. Thumm, M. and
    6. Kühnel, K.
    (2016). Analyzing protein-phosphoinositide interactions with liposome flotation assays. Methods Mol. Biol. 1376, 155-162. doi:10.1007/978-1-4939-3170-5_13
    OpenUrlCrossRef
  19. ↵
    1. Cardoso, C. M. P.,
    2. Groth-Pedersen, L.,
    3. Høyer-Hansen, M.,
    4. Kirkegaard, T.,
    5. Corcelle, E.,
    6. Andersen, J. S.,
    7. Jäättelä, M. and
    8. Nylandsted, J.
    (2009). Depletion of kinesin 5B affects lysosomal distribution and stability and induces peri-nuclear accumulation of autophagosomes in cancer cells. PLoS ONE 4, e4424. doi:10.1371/journal.pone.0004424
    OpenUrlCrossRefPubMed
  20. ↵
    1. Cebollero, E.,
    2. van der Vaart, A.,
    3. Zhao, M.,
    4. Rieter, E.,
    5. Klionsky, D. J.,
    6. Helms, J. B. and
    7. Reggiori, F.
    (2012). Phosphatidylinositol-3-phosphate clearance plays a key role in autophagosome completion. Curr. Biol. 22, 1545-1553. doi:10.1016/j.cub.2012.06.029
    OpenUrlCrossRefPubMed
  21. ↵
    1. Chen, Y. and
    2. Yu, L.
    (2017). Recent progress in autophagic lysosome reformation. Traffic 18, 358-361. doi:10.1111/tra.12484
    OpenUrlCrossRef
  22. ↵
    1. Cheng, J.,
    2. Fujita, A.,
    3. Yamamoto, H.,
    4. Tatematsu, T.,
    5. Kakuta, S.,
    6. Obara, K.,
    7. Ohsumi, Y. and
    8. Fujimoto, T.
    (2014). Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries. Nat. Commun. 5, 3207. doi:10.1038/ncomms4207
    OpenUrlCrossRefPubMed
  23. ↵
    1. Cheong, H. and
    2. Klionsky, D. J.
    (2008). Dual role of Atg1 in regulation of autophagy-specific PAS assembly in Saccharomyces cerevisiae. Autophagy 4, 724-726. doi:10.4161/auto.6375
    OpenUrlCrossRefPubMed
  24. ↵
    1. Cheong, H.,
    2. Yorimitsu, T.,
    3. Reggiori, F.,
    4. Legakis, J. E.,
    5. Wang, C.-W. and
    6. Klionsky, D. J.
    (2005). Atg17 regulates the magnitude of the autophagic response. Mol. Biol. Cell 16, 3438-3453. doi:10.1091/mbc.e04-10-0894
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Cheong, H.,
    2. Nair, U.,
    3. Geng, J. and
    4. Klionsky, D. J.
    (2008). The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae. Mol. Biol. Cell 19, 668-681. doi:10.1091/mbc.e07-08-0826
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Chiaruttini, N.,
    2. Redondo-Morata, L.,
    3. Colom, A.,
    4. Humbert, F.,
    5. Lenz, M.,
    6. Scheuring, S. and
    7. Roux, A.
    (2015). Relaxation of loaded ESCRT-III spiral springs drives membrane deformation. Cell 163, 866-879. doi:10.1016/j.cell.2015.10.017
    OpenUrlCrossRefPubMed
  27. ↵
    1. Christ, L.,
    2. Raiborg, C.,
    3. Wenzel, E. M.,
    4. Campsteijn, C. and
    5. Stenmark, H.
    (2017). Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery. Trends Biochem. Sci. 42, 42-56. doi:10.1016/j.tibs.2016.08.016
    OpenUrlCrossRefPubMed
  28. ↵
    1. Coyle, J. E.,
    2. Qamar, S.,
    3. Rajashankar, K. R. and
    4. Nikolov, D. B.
    (2002). Structure of GABARAP in two conformations: implications for GABA(A) receptor localization and tubulin binding. Neuron 33, 63-74. doi:10.1016/S0896-6273(01)00558-X
    OpenUrlCrossRefPubMedWeb of Science
  29. ↵
    1. De Duve, C.
    (1963). The lysosome. Sci. Am. 208, 64-73. doi:10.1038/scientificamerican0563-64
    OpenUrlCrossRefPubMed
  30. ↵
    1. Davis, S.,
    2. Wang, J. and
    3. Ferro-Novick, S.
    (2017). Crosstalk between the Secretory and Autophagy Pathways Regulates Autophagosome Formation. Dev. Cell. 41, 23-32. doi:10.1016/j.devcel.2017.03.015
    OpenUrlCrossRef
  31. ↵
    1. Diao, J.,
    2. Liu, R.,
    3. Rong, Y.,
    4. Zhao, M.,
    5. Zhang, J.,
    6. Lai, Y.,
    7. Zhou, Q.,
    8. Wilz, L. M.,
    9. Li, J.,
    10. Vivona, S. et al.
    (2015). ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature 520, 563-566. doi:10.1038/nature14147
    OpenUrlCrossRefPubMed
  32. ↵
    1. Dikic, I. and
    2. Elazar, Z.
    (2018). Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol. 19, 349-364. doi:10.1038/s41580-018-0003-4
    OpenUrlCrossRef
  33. ↵
    1. Domanov, Y. A.,
    2. Aimon, S.,
    3. Toombes, G. E. S.,
    4. Renner, M.,
    5. Quemeneur, F.,
    6. Triller, A.,
    7. Turner, M. S. and
    8. Bassereau, P.
    (2011). Mobility in geometrically confined membranes. Proc. Natl. Acad. Sci. USA 108, 12605-12610. doi:10.1073/pnas.1102646108
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Dooley, H. C.,
    2. Razi, M.,
    3. Polson, H. E. J.,
    4. Girardin, S. E.,
    5. Wilson, M. I. and
    6. Tooze, S. A.
    (2014). WIPI2 Links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1. Mol. Cell 55, 238-252. doi:10.1016/j.molcel.2014.05.021
    OpenUrlCrossRefPubMed
  35. ↵
    1. Fan, W.,
    2. Nassiri, A. and
    3. Zhong, Q.
    (2011). Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L). Proc. Natl. Acad. Sci. USA 108, 7769-7774. doi:10.1073/pnas.1016472108
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Folch, J.,
    2. Lees, M. and
    3. Sloane Stanley, G. H.
    (1957). A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497-509.
    OpenUrlFREE Full Text
  37. ↵
    1. Fracchiolla, D.,
    2. Sawa-Makarska, J.,
    3. Zens, B.,
    4. Ruiter, A.
    5. de, Zaffagnini, G.,
    6. Brezovich, A.,
    7. Romanov, J.,
    8. Runggatscher, K.,
    9. Kraft, C.
    , et al. (2016). Mechanism of cargo-directed Atg8 conjugation during selective autophagy. eLife 5, 18799. doi:10.7554/eLife.18544
    OpenUrlCrossRef
  38. ↵
    1. Fujita, N.,
    2. Hayashi-Nishino, M.,
    3. Fukumoto, H.,
    4. Omori, H.,
    5. Yamamoto, A.,
    6. Noda, T. and
    7. Yoshimori, T.
    (2008). An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol. Biol. Cell 19, 4651-4659. doi:10.1091/mbc.e08-03-0312
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Ge, L.,
    2. Melville, D.,
    3. Zhang, M. and
    4. Schekman, R.
    (2013). The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis. eLife 2013, e00947. doi:10.7554/eLife.00947
    OpenUrlCrossRef
  40. ↵
    1. Ge, L.,
    2. Zhang, M. and
    3. Schekman, R.
    (2014). Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment. eLife 3, e04135. doi:10.7554/eLife.04135
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Ge, L.,
    2. Zhang, M.,
    3. Kenny, S. J.,
    4. Liu, D.,
    5. Maeda, M.,
    6. Saito, K.,
    7. Mathur, A.,
    8. Xu, K. and
    9. Schekman, R.
    (2017). Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis. EMBO Rep. 18, 1586-1603. doi:10.15252/embr.201744559
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Geng, J. and
    2. Klionsky, D. J.
    (2008). The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. “Protein modifications: beyond the usual suspects” review series. EMBO Rep. 9, 859-864. doi:10.1038/embor.2008.163
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Gerber, S. H.,
    2. Rah, J.-C.,
    3. Min, S.-W.,
    4. Liu, X.,
    5. de Wit, H.,
    6. Dulubova, I.,
    7. Meyer, A. C.,
    8. Rizo, J.,
    9. Arancillo, M.,
    10. Hammer, R. E. et al.
    (2008). Conformational switch of syntaxin-1 controls synaptic vesicle fusion. Science 321, 1507-1510. doi:10.1126/science.1163174
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Gómez-Sánchez, R.,
    2. Rose, J.,
    3. Guimarães, R.,
    4. Mari, M.,
    5. Papinski, D.,
    6. Rieter, E.,
    7. Geerts, W. J.,
    8. Hardenberg, R.,
    9. Kraft, C.,
    10. Ungermann, C. et al.
    (2018). Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores. J. Cell Biol. 144, jcb.201710116.doi:10.1083/jcb.201710116
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Graef, M.,
    2. Friedman, J. R.,
    3. Graham, C.,
    4. Babu, M. and
    5. Nunnari, J.
    (2013). ER exit sites are physical and functional core autophagosome biogenesis components. Mol. Biol. Cell 24, 2918-2931. doi:10.1091/mbc.e13-07-0381
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Hanada, T.,
    2. Noda, N. N.,
    3. Satomi, Y.,
    4. Ichimura, Y.,
    5. Fujioka, Y.,
    6. Takao, T.,
    7. Inagaki, F. and
    8. Ohsumi, Y.
    (2007). The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J. Biol. Chem. 282, 37298-37302. doi:10.1074/jbc.C700195200
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Harding, T. M.,
    2. Morano, K. A.,
    3. Scott, S. V. and
    4. Klionsky, D. J.
    (1995). Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway. J. Cell Biol. 131, 591-602. doi:10.1083/jcb.131.3.591
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Hardy, G. J.,
    2. Nayak, R. and
    3. Zauscher, S.
    (2013). Model cell membranes: techniques to form complex biomimetic supported lipid bilayers via vesicle fusion. Curr. Opin. Colloid. Interface Sci. 18, 448-458. doi:10.1016/j.cocis.2013.06.004
    OpenUrlCrossRef
  49. ↵
    1. Hayashi-Nishino, M.,
    2. Fujita, N.,
    3. Noda, T.,
    4. Yamaguchi, A.,
    5. Yoshimori, T. and
    6. Yamamoto, A.
    (2009). A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat. Cell Biol. 11, 1433-1437. doi:10.1038/ncb1991
    OpenUrlCrossRefPubMedWeb of Science
  50. ↵
    1. He, C.,
    2. Song, H.,
    3. Yorimitsu, T.,
    4. Monastyrska, I.,
    5. Yen, W.-L.,
    6. Legakis, J. E. and
    7. Klionsky, D. J.
    (2006). Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast. J. Cell Biol. 175, 925-935. doi:10.1083/jcb.200606084
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Hernandez, J. M.,
    2. Stein, A.,
    3. Behrmann, E.,
    4. Riedel, D.,
    5. Cypionka, A.,
    6. Farsi, Z.,
    7. Walla, P. J.,
    8. Raunser, S. and
    9. Jahn, R.
    (2012). Membrane fusion intermediates via directional and full assembly of the SNARE complex. Science 336, 1581-1584. doi:10.1126/science.1221976
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Hirata, E.,
    2. Ohya, Y. and
    3. Suzuki, K.
    (2017). Atg4 plays an important role in efficient expansion of autophagic isolation membranes by cleaving lipidated Atg8 in Saccharomyces cerevisiae. PLoS ONE 12, e0181047. doi:10.1371/journal.pone.0181047
    OpenUrlCrossRef
  53. ↵
    1. Ho, R. and
    2. Stroupe, C.
    (2015). The HOPS/class C Vps complex tethers membranes by binding to one Rab GTPase in each apposed membrane. Mol. Biol. Cell 26, 2655-2663. doi:10.1091/mbc.e14-04-0922
    OpenUrlAbstract/FREE Full Text
  54. ↵
    1. Ho, R. and
    2. Stroupe, C.
    (2016). The HOPS/class C Vps complex tethers high-curvature membranes via a direct protein–membrane interaction. Traffic 17, 1078-1090. doi:10.1111/tra.12421
    OpenUrlCrossRef
  55. ↵
    1. Hosokawa, N.,
    2. Hara, T.,
    3. Kaizuka, T.,
    4. Kishi, C.,
    5. Takamura, A.,
    6. Miura, Y.,
    7. Iemura, S.-I.,
    8. Natsume, T.,
    9. Takehana, K.,
    10. Yamada, N. et al.
    (2009). Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol. Biol. Cell 20, 1981-1991. doi:10.1091/mbc.e08-12-1248
    OpenUrlAbstract/FREE Full Text
  56. ↵
    1. Ichimura, Y.,
    2. Kirisako, T.,
    3. Takao, T.,
    4. Satomi, Y.,
    5. Shimonishi, Y.,
    6. Ishihara, N.,
    7. Mizushima, N.,
    8. Tanida, I.,
    9. Kominami, E.,
    10. Ohsumi, M. et al.
    (2000). A ubiquitin-like system mediates protein lipidation. Nature 408, 488-492. doi:10.1038/35044114
    OpenUrlCrossRefPubMedWeb of Science
  57. ↵
    1. Ichimura, Y.,
    2. Imamura, Y.,
    3. Emoto, K.,
    4. Umeda, M.,
    5. Noda, T. and
    6. Ohsumi, Y.
    (2004). In vivo and in vitro reconstitution of Atg8 conjugation essential for autophagy. J. Biol. Chem. 279, 40584-40592. doi:10.1074/jbc.M405860200
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Itakura, E.,
    2. Kishi-Itakura, C. and
    3. Mizushima, N.
    (2012). The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151, 1256-1269. doi:10.1016/j.cell.2012.11.001
    OpenUrlCrossRefPubMedWeb of Science
  59. ↵
    1. Jahreiss, L.,
    2. Menzies, F. M. and
    3. Rubinsztein, D. C.
    (2008). The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes. Traffic 9, 574-587. doi:10.1111/j.1600-0854.2008.00701.x
    OpenUrlCrossRefPubMedWeb of Science
  60. ↵
    1. Jakhanwal, S.,
    2. Lee, C.-T.,
    3. Urlaub, H. and
    4. Jahn, R.
    (2017). An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly. EMBO J. 36, 1788-1802. doi:10.15252/embj.201696270
    OpenUrlCrossRef
  61. ↵
    1. Jao, C. C.,
    2. Ragusa, M. J.,
    3. Stanley, R. E. and
    4. Hurley, J. H.
    (2013). A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy. Proc. Natl. Acad. Sci. USA 110, 5486-5491. doi:10.1073/pnas.1220306110
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Jiang, P.,
    2. Nishimura, T.,
    3. Sakamaki, Y.,
    4. Itakura, E.,
    5. Hatta, T.,
    6. Natsume, T. and
    7. Mizushima, N.
    (2014). The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol. Biol. Cell 25, 1327-1337. doi:10.1091/mbc.e13-08-0447
    OpenUrlAbstract/FREE Full Text
  63. ↵
    1. Joachim, J.,
    2. Razi, M.,
    3. Judith, D.,
    4. Wirth, M.,
    5. Calamita, E.,
    6. Encheva, V.,
    7. Dynlacht, B. D.,
    8. Snijders, A. P.,
    9. O'Reilly, N.,
    10. Jefferies, H. B. J. et al.
    (2017). Centriolar satellites control GABARAP ubiquitination and GABARAP-mediated autophagy. Curr. Biol. 27, 2123-2136.e7. doi:10.1016/j.cub.2017.06.021
    OpenUrlCrossRef
  64. ↵
    1. Julkowska, M. M.,
    2. Rankenberg, J. M. and
    3. Testerink, C.
    (2013). Liposome-binding assays to assess specificity and affinity of phospholipid-protein interactions. Methods Mol. Biol. 1009, 261-271. doi:10.1007/978-1-62703-401-2_24
    OpenUrlCrossRefPubMed
  65. ↵
    1. Juris, L.,
    2. Montino, M.,
    3. Rube, P.,
    4. Schlotterhose, P.,
    5. Thumm, M. and
    6. Krick, R.
    (2015). PI3P binding by Atg21 organises Atg8 lipidation. EMBO J. 34, 955-973. doi:10.15252/embj.201488957
    OpenUrlCrossRefPubMed
  66. ↵
    1. Kabeya, Y.,
    2. Mizushima, N.,
    3. Ueno, T.,
    4. Yamamoto, A.,
    5. Kirisako, T.,
    6. Noda, T.,
    7. Kominami, E.,
    8. Ohsumi, Y. and
    9. Yoshimori, T.
    (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720-5728. doi:10.1093/emboj/19.21.5720
    OpenUrlAbstract
  67. ↵
    1. Kabeya, Y.,
    2. Mizushima, N.,
    3. Yamamoto, A.,
    4. Oshitani-Okamoto, S.,
    5. Ohsumi, Y. and
    6. Yoshimori, T.
    (2004). LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J. Cell. Sci. 117, 2805-2812. doi:10.1242/jcs.01131
    OpenUrlAbstract/FREE Full Text
  68. ↵
    1. Kabeya, Y.,
    2. Kamada, Y.,
    3. Baba, M.,
    4. Takikawa, H.,
    5. Sasaki, M. and
    6. Ohsumi, Y.
    (2005). Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy. Mol. Biol. Cell 16, 2544-2553. doi:10.1091/mbc.e04-08-0669
    OpenUrlAbstract/FREE Full Text
  69. ↵
    1. Kabeya, Y.,
    2. Noda, N. N.,
    3. Fujioka, Y.,
    4. Suzuki, K.,
    5. Inagaki, F. and
    6. Ohsumi, Y.
    (2009). Characterization of the Atg17-Atg29-Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 389, 612-615. doi:10.1016/j.bbrc.2009.09.034
    OpenUrlCrossRefPubMed
  70. ↵
    1. Kamber, R. A.,
    2. Shoemaker, C. J. and
    3. Denic, V.
    (2015). Receptor-bound targets of selective autophagy use a scaffold protein to activate the Atg1 kinase. Mol. Cell 59, 372-381. doi:10.1016/j.molcel.2015.06.009
    OpenUrlCrossRef
  71. ↵
    1. Karanasios, E.,
    2. Walker, S. A.,
    3. Okkenhaug, H.,
    4. Manifava, M.,
    5. Hummel, E.,
    6. Zimmermann, H.,
    7. Ahmed, Q.,
    8. Domart, M.-C.,
    9. Collinson, L. and
    10. Ktistakis, N. T.
    (2016). Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles. Nat. Commun. 7, 12420. doi:10.1038/ncomms12420
    OpenUrlCrossRefPubMed
  72. ↵
    1. Kaufmann, A.,
    2. Beier, V.,
    3. Franquelim, H. G. and
    4. Wollert, T.
    (2014). Molecular mechanism of autophagic membrane-scaffold assembly and disassembly. Cell 156, 469-481. doi:10.1016/j.cell.2013.12.022
    OpenUrlCrossRefPubMedWeb of Science
  73. ↵
    1. Kauffman, K. J.,
    2. Yu, S.,
    3. Jin, J.,
    4. Mugo, B.,
    5. Nguyen, N.,
    6. O'Brien, A.,
    7. Nag, S.,
    8. Lystad, A. H. and
    9. Melia, T. J.
    (2018). Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases. Autophagy 215, 1-19. doi:10.1080/15548627.2018.1437341
    OpenUrlCrossRef
  74. ↵
    1. Khaminets, A.,
    2. Behl, C. and
    3. Dikic, I.
    (2016). Ubiquitin-dependent and independent signals in selective autophagy. Trends Cell Biol. 26, 6-16. doi:10.1016/j.tcb.2015.08.010
    OpenUrlCrossRefPubMed
  75. ↵
    1. Kiessling, V.,
    2. Liang, B.,
    3. Kreutzberger, A. J. B. and
    4. Tamm, L. K.
    (2017). Planar supported membranes with mobile SNARE proteins and quantitative fluorescence microscopy assays to study synaptic vesicle fusion. Front. Mol. Neurosci. 10, 72. doi:10.3389/fnmol.2017.00072
    OpenUrlCrossRef
  76. ↵
    1. Kim, J.,
    2. Kamada, Y.,
    3. Stromhaug, P. E.,
    4. Guan, J.,
    5. Hefner-Gravink, A.,
    6. Baba, M.,
    7. Scott, S. V.,
    8. Ohsumi, Y.,
    9. Dunn, W. A. and
    10. Klionsky, D. J.
    (2001). Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole. J. Cell Biol. 153, 381-396. doi:10.1083/jcb.153.2.381
    OpenUrlAbstract/FREE Full Text
  77. ↵
    1. Kim, J.,
    2. Kundu, M.,
    3. Viollet, B. and
    4. Guan, K.-L.
    (2011). AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 1-22. doi:10.1038/ncb0111-1
    OpenUrlCrossRefPubMedWeb of Science
  78. ↵
    1. Kim, B.-W.,
    2. Kwon, D. H. and
    3. Song, H. K.
    (2016). Structure biology of selective autophagy receptors. BMB Rep. 49, 73-80. doi:10.5483/BMBRep.2016.49.2.265
    OpenUrlCrossRef
  79. ↵
    1. Kimura, S.,
    2. Noda, T. and
    3. Yoshimori, T.
    (2008). Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes. Cell Struct. Funct. 33, 109-122. doi:10.1247/csf.08005
    OpenUrlCrossRefPubMedWeb of Science
  80. ↵
    1. King, S. J. and
    2. Schroer, T. A.
    (2000). Dynactin increases the processivity of the cytoplasmic dynein motor. Nat. Cell Biol 2, 20-24. doi:10.1038/71338
    OpenUrlCrossRefPubMedWeb of Science
  81. ↵
    1. Kirisako, T.,
    2. Baba, M.,
    3. Ishihara, N.,
    4. Miyazawa, K.,
    5. Ohsumi, M.,
    6. Yoshimori, T.,
    7. Noda, T. and
    8. Ohsumi, Y.
    (1999). Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J. Cell Biol. 147, 435-446. doi:10.1083/jcb.147.2.435
    OpenUrlAbstract/FREE Full Text
  82. ↵
    1. Kirisako, T.,
    2. Ichimura, Y.,
    3. Okada, H.,
    4. Kabeya, Y.,
    5. Mizushima, N.,
    6. Yoshimori, T.,
    7. Ohsumi, M.,
    8. Takao, T.,
    9. Noda, T. and
    10. Ohsumi, Y.
    (2000). The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J. Cell Biol. 151, 263-276. doi:10.1083/jcb.151.2.263
    OpenUrlAbstract/FREE Full Text
  83. ↵
    1. Knorr, R. L.,
    2. Lipowsky, R. and
    3. Dimova, R.
    (2015). Autophagosome closure requires membrane scission. Autophagy 11, 2134-2137. doi:10.1080/15548627.2015.1091552
    OpenUrlCrossRefPubMed
  84. ↵
    1. Köchl, R.,
    2. Hu, X. W.,
    3. Chan, E. Y. W. and
    4. Tooze, S. A.
    (2006). Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. Traffic 7, 129-145. doi:10.1111/j.1600-0854.2005.00368.x
    OpenUrlCrossRefPubMedWeb of Science
  85. ↵
    1. Köfinger, J.,
    2. Ragusa, M. J.,
    3. Lee, I.-H.,
    4. Hummer, G., and
    5. Hurley, J. H.
    (2015). Solution structure of the Atg1 complex: implications for the architecture of the phagophore assembly site. Structure 23, 809-818. doi:10.1016/j.str.2015.02.012
    OpenUrlCrossRef
  86. ↵
    1. Kondo-Okamoto, N.,
    2. Noda, N. N.,
    3. Suzuki, S. W.,
    4. Nakatogawa, H.,
    5. Takahashi, I.,
    6. Matsunami, M.,
    7. Hashimoto, A.,
    8. Inagaki, F.,
    9. Ohsumi, Y. and
    10. Okamoto, K.
    (2012). Autophagy-related protein 32 acts as autophagic degron and directly initiates mitophagy. J. Biol. Chem. 287, 10631-10638. doi:10.1074/jbc.M111.299917
    OpenUrlAbstract/FREE Full Text
  87. ↵
    1. Korolchuk, V. I.,
    2. Saiki, S.,
    3. Lichtenberg, M.,
    4. Siddiqi, F. H.,
    5. Roberts, E. A.,
    6. Imarisio, S.,
    7. Jahreiss, L.,
    8. Sarkar, S.,
    9. Futter, M.,
    10. Menzies, F. M. et al.
    (2011). Lysosomal positioning coordinates cellular nutrient responses. Nat. Cell Biol. 13, 453-460. doi:10.1038/ncb2204
    OpenUrlCrossRefPubMedWeb of Science
  88. ↵
    1. Kozlovsky, Y. and
    2. Kozlov, M. M.
    (2003). Membrane fission: model for intermediate structures. Biophys. J. 85, 85-96. doi:10.1016/S0006-3495(03)74457-9
    OpenUrlCrossRefPubMedWeb of Science
  89. ↵
    1. Kumeta, H.,
    2. Watanabe, M.,
    3. Nakatogawa, H.,
    4. Yamaguchi, M.,
    5. Ogura, K.,
    6. Adachi, W.,
    7. Fujioka, Y.,
    8. Noda, N. N.,
    9. Ohsumi, Y. and
    10. Inagaki, F.
    (2010). The NMR structure of the autophagy-related protein Atg8. J. Biomol. NMR 47, 237-241. doi:10.1007/s10858-010-9420-1
    OpenUrlCrossRefPubMedWeb of Science
  90. ↵
    1. Lamark, T.,
    2. Svenning, S. and
    3. Johansen, T.
    (2017). Regulation of selective autophagy: the p62/SQSTM1 paradigm. Essays Biochem. 61, 609-624. doi:10.1042/EBC20170035
    OpenUrlAbstract/FREE Full Text
  91. ↵
    1. Li, M.,
    2. Hou, Y.,
    3. Wang, J.,
    4. Chen, X.,
    5. Shao, Z.-M. and
    6. Yin, X.-M.
    (2011). Kinetics comparisons of mammalian Atg4 homologues indicate selective preferences toward diverse Atg8 substrates. J. Biol. Chem. 286, 7327-7338. doi:10.1074/jbc.M110.199059
    OpenUrlCrossRefPubMed
  92. ↵
    1. Liang, X. H.,
    2. Jackson, S.,
    3. Seaman, M.,
    4. Brown, K.,
    5. Kempkes, B.,
    6. Hibshoosh, H. and
    7. Levine, B.
    (1999). Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402, 672-676. doi:10.1038/45257
    OpenUrlCrossRefPubMedWeb of Science
  93. ↵
    1. Maday, S. and
    2. Holzbaur, E. L. F.
    (2014). Autophagosome biogenesis in primary neurons follows an ordered and spatially regulated pathway. Dev. Cell. 30, 71-85. doi:10.1016/j.devcel.2014.06.001
    OpenUrlCrossRefPubMed
  94. ↵
    1. Mari, M.,
    2. Griffith, J.,
    3. Rieter, E.,
    4. Krishnappa, L.,
    5. Klionsky, D. J. and
    6. Reggiori, F.
    (2010). An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis. J. Cell Biol. 190, 1005-1022. doi:10.1083/jcb.200912089
    OpenUrlAbstract/FREE Full Text
  95. ↵
    1. Meiling-Wesse, K.,
    2. Epple, U. D.,
    3. Krick, R.,
    4. Barth, H.,
    5. Appelles, A.,
    6. Voss, C.,
    7. Eskelinen, E.-L. and
    8. Thumm, M.
    (2005). Trs85 (Gsg1), a component of the TRAPP complexes, is required for the organization of the preautophagosomal structure during selective autophagy via the Cvt pathway. J. Biol. Chem. 280, 33669-33678. doi:10.1074/jbc.M501701200
    OpenUrlAbstract/FREE Full Text
  96. ↵
    1. Mercer, T. J.,
    2. Gubas, A. and
    3. Tooze, S. A.
    (2018). A molecular perspective of mammalian autophagosome biogenesis. J. Biol. Chem. 293, 5386-5395. doi:10.1074/jbc.R117.810366
    OpenUrlAbstract/FREE Full Text
  97. ↵
    1. Mierzwa, B. E.,
    2. Chiaruttini, N.,
    3. Redondo-Morata, L.,
    4. Moser von Filseck, J.,
    5. König, J.,
    6. Larios, J.,
    7. Poser, I.,
    8. Müller-Reichert, T.,
    9. Scheuring, S.,
    10. Roux, A. et al.
    (2017). Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis. Nat. Cell Biol. 19, 787-798. doi:10.1038/ncb3559
    OpenUrlCrossRef
  98. ↵
    1. Miller, S.,
    2. Tavshanjian, B.,
    3. Oleksy, A.,
    4. Perisic, O.,
    5. Houseman, B. T.,
    6. Shokat, K. M. and
    7. Williams, R. L.
    (2010). Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34. Science 327, 1638-1642. doi:10.1126/science.1184429
    OpenUrlAbstract/FREE Full Text
  99. ↵
    1. Mizushima, N.,
    2. Noda, T.,
    3. Yoshimori, T.,
    4. Tanaka, Y.,
    5. Ishii, T.,
    6. George, M. D.,
    7. Klionsky, D. J.,
    8. Ohsumi, M. and
    9. Ohsumi, Y.
    (1998). A protein conjugation system essential for autophagy. Nature 395, 395-398. doi:10.1038/26506
    OpenUrlCrossRefPubMedWeb of Science
  100. ↵
    1. Mizushima, N.,
    2. Kuma, A.,
    3. Kobayashi, Y.,
    4. Yamamoto, A.,
    5. Matsubae, M.,
    6. Takao, T.,
    7. Natsume, T.,
    8. Ohsumi, Y. and
    9. Yoshimori, T.
    (2003). Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate. J. Cell. Sci. 116, 1679-1688. doi:10.1242/jcs.00381
    OpenUrlAbstract/FREE Full Text
  101. ↵
    1. Mui, B. and
    2. Hope, M.
    (2009). Formation of large unilamellar vesicles by extrusion. In Liposome Technology, Vol. I, pp. 55-65 (Ed: Gregory Gregoriadis). Informa Healthcare.
  102. ↵
    1. Nair, U.,
    2. Jotwani, A.,
    3. Geng, J.,
    4. Gammoh, N.,
    5. Richerson, D.,
    6. Yen, W.-L.,
    7. Griffith, J.,
    8. Nag, S.,
    9. Wang, K.,
    10. Moss, T. et al.
    (2011). SNARE proteins are required for macroautophagy. Cell 146, 290-302. doi:10.1016/j.cell.2011.06.022
    OpenUrlCrossRefPubMedWeb of Science
  103. ↵
    1. Nair, U.,
    2. Yen, W.-L.,
    3. Mari, M.,
    4. Cao, Y.,
    5. Xie, Z.,
    6. Baba, M.,
    7. Reggiori, F. and
    8. Klionsky, D. J.
    (2012). A role for Atg8–PE deconjugation in autophagosome biogenesis. Autophagy 8, 780-793. doi:10.4161/auto.19385
    OpenUrlCrossRefPubMedWeb of Science
  104. ↵
    1. Nakatogawa, H.,
    2. Ichimura, Y. and
    3. Ohsumi, Y.
    (2007). Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130, 165-178. doi:10.1016/j.cell.2007.05.021
    OpenUrlCrossRefPubMedWeb of Science
  105. ↵
    1. Nascimbeni, A. C.,
    2. Codogno, P. and
    3. Morel, E.
    (2017). Phosphatidylinositol-3-phosphate in the regulation of autophagy membrane dynamics. FEBS J. 284, 1267-1278. doi:10.1111/febs.13987
    OpenUrlCrossRef
  106. ↵
    1. Nath, S.,
    2. Dancourt, J.,
    3. Shteyn, V.,
    4. Puente, G.,
    5. Fong, W. M.,
    6. Nag, S.,
    7. Bewersdorf, J.,
    8. Yamamoto, A.,
    9. Antonny, B. and
    10. Melia, T. J.
    (2014). Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3. Nat. Cell Biol. 16, 415-424. doi:10.1038/ncb2940
    OpenUrlCrossRefPubMed
  107. ↵
    1. Ngu, M.,
    2. Hirata, E. and
    3. Suzuki, K.
    (2015). Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae. J. Biol. Chem. 290, 8146-8153. doi:10.1074/jbc.M114.626952
    OpenUrlAbstract/FREE Full Text
  108. ↵
    1. Nguyen, T. N.,
    2. Padman, B. S.,
    3. Usher, J.,
    4. Oorschot, V.,
    5. Ramm, G. and
    6. Lazarou, M.
    (2016). Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J. Cell Biol. 215, 857-874. doi:10.1083/jcb.201607039
    OpenUrlAbstract/FREE Full Text
  109. ↵
    1. Nguyen, N.,
    2. Shteyn, V. and
    3. Melia, T. J.
    (2017). Sensing membrane curvature in macroautophagy. J. Mol. Biol. 429, 457-472. doi:10.1016/j.jmb.2017.01.006
    OpenUrlCrossRef
  110. ↵
    1. Noda, N. N.,
    2. Ohsumi, Y. and
    3. Inagaki, F.
    (2010). Atg8-family interacting motif crucial for selective autophagy. FEBS Lett. 584, 1379-1385. doi:10.1016/j.febslet.2010.01.018
    OpenUrlCrossRefPubMedWeb of Science
  111. ↵
    1. Ohashi, Y. and
    2. Munro, S.
    (2010). Membrane delivery to the yeast autophagosome from the Golgi-endosomal system. Mol. Biol. Cell 21, 3998-4008. doi:10.1091/mbc.e10-05-0457
    OpenUrlAbstract/FREE Full Text
  112. ↵
    1. Park, J.-M.,
    2. Jung, C. H.,
    3. Seo, M.,
    4. Otto, N. M.,
    5. Grunwald, D.,
    6. Kim, K. H.,
    7. Moriarity, B.,
    8. Kim, Y.-M.,
    9. Starker, C.,
    10. Nho, R. S. et al.
    (2016). The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14. Autophagy 12, 547-564. doi:10.1080/15548627.2016.1140293
    OpenUrlCrossRefPubMed
  113. ↵
    1. Park, J.-M.,
    2. Seo, M.,
    3. Jung, C. H.,
    4. Grunwald, D.,
    5. Stone, M.,
    6. Otto, N. M.,
    7. Toso, E.,
    8. Ahn, Y.,
    9. Kyba, M.,
    10. Griffin, T. J. et al.
    (2018). ULK1 phosphorylates Ser30 of BECN1 in association with ATG14 to stimulate autophagy induction. Autophagy 14, 584-597. doi:10.1080/15548627.2017.1422851
    OpenUrlCrossRef
  114. ↵
    1. Paumet, F.,
    2. Rahimian, V. and
    3. Rothman, J. E.
    (2004). The specificity of SNARE-dependent fusion is encoded in the SNARE motif. Proc. Natl. Acad. Sci. USA 101, 3376-3380. doi:10.1073/pnas.0400271101
    OpenUrlAbstract/FREE Full Text
  115. ↵
    1. Pengo, N.,
    2. Agrotis, A.,
    3. Prak, K.,
    4. Jones, J. and
    5. Ketteler, R.
    (2017). A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B. Nat. Commun. 8, 9. doi:10.1038/s41467-017-00303-2
    OpenUrlCrossRefPubMed
  116. ↵
    1. Poirier, M. A.,
    2. Xiao, W.,
    3. Macosko, J. C.,
    4. Chan, C.,
    5. Shin, Y.-K. and
    6. Bennett, M. K.
    (1998). The synaptic SNARE complex is a parallel four-stranded helical bundle. Nat. Struct. Biol. 5, 765-769. doi:10.1038/1799
    OpenUrlCrossRefPubMedWeb of Science
  117. ↵
    1. Proikas-Cezanne, T.,
    2. Waddell, S.,
    3. Gaugel, A.,
    4. Frickey, T.,
    5. Lupas, A. and
    6. Nordheim, A.
    (2004). WIPI-1α (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy. Oncogene 23, 9314-9325. doi:10.1038/sj.onc.1208331
    OpenUrlCrossRefPubMedWeb of Science
  118. ↵
    1. Proikas-Cezanne, T.,
    2. Takacs, Z.,
    3. Dönnes, P. and
    4. Kohlbacher, O.
    (2015). WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome. J. Cell. Sci. 128, 207-217. doi:10.1242/jcs.146258
    OpenUrlAbstract/FREE Full Text
  119. ↵
    1. Puente, C.,
    2. Hendrickson, R. C. and
    3. Jiang, X.
    (2016). Nutrient-regulated phosphorylation of ATG13 inhibits starvation-induced autophagy. J. Biol. Chem. 291, 6026-6035. doi:10.1074/jbc.M115.689646
    OpenUrlAbstract/FREE Full Text
  120. ↵
    1. Ragusa, M. J.,
    2. Stanley, R. E. and
    3. Hurley, J. H.
    (2012). Architecture of the Atg17 complex as a scaffold for autophagosome biogenesis. Cell 151, 1501-1512. doi:10.1016/j.cell.2012.11.028
    OpenUrlCrossRefPubMedWeb of Science
  121. ↵
    1. Rao, Y.,
    2. Perna, M. G.,
    3. Hofmann, B.,
    4. Beier, V. and
    5. Wollert, T.
    (2016). The Atg1-kinase complex tethers Atg9-vesicles to initiate autophagy. Nat. Commun. 7, 10338. doi:10.1038/ncomms10338
    OpenUrlCrossRefPubMed
  122. ↵
    1. Ravikumar, B.,
    2. Duden, R. and
    3. Rubinsztein, D. C.
    (2002). Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. Mol. Genet. 11, 1107-1117. doi:10.1093/hmg/11.9.1107
    OpenUrlCrossRefPubMedWeb of Science
  123. ↵
    1. Rayan, G.,
    2. Guet, J.-E.,
    3. Taulier, N.,
    4. Pincet, F. and
    5. Urbach, W.
    (2010). Recent applications of fluorescence recovery after photobleaching (FRAP) to membrane bio-macromolecules. Sensors 10, 5927-5948. doi:10.3390/s100605927
    OpenUrlCrossRef
  124. ↵
    1. Reggiori, F. and
    2. Ungermann, C.
    (2017). Autophagosome maturation and fusion. J. Mol. Biol. 429, 486-496. doi:10.1016/j.jmb.2017.01.002
    OpenUrlCrossRef
  125. ↵
    1. Reggiori, F.,
    2. Wang, C.-W.,
    3. Nair, U.,
    4. Shintani, T.,
    5. Abeliovich, H. and
    6. Klionsky, D. J.
    (2004). Early stages of the secretory pathway, but not endosomes, are required for Cvt vesicle and autophagosome assembly in Saccharomyces cerevisiae. Mol. Biol. Cell 15, 2189-2204. doi:10.1091/mbc.e03-07-0479
    OpenUrlAbstract/FREE Full Text
  126. ↵
    1. Reggiori, F.,
    2. Shintani, T.,
    3. Nair, U. and
    4. Klionsky, D. J.
    (2005). Atg9 cycles between mitochondria and the pre-autophagosomal structure in yeasts. Autophagy 1, 101-109. doi:10.4161/auto.1.2.1840
    OpenUrlCrossRefPubMedWeb of Science
  127. ↵
    1. Renard, H.-F.,
    2. Johannes, L. and
    3. Morsomme, P.
    (2018). Increasing diversity of biological membrane fission mechanisms. Trends Cell Biol. 28, 274-286. doi:10.1016/j.tcb.2017.12.001
    OpenUrlCrossRef
  128. ↵
    1. Rexach, M. F. and
    2. Schekman, R. W.
    (1991). Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J. Cell Biol. 114, 219-229. doi:10.1083/jcb.114.2.219
    OpenUrlAbstract/FREE Full Text
  129. ↵
    1. Rieder, S. E. and
    2. Emr, S. D.
    (1997). A novel RING finger protein complex essential for a late step in protein transport to the yeast vacuole. Mol. Biol. Cell 8, 2307-2327. doi:10.1091/mbc.8.11.2307
    OpenUrlAbstract/FREE Full Text
  130. ↵
    1. Ries, J. and
    2. Schwille, P.
    (2008). New concepts for fluorescence correlation spectroscopy on membranes. Phys. Chem. Chem. Phys. 10, 3487-3497. doi:10.1039/b718132a
    OpenUrlCrossRefPubMed
  131. ↵
    1. Rogov, V.,
    2. Dötsch, V.,
    3. Johansen, T. and
    4. Kirkin, V.
    (2014). Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy. Mol. Cell 53, 167-178. doi:10.1016/j.molcel.2013.12.014
    OpenUrlCrossRefPubMedWeb of Science
  132. ↵
    1. Rogov, V. V.,
    2. Stolz, A.,
    3. Ravichandran, A. C.,
    4. Rios-Szwed, D. O.,
    5. Suzuki, H.,
    6. Kniss, A.,
    7. Löhr, F.,
    8. Wakatsuki, S.,
    9. Dötsch, V.,
    10. Dikic, I. et al.
    (2017). Structural and functional analysis of the GABARAP interaction motif (GIM). EMBO Rep. 18, 1382-1396. doi:10.15252/embr.201643587
    OpenUrlCrossRef
  133. ↵
    1. Romanov, J.,
    2. Walczak, M.,
    3. Ibiricu, I.,
    4. Schüchner, S.,
    5. Ogris, E.,
    6. Kraft, C. and
    7. Martens, S.
    (2012). Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation. EMBO J. 31, 4304-4317. doi:10.1038/emboj.2012.278
    OpenUrlCrossRefPubMed
  134. ↵
    1. Sánchez-Wandelmer, J.,
    2. Kriegenburg, F.,
    3. Rohringer, S.,
    4. Schuschnig, M.,
    5. Gómez-Sánchez, R.,
    6. Zens, B.,
    7. Abreu, S.,
    8. Hardenberg, R.,
    9. Hollenstein, D.,
    10. Gao, J. et al.
    (2017). Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation. Nat. Commun. 8, 295. doi:10.1038/s41467-017-00302-3
    OpenUrlCrossRef
  135. ↵
    1. Sawa-Makarska, J.,
    2. Abert, C.,
    3. Romanov, J.,
    4. Zens, B.,
    5. Ibiricu, I. and
    6. Martens, S.
    (2014). Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane-cargo apposition during selective autophagy. Nat. Cell Biol. 16, 425-433. doi:10.1038/ncb2935
    OpenUrlCrossRefPubMed
  136. ↵
    1. Schaaf, M. B. E.,
    2. Keulers, T. G.,
    3. Vooijs, M. A. and
    4. Rouschop, K. M. A.
    (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. FASEB J. 30, 3961-3978. doi:10.1096/fj.201600698R
    OpenUrlCrossRefPubMed
  137. ↵
    1. Schott, D. H.,
    2. Collins, R. N. and
    3. Bretscher, A.
    (2002). Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length. J. Cell Biol. 156, 35-39. doi:10.1083/jcb.200110086
    OpenUrlAbstract/FREE Full Text
  138. ↵
    1. Sekito, T.,
    2. Kawamata, T.,
    3. Ichikawa, R.,
    4. Suzuki, K. and
    5. Ohsumi, Y.
    (2009). Atg17 recruits Atg9 to organize the pre-autophagosomal structure. Genes Cells 14, 525-538. doi:10.1111/j.1365-2443.2009.01299.x
    OpenUrlCrossRefPubMed
  139. ↵
    1. Sezgin, E. and
    2. Schwille, P.
    (2012). Model membrane platforms to study protein-membrane interactions. Mol. Membr. Biol. 29, 144-154. doi:10.3109/09687688.2012.700490
    OpenUrlCrossRefPubMed
  140. ↵
    1. Shintani, T.,
    2. Huang, W.-P.,
    3. Stromhaug, P. E. and
    4. Klionsky, D. J.
    (2002). Mechanism of cargo selection in the cytoplasm to vacuole targeting pathway. Dev. Cell 3, 825-837. doi:10.1016/S1534-5807(02)00373-8
    OpenUrlCrossRefPubMedWeb of Science
  141. ↵
    1. Simmons, R.
    (1996). Molecular motors: single-molecule mechanics. Curr. Biol. 6, 392-394. doi:10.1016/S0960-9822(02)00504-3
    OpenUrlCrossRefPubMed
  142. ↵
    1. Sou, Y.,
    2. Tanida, I.,
    3. Komatsu, M.,
    4. Ueno, T. and
    5. Kominami, E.
    (2006). Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE-16. J. Biol. Chem. 281, 3017-3024. doi:10.1074/jbc.M505888200
    OpenUrlAbstract/FREE Full Text
  143. ↵
    1. Stadel, D.,
    2. Millarte, V.,
    3. Tillmann, K. D.,
    4. Huber, J.,
    5. Tamin-Yecheskel, B.-C.,
    6. Akutsu, M.,
    7. Demishtein, A.,
    8. Ben-Zeev, B.,
    9. Anikster, Y.,
    10. Perez, F. et al.
    (2015). TECPR2 cooperates with LC3C to regulate COPII-dependent ER export. Mol. Cell 60, 89-104. doi:10.1016/j.molcel.2015.09.010
    OpenUrlCrossRefPubMed
  144. ↵
    1. Stein, A.,
    2. Weber, G.,
    3. Wahl, M. C. and
    4. Jahn, R.
    (2009). Helical extension of the neuronal SNARE complex into the membrane. Nature 460, 525-528. doi:10.1038/nature08156
    OpenUrlCrossRefPubMedWeb of Science
  145. ↵
    1. Stolz, A.,
    2. Putyrski, M.,
    3. Kutle, I.,
    4. Huber, J.,
    5. Wang, C.,
    6. Major, V.,
    7. Sidhu, S. S.,
    8. Youle, R. J.,
    9. Rogov, V. V.,
    10. Dötsch, V. et al.
    (2017). Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins. EMBO J. 36, 549-564. doi:10.15252/embj.201695063
    OpenUrlCrossRef
  146. ↵
    1. Stroupe, C.,
    2. Hickey, C. M.,
    3. Mima, J.,
    4. Burfeind, A. S. and
    5. Wickner, W.
    (2009). Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components. Proc. Natl. Acad. Sci. USA 106, 17626-17633. doi:10.1073/pnas.0903801106
    OpenUrlAbstract/FREE Full Text
  147. ↵
    1. Suzuki, K.,
    2. Kirisako, T.,
    3. Kamada, Y.,
    4. Mizushima, N.,
    5. Noda, T. and
    6. Ohsumi, Y.
    (2001). The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO J. 20, 5971-5981. doi:10.1093/emboj/20.21.5971
    OpenUrlAbstract
  148. ↵
    1. Suzuki, K.,
    2. Kubota, Y.,
    3. Sekito, T. and
    4. Ohsumi, Y.
    (2007). Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes Cells 12, 209-218. doi:10.1111/j.1365-2443.2007.01050.x
    OpenUrlCrossRefPubMedWeb of Science
  149. ↵
    1. Suzuki, K.,
    2. Kondo, C.,
    3. Morimoto, M. and
    4. Ohsumi, Y.
    (2010). Selective transport of alpha-mannosidase by autophagic pathways: identification of a novel receptor, Atg34p. J. Biol. Chem. 285, 30019-30025. doi:10.1074/jbc.M110.143511
    OpenUrlAbstract/FREE Full Text
  150. ↵
    1. Suzuki, K.,
    2. Akioka, M.,
    3. Kondo-Kakuta, C.,
    4. Yamamoto, H. and
    5. Ohsumi, Y.
    (2013). Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae. J. Cell. Sci. 126, 2534-2544. doi:10.1242/jcs.122960
    OpenUrlAbstract/FREE Full Text
  151. ↵
    1. Suzuki, S. W.,
    2. Yamamoto, H.,
    3. Oikawa, Y.,
    4. Kondo-Kakuta, C.,
    5. Kimura, Y.,
    6. Hirano, H. and
    7. Ohsumi, Y.
    (2015). Atg13 HORMA domain recruits Atg9 vesicles during autophagosome formation. Proc. Natl. Acad. Sci. USA 112, 3350-3355. doi:10.1073/pnas.1421092112
    OpenUrlAbstract/FREE Full Text
  152. ↵
    1. Svoboda, K. and
    2. Block, S. M.
    (1994). Force and velocity measured for single kinesin molecules. Cell 77, 773-784. doi:10.1016/0092-8674(94)90060-4
    OpenUrlCrossRefPubMedWeb of Science
  153. ↵
    1. Sweeney, H. L. and
    2. Holzbaur, E. L. F.
    (2018). Motor proteins. Cold Spring Harb Perspect Biol 10, a021931. doi:10.1101/cshperspect.a021931
    OpenUrlAbstract/FREE Full Text
  154. ↵
    1. Takahashi, Y.,
    2. He, H.,
    3. Tang, Z.,
    4. Hattori, T.,
    5. Liu, Y.,
    6. Young, M. M.,
    7. Serfass, J. M.,
    8. Chen, L.,
    9. Gebru, M.,
    10. Chen, C. et al.
    (2018). An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure. Nat. Commun. 9, 2855. doi:10.1038/s41467-018-05254-w
    OpenUrlCrossRef
  155. ↵
    1. Thonghin, N.,
    2. Kargas, V.,
    3. Clews, J. and
    4. Ford, R. C.
    (2018). Cryo-electron microscopy of membrane proteins. Methods 147, 176-186. doi:10.1016/j.ymeth.2018.04.018
    OpenUrlCrossRef
  156. ↵
    1. Thumm, M.,
    2. Egner, R.,
    3. Koch, B.,
    4. Schlumpberger, M.,
    5. Straub, M.,
    6. Veenhuis, M. and
    7. Wolf, D. H.
    (1994). Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Lett. 349, 275-280. doi:10.1016/0014-5793(94)00672-5
    OpenUrlCrossRefPubMedWeb of Science
  157. ↵
    1. Toba, S.,
    2. Watanabe, T. M.,
    3. Yamaguchi-Okimoto, L.,
    4. Toyoshima, Y. Y., and
    5. Higuchi, H.
    (2006). Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein. Proc. Natl. Acad. Sci. USA 103, 5741-5745. doi:10.1073/pnas.0508511103
    OpenUrlAbstract/FREE Full Text
  158. ↵
    1. Tsuboyama, K.,
    2. Koyama-Honda, I.,
    3. Sakamaki, Y.,
    4. Koike, M.,
    5. Morishita, H. and
    6. Mizushima, N.
    (2016). The ATG conjugation systems are important for degradation of the inner autophagosomal membrane. Science 354, 1036-1041. doi:10.1126/science.aaf6136
    OpenUrlAbstract/FREE Full Text
  159. ↵
    1. Tsukada, M. and
    2. Ohsumi, Y.
    (1993). Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 333, 169-174. doi:10.1016/0014-5793(93)80398-E
    OpenUrlCrossRefPubMedWeb of Science
  160. ↵
    1. von Muhlinen, N.,
    2. Akutsu, M.,
    3. Ravenhill, B. J.,
    4. Foeglein, Á.,
    5. Bloor, S.,
    6. Rutherford, T. J.,
    7. Freund, S. M. V.,
    8. Komander, D. and
    9. Randow, F.
    (2012). LC3C, Bound Selectively by a Noncanonical LIR Motif in NDP52, Is Required for Antibacterial Autophagy. Mol. Cell 48, 329-342. doi:10.1016/j.molcel.2012.08.024
    OpenUrlCrossRefPubMedWeb of Science
  161. ↵
    1. Wang, H.,
    2. Sun, H.-Q.,
    3. Zhu, X.,
    4. Zhang, L.,
    5. Albanesi, J.,
    6. Levine, B. and
    7. Yin, H.
    (2015). GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion. Proc. Natl. Acad. Sci. USA 112, 7015-7020. doi:10.1073/pnas.1507263112
    OpenUrlAbstract/FREE Full Text
  162. ↵
    1. Weidberg, H.,
    2. Shvets, E.,
    3. Shpilka, T.,
    4. Shimron, F.,
    5. Shinder, V. and
    6. Elazar, Z.
    (2010). LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. EMBO J. 29, 1792-1802. doi:10.1038/emboj.2010.74
    OpenUrlCrossRefPubMedWeb of Science
  163. ↵
    1. Weidberg, H.,
    2. Shpilka, T.,
    3. Shvets, E.,
    4. Abada, A.,
    5. Shimron, F. and
    6. Elazar, Z.
    (2011). LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis. Dev. Cell 20, 444-454. doi:10.1016/j.devcel.2011.02.006
    OpenUrlCrossRefPubMedWeb of Science
  164. ↵
    1. Wenzel, E. M.,
    2. Schultz, S. W.,
    3. Schink, K. O.,
    4. Pedersen, N. M.,
    5. Nähse, V.,
    6. Carlson, A.,
    7. Brech, A.,
    8. Stenmark, H. and
    9. Raiborg, C.
    (2018). Concerted ESCRT and clathrin recruitment waves define the timing and morphology of intraluminal vesicle formation. Nat. Commun. 9, 2932. doi:10.1038/s41467-018-05345-8
    OpenUrlCrossRef
  165. ↵
    1. Wild, P.,
    2. McEwan, D. G. and
    3. Dikic, I.
    (2013). The LC3 interactome at a glance. J. Cell. Sci. 127, 3-9. doi:10.1242/jcs.140426
    OpenUrlAbstract/FREE Full Text
  166. ↵
    1. Wollert, T.
    (2012). Reconstituting multivesicular body biogenesis with purified components. Methods Cell Biol. 108, 73-92. doi:10.1016/B978-0-12-386487-1.00004-3
    OpenUrlCrossRefPubMed
  167. ↵
    1. Wollert, T. and
    2. Hurley, J. H.
    (2010). Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464, 864-869. doi:10.1038/nature08849
    OpenUrlCrossRefPubMedWeb of Science
  168. ↵
    1. Wollert, T.,
    2. Wunder, C.,
    3. Lippincott-Schwartz, J. and
    4. Hurley, J. H.
    (2009). Membrane scission by the ESCRT-III complex. Nature 458, 172-177. doi:10.1038/nature07836
    OpenUrlCrossRefPubMedWeb of Science
  169. ↵
    1. Wurzer, B.,
    2. Zaffagnini, G.,
    3. Fracchiolla, D.,
    4. Turco, E.,
    5. Abert, C.,
    6. Romanov, J. and
    7. Martens, S.
    (2015). Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy. eLife 4, e08941. doi:10.7554/eLife.08941
    OpenUrlAbstract/FREE Full Text
  170. ↵
    1. Xie, Z.,
    2. Xie, Z.,
    3. Nair, U.,
    4. Nair, U.,
    5. Klionsky, D. J. and
    6. Klionsky, D. J.
    (2008). Atg8 controls phagophore expansion during autophagosome formation. Mol. Biol. Cell 19, 3290-3298. doi:10.1091/mbc.e07-12-1292
    OpenUrlAbstract/FREE Full Text
  171. ↵
    1. Yamamoto, H.,
    2. Kakuta, S.,
    3. Watanabe, T. M.,
    4. Kitamura, A.,
    5. Sekito, T.,
    6. Kondo-Kakuta, C.,
    7. Ichikawa, R.,
    8. Kinjo, M. and
    9. Ohsumi, Y.
    (2012). Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J. Cell Biol. 198, 219-233. doi:10.1083/jcb.201202061
    OpenUrlAbstract/FREE Full Text
  172. ↵
    1. Yamamoto, H.,
    2. Fujioka, Y.,
    3. Suzuki, S. W.,
    4. Noshiro, D.,
    5. Suzuki, H.,
    6. Kondo-Kakuta, C.,
    7. Kimura, Y.,
    8. Hirano, H.,
    9. Ando, T.,
    10. Noda, N. N. et al.
    (2016). The intrinsically disordered protein Atg13 mediates supramolecular assembly of autophagy initiation complexes. Dev. Cell 38, 86-99. doi:10.1016/j.devcel.2016.06.015
    OpenUrlCrossRef
  173. ↵
    1. Yang, J.,
    2. Chai, X.-Q.,
    3. Zhao, X.-X. and
    4. Li, X.
    (2017). Comparative genomics revealed the origin and evolution of autophagy pathway. J. Syst. Evol. 55, 71-82. doi:10.1111/jse.12212
    OpenUrlCrossRef
  174. ↵
    1. Yorimitsu, T. and
    2. Klionsky, D. J.
    (2005). Atg11 links cargo to the vesicle-forming machinery in the cytoplasm to vacuole targeting pathway. Mol. Biol. Cell 16, 1593-1605. doi:10.1091/mbc.e04-11-1035
    OpenUrlAbstract/FREE Full Text
  175. ↵
    1. Yu, Z.-Q.,
    2. Ni, T.,
    3. Hong, B.,
    4. Wang, H.-Y.,
    5. Jiang, F.-J.,
    6. Zou, S.,
    7. Chen, Y.,
    8. Zheng, X.-L.,
    9. Klionsky, D. J.,
    10. Liang, Y. et al.
    (2012). Dual roles of Atg8-PE deconjugation by Atg4 in autophagy. Autophagy 8, 883-892. doi:10.4161/auto.19652
    OpenUrlCrossRef
View Abstract
Previous ArticleNext Article
Back to top
Previous ArticleNext Article

This Issue

Keywords

  • Autopaghy
  • In vitro reconstitution
  • Membrane
  • Atg protein

 Download PDF

Email

Thank you for your interest in spreading the word on Journal of Cell Science.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Reconstruction of destruction – in vitro reconstitution methods in autophagy research
(Your Name) has sent you a message from Journal of Cell Science
(Your Name) thought you would like to see the Journal of Cell Science web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Review
Reconstruction of destruction – in vitro reconstitution methods in autophagy research
Satish Babu Moparthi, Thomas Wollert
Journal of Cell Science 2019 132: jcs223792 doi: 10.1242/jcs.223792 Published 31 October 2018
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Review
Reconstruction of destruction – in vitro reconstitution methods in autophagy research
Satish Babu Moparthi, Thomas Wollert
Journal of Cell Science 2019 132: jcs223792 doi: 10.1242/jcs.223792 Published 31 October 2018

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Alerts

Please log in to add an alert for this article.

Sign in to email alerts with your email address

Article navigation

  • Top
  • Article
    • ABSTRACT
    • Introduction
    • Out of nowhere – nucleation of the phagophore in yeast and humans
    • A lipid diet for autophagy – expansion of the phagophore membrane
    • Fusion or fission? Maturation and sealing of the phagophore
    • Born to die – fusion of autophagosomes with lysosomes
    • Insights into the inside – cargo selection in autophagy
    • Conclusions and perspectives
    • Acknowledgements
    • Footnotes
    • References
  • Figures & tables
  • Info & metrics
  • PDF

Related articles

Cited by...

More in this TOC section

  • Polarisome assembly mediates actin remodeling during polarized yeast and fungal growth
  • The journey of Ca2+ through the cell – pulsing through the network of ER membrane contact sites
  • Notch in mechanotransduction – from molecular mechanosensitivity to tissue mechanostasis
Show more REVIEW

Similar articles

Subject collections

  • Autophagy

Other journals from The Company of Biologists

Development

Journal of Experimental Biology

Disease Models & Mechanisms

Biology Open

Advertisement

2020 at The Company of Biologists

Despite the challenges of 2020, we were able to bring a number of long-term projects and new ventures to fruition. While we look forward to a new year, join us as we reflect on the triumphs of the last 12 months.


Mole – The Corona Files

"This is not going to go away, 'like a miracle.' We have to do magic. And I know we can."

Mole continues to offer his wise words to researchers on how to manage during the COVID-19 pandemic.


Cell scientist to watch – Christine Faulkner

In an interview, Christine Faulkner talks about where her interest in plant science began, how she found the transition between Australia and the UK, and shares her thoughts on virtual conferences.


Read & Publish participation extends worldwide

“The clear advantages are rapid and efficient exposure and easy access to my article around the world. I believe it is great to have this publishing option in fast-growing fields in biomedical research.”

Dr Jaceques Behmoaras (Imperial College London) shares his experience of publishing Open Access as part of our growing Read & Publish initiative. We now have over 60 institutions in 12 countries taking part – find out more and view our full list of participating institutions.


JCS and COVID-19

For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.

If you have any questions or concerns, please do not hestiate to contact the Editorial Office.

Articles

  • Accepted manuscripts
  • Latest complete issue
  • Issue archive
  • Archive by article type
  • Special issues
  • Subject collections
  • Interviews
  • Sign up for alerts

About us

  • About Journal of Cell Science
  • Editors and Board
  • Editor biographies
  • Travelling Fellowships
  • Grants and funding
  • Journal Meetings
  • Workshops
  • The Company of Biologists

For Authors

  • Submit a manuscript
  • Aims and scope
  • Presubmission enquiries
  • Fast-track manuscripts
  • Article types
  • Manuscript preparation
  • Cover suggestions
  • Editorial process
  • Promoting your paper
  • Open Access
  • JCS Prize
  • Manuscript transfer network
  • Biology Open transfer

Journal Info

  • Journal policies
  • Rights and permissions
  • Media policies
  • Reviewer guide
  • Sign up for alerts

Contacts

  • Contact JCS
  • Subscriptions
  • Advertising
  • Feedback

Twitter   YouTube   LinkedIn

© 2021   The Company of Biologists Ltd   Registered Charity 277992