Dynein light chain interaction with the peroxisomal import docking complex modulates peroxisome biogenesis in yeast

The molecular motor dynein has long been implicated in mitotic spindle positioning and the transport of organelles along microtubules (Kardon and Vale, 2009; Moore et al., 2009). Non-motor functions for components of dynein have been suggested by their interactions with proteins that have no evident link to subcellular motility (Fan et al., 1998; Navarro-Lérida et al., 2004). For example, dynein light chain, Dyn2p, was shown to help organize assembly of the nuclear pore complex in a motor-independent manner in the yeast Saccharomyces cerevisiae (Stelter et al., 2007). This study also reported that Dyn2p localized in part to peroxisomes through interaction with Pex14p, a component of the peroxisomal matrix protein import docking complex. Components of the dynein complex have also been shown to interact with Pex14 in human cells (Bharti et al., 2011), and a large-scale study of S. cerevisiae showed that cells deleted for the DYN2 gene were unable to use oleic acid as a carbon source, the metabolism of which requires functional peroxisomes (Smith et al., 2006). Together, these findings suggest a role for Dyn2p in peroxisome biology.

Peroxisomes are ubiquitous membrane-bounded organelles involved in a variety of important biochemical and metabolic processes, notably the β-oxidation of fatty acids and the detoxification of reactive oxygen species (Fidaleo, 2010; Islinger et al., 2010). Peroxisomes also function as platforms for complex cellular signaling pathways such as those acting in antiviral innate immunity (Berg et al., 2012; Dixit et al., 2010; Horner et al., 2011). Several inborn human disorders are caused by peroxisome dysfunction. Patients with these disorders, collectively called the peroxisome biogenesis disorders, exhibit a variety of physiological abnormalities due to the absence of functional peroxisomes, and usually die within their first year (Fidaleo, 2010; Steinberg et al., 2006).

Peroxisomes arise by two different pathways: de novo biogenesis at the ER, and the growth and division of pre-existing peroxisomes (Ma et al., 2011; Mast et al., 2010; Schrader et al., 2012; Tabak et al., 2008). These two pathways work in concert to maintain the peroxisome population of a cell. Which pathway predominates depends on the type of cell and the internal and external environmental conditions to which it is exposed (Hoepfner et al., 2005; Kim et al., 2006; Motley and Hettema, 2007). The dynamic nature of peroxisome biogenesis leads to a heterogeneous population of peroxisomes at different stages of assembly (Titorenko and Rachubinski, 2000; Titorenko et al., 2000; van der Zand et al., 2012).

Proper import of peroxisomal matrix proteins is essential to peroxisome biogenesis. Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally imported into peroxisomes. Matrix proteins contain either a C-terminal peroxisome targeting signal type 1 (PTS1) or a PTS2 at or near their N-terminus (Gould et al., 1989; Swinkels et al., 1991). Translocation of matrix proteins across the peroxisomal membrane depends on the cycling receptors Pex5p and Pex7p (Marzioch et al., 1994; McCollum et al., 1993), which recognize PTS1- and PTS2-containing cargoes, respectively. Pex5p and Pex7p bind their cargo proteins in the cytosol and deliver them to the peroxisome through interactions with the peroxisomal membrane proteins Pex13p, Pex14p and Pex17p, which make up the peroxisomal matrix protein import docking complex (Rucktäschel et al., 2011). Translocation of PTS1-containing proteins into the peroxisomal matrix is accomplished through a transient peroxisomal pore composed in part by Pex14p and cargo-laden Pex5p (Meinecke et al., 2010). The interaction of Dyn2p with Pex14p suggests a potential role for Dyn2p in matrix protein import into the peroxisome.

Here we show that absence of Dyn2p in the yeast Yarrowia lipolytica results in impaired peroxisome function and biogenesis, abnormal peroxisome morphology, and mislocalization of peroxisomal matrix proteins. We also demonstrate that Dyn2p physically and genetically interacts with docking complex proteins, suggesting that Dyn2p works in concert with the docking complex for efficient import of matrix proteins into the peroxisome.

The association of components of the dynein complex with peroxisomes in both yeast and mammalian cells and the demonstration that the dynein light chain protein, Dyn2p, interacts with the peroxisomal matrix protein import docking complex component Pex14p (Bharti et al., 2011; Stelter et al., 2007) suggest a possible role for components of dynein in peroxisome biogenesis. We chose to investigate the role of dynein light chain in peroxisome biogenesis using the heterothallic yeast Yarrowia lipolytica because of its robust growth and peroxisome proliferative capacity on medium containing fatty acid, the metabolism of which requires functional peroxisomes. A BLAST search of the Y. lipolytica proteome uncovered one homologue of S. cerevisiae Dyn2p: YALI0D07700p. Y. lipolytica Dyn2p exhibits 52% identity and 25% similarity to S. cerevisiae Dyn2p (Fig. 1A).

Deletion of the DYN2 gene led to compromised growth of Y. lipolytica on oleic-acid-containing YPBO agar medium compared with that of the wild-type strain, although the growth defect was not as dramatic as that observed for cells deleted for the PEX14 or PEX17 gene encoding a component of the peroxisomal matrix protein import docking machinery (Fig. 1B). Transformation of the dyn2Δ strain with plasmid expressing the wild-type DYN2 gene or a chimeric gene encoding fluorescent Dyn2p–mCherry re-established growth of the strain on YPBO, showing that the reduced ability of cells to grow on oleic-acid-containing medium was due specifically to deletion of the DYN2 gene and that Dyn2p–mCherry functions similarly to Dyn2p (Fig. 1C). Y. lipolytica Dyn2p could also rescue the growth defect of a S. cerevisiae strain deleted for DYN2 on oleic-acid-containing medium (supplementary material Fig. S1).

A reduced ability of Y. lipolytica to grow on oleic-acid-containing medium is often due to compromised peroxisome biogenesis, which in turn often results in abnormal peroxisome morphology. To see whether deletion of DYN2 affects peroxisome biogenesis and/or morphology in Y. lipolytica, wild-type and dyn2Δ strains expressing the fluorescent PTS1-containing peroxisomal marker protein mRFP–SKL or the fluorescent PTS2-containing peroxisomal protein 3-ketoacyl-CoA thiolase tagged at its C-terminus with mRFP (Pot1p–mRFP) were cultured in glucose-containing YPD medium and then transferred to oleic-acid-containing YPBO medium to promote peroxisome proliferation. Cells were observed by confocal microscopy at various times after transfer (Fig. 2A). With increasing time of incubation in YPBO medium, dyn2Δ cells exhibited localization of mRFP–SKL to discrete punctate structures similar to those observed in wild-type cells. Pot1p–mRFP also showed a punctate pattern of fluorescence in wild-type cells. Strikingly in contrast, Pot1p–mRFP in dyn2Δ cells showed predominantly a diffuse pattern of fluorescence characteristic of a cytosolic localization, although some fluorescent punctate structures were present. Therefore, fluorescence microscopy provides evidence of compromised matrix protein import in dyn2Δ cells.

The ultrastructure of peroxisomes in wild-type and dyn2Δ cells was compared by electron microscopy of cells incubated in YPBO medium for 10 hours (Fig. 2B). Wild-type cells showed typical round peroxisomes surrounded by a single unit membrane and containing a homogenous granular matrix. dyn2Δ cells occasionally contained peroxisomes similar to those seen in wild-type cells or large elongated peroxisomes (Fig. 2B,i) but usually contained small vesicular structures, some of which resembled peroxisomes (Fig. 2B,ii,iii). These vesicular structures were often observed arranged in tandem (Fig. 2B,ii) or clustered (Fig. 2B,iii). Together our data show that Dyn2p is required for normal peroxisomal matrix protein import and normal peroxisome function and formation under peroxisome proliferative conditions in oleic-acid-containing medium.

One current model of peroxisome assembly in yeast proposes that peroxisomes are made de novo by the early fusion of distinct peroxisomal precursors to produce another peroxisomal precursor that in turn is converted through the step-wise import of subsets of matrix proteins and phospholipids to eventually form a ‘mature’ peroxisome (Titorenko and Rachubinski, 2001; Titorenko et al., 2000; van der Zand et al., 2012). If matrix protein import along this pathway is compromised, peroxisome assembly and function are also usually compromised. In light of this link between peroxisome assembly/function and matrix protein import, and also considering the finding that Dyn2p co-purifies with the matrix protein import docking complex component, Pex14p (Stelter et al., 2007), we first examined matrix protein localization in dyn2Δ cells under conditions of peroxisome proliferation in oleic-acid-containing medium. Subcellular fractions from wild-type and dyn2Δ cells incubated in YPBO medium for 10 hours were subjected to immunoblot analysis for different matrix proteins, including the PTS1-containing proteins malate synthase (MLS) and isocitrate lyase (ICL); a 62-kDa protein reactive to antibodies against the tripeptide PTS1, Ser-Lys-Leu; the PTS2-containing protein Pot1p; and the five isoforms of the enzyme acyl-CoA oxidase (Aox1p to Aox5p), which contain neither a conventional PTS1 sequence nor a PTS2 sequence (Fig. 3A). As expected, matrix proteins in wild-type cells enriched primarily in the 20,000 g pellet (20KgP), which is enriched for ‘mature’ peroxisomes (Titorenko and Rachubinski, 2000; Titorenko et al., 2000). In contrast, matrix proteins in dyn2Δ cells were often observed across all fractions, with several enriched preferentially in the 200,000 g pellet (200KgP), which contains small peroxisomal vesicles (Titorenko and Rachubinski, 2000; Titorenko et al., 2000), and the 200,000 g supernatant (200KgS), which is enriched for cytosol. The peroxisomal membrane proteins Pex2p, Pex3Bp and Pex19p were found to be similarly enriched in the 20KgP and 200KgP fractions from both wild-type and dyn2Δ cells. These results indicate that under peroxisome proliferating conditions in oleic-acid-containing medium, a fraction of individual matrix proteins is found in peroxisomes in dyn2Δ cells, but unlike in wild-type cells, different matrix proteins in dyn2Δ cells were enriched more in the 200KgP fraction containing small peroxisomal structures and less in the 20KgP fraction containing peroxisomes. Deletion of the DYN2 gene apparently has no or little effect on the localization of peroxisomal membrane proteins.

Protease protection was performed to confirm that matrix protein import into membrane-enclosed peroxisomal structures is compromised in dyn2Δ cells (supplementary material Fig. S2). Postnuclear supernatant (PNS) fractions containing immature and mature peroxisomes and cytosol from wild-type and dyn2Δ cells were subjected to treatment with increasing amounts of trypsin and analyzed by immunoblotting with antibodies to different peroxisomal matrix proteins. Matrix proteins from dyn2Δ cells were preferentially degraded with trypsin. Taken altogether, our findings strongly support that peroxisome maturation and peroxisomal matrix protein import are compromised in dyn2Δ cells under peroxisome proliferative conditions in oleic-acid-containing medium.

Interestingly, we also observed a defect in the post-translational processing of Pot1p in dyn2Δ cells (Fig. 3B). Normally, the 45-kDa precursor of Pot1p (p-Pot1p) is translocated into the peroxisomal matrix where it is proteolytically processed to its mature 43-kDa form (m-Pot1p) (Szilard et al., 1995). In dyn2Δ cells, Pot1p remained in precursor form. The inability to proteolytically process Pot1p was not due simply to flawed Pot1p import in dyn2Δ cells, as Pot1p was found in both the 20KgP and 200KgP fractions containing peroxisomal structures from dyn2Δ cells (Fig. 3A,C). Furthermore, hypotonic lysis of the organelles in the 20KgP and 200KgP fractions with dilute alkaline Tris buffer followed by ultracentrifugation liberated Pot1p to the Ti8S fraction enriched for matrix proteins and not the Ti8P fraction enriched for membrane proteins of both wild-type and dyn2Δ cells (Fig. 3C). Therefore, Pot1p found in the matrix of peroxisomal structures in dyn2Δ cells is primarily in the precursor form, p-Pot1p.

The limited growth of the dyn2Δ strain on YPBO medium containing oleic acid (Fig. 1B) and the localization of a fraction of several peroxisomal matrix proteins to the 20KgP fraction (Fig. 3A) suggested that the dyn2Δ strain contains some functional peroxisomes. Isopycnic density gradient centrifugation of the 20KgP fractions from the wild-type and dyn2Δ strains and immunoblotting with antibodies to different matrix proteins showed several matrix proteins to be enriched in different fractions in extracts of the two cell types (Fig. 4). In wild-type cells, matrix proteins peaked in fractions 3 and 4, as observed previously (Szilard et al., 1995; Titorenko et al., 1996). Matrix proteins from dyn2Δ cells were also enriched in fractions 3 and 4, suggesting that dyn2Δ cells contain some membrane-enclosed structures that resemble biochemically wild-type peroxisomes with regard to their density and matrix protein composition. Matrix proteins from dyn2Δ cells were also substantially present in fractions of greater and lesser density than fractions 3 and 4. Interestingly, not all matrix proteins in dyn2Δ cells exhibited the same shift in distribution to other fractions. For example, in dyn2Δ cells there was a shift of MLS and the 62-kDa SKL-containing protein to fractions of higher density, whereas other matrix proteins such as Aox3p and Aox5p shifted to fractions of lower density. Consistent with the results of fluorescence confocal microscopy and electron microscopy (Fig. 2), these data suggest that the peroxisome population of dyn2Δ cells is more heterogeneous in its composition than is the peroxisome population of wild-type cells. These findings are also consistent with previous observations that peroxisomal structures accumulate in yeast cells compromised in peroxisome biogenesis and that the matrix protein composition of these peroxisomal structures are different (Titorenko and Rachubinski, 2000; Titorenko et al., 2000; van der Zand et al., 2012).

We also compared matrix protein localization in wild-type and dyn2Δ cells incubated under peroxisome non-proliferating conditions in glucose-containing YPD medium. Immunoblot analysis showed that matrix proteins were distributed similarly in subcellular fractions from wild-type and dyn2Δ cells (supplementary material Fig. S3). The different peroxisomal matrix and membrane proteins were enriched preferentially in the 20KgP and 200KgP fractions from both wild-type and dyn2Δ cells. Our data suggest that dyn2Δ cells can import matrix proteins into peroxisomes as efficiently as wild-type cells under peroxisome non-proliferating conditions but are less efficient than wild-type cells when there is a demand for increased matrix protein import imposed by increases in peroxisome size and number under peroxisome proliferating conditions.

In S. cerevisiae, Dyn2p localization to the peroxisome is eliminated by deletion of the PEX14 gene, and Pex14p is co-precipitated by Dyn2p in affinity purification (Stelter et al., 2007). Using bimolecular fluorescence complementation (BiFC; also known as split GFP) (Munck et al., 2009), we showed that Dyn2p associates with Pex14p in vivo in Y. lipolytica (Fig. 5A). The N- and C-terminal-GFP-tagged constructs of Dyn2p and Pex14p were shown to function similarly to their wild-type counterparts as they were sufficient to restore growth of strains deleted for DYN2 and PEX14, respectively, on oleic-acid-containing medium (supplementary material Fig. S4). The interaction of Dyn2p with Pex14p in vivo is independent of the import of most matrix proteins, as deletion of the gene PEX5 encoding the PTS1 receptor did not eliminate the fluorescence resulting from interaction of the split GFPs fused to Dyn2p and Pex14p (Fig. 5A). In contrast, deletion of the PEX17 gene encoding another component of the matrix protein import docking complex did eliminate the interaction between Dyn2p and Pex14p, suggesting that Pex17p helps to mediate the association between Dyn2p and Pex14p (Fig. 5A).

The association between Dyn2p and Pex14p occurred at the level of peroxisomes, as the GFP fluorescence resulting from Dyn2p interaction with Pex14p colocalized with the fluorescent peroxisomal marker, mRFP–SKL (Fig. 5B). Not all peroxisomes labeled with mRFP–SKL colocalized with the GFP signal, suggesting that Dyn2p may associate with a specific peroxisome subpopulation or that its interaction with peroxisomes is highly dynamic.

The in vivo detection of association between Dyn2p and Pex14p at the peroxisome predicts that some Dyn2p would co-purify with peroxisomes isolated by subcellular fractionation. As expected, a significant fraction of Dyn2p–mCherry localized to the 200KgS fraction enriched for cytosolic proteins (Fig. 5C). However, Dyn2p–mCherry was also found in the 20KgP fraction enriched for peroxisomes and mitochondria and in the 200KgP fraction enriched for small vesicles, including small peroxisomal vesicles (Fig. 5C). Interestingly, Dyn2p–mCherry migrates as a doublet in all fractions, with the lower molecular mass species most enriched in the 200KgP fraction. The origin and significance of this Dyn2p doublet are unknown but could be the result of a post-translational modification of Dyn2p, as has been reported for human dynein light chain 1 (Song et al., 2008), which could, for example, promote the association of Dyn2p with peroxisomes or peroxisomal vesicles. Isopycnic density gradient centrifugation of the 20KgP fraction indicated that Dyn2p–mCherry co-enriched with the peroxisomal matrix enzyme Pot1p but not with the mitochondrial protein Sdh2p (Fig. 5D), indicating that a portion of the Dyn2p localizes to peroxisomes.

We performed GST pull-down assays to differentiate between direct and bridged protein interactions with Dyn2p (Fig. 6A). GST alone, or GST fusions to Dyn2p and to the peroxisomal matrix protein import docking components Pex14p and Pex17p, immobilized on glutathione–Sepharose beads were used as bait for maltose binding protein (MBP) or MBP fusions to Dyn2p, Pex14p and Pex17p. MBP fusions were detected by immunoblotting with anti-MBP antibody. Expression of a MBP fusion to the docking complex protein Pex13p failed because of its cytotoxicity to Escherichia coli. Direct interaction was observed between Pex14p and Pex17p and between Pex14p and itself. GST–Dyn2p pulled down MBP–Dyn2p, MBP–Pex14p and MBP–Pex17p but not MBP alone. The interaction of GST–Dyn2p with MBP–Pex17p was particularly robust, suggesting that the interaction of Dyn2p with the peroxisomal matrix protein import complex is chiefly mediated through Pex17p.

The amino acid sequence VDAQTQTE (residues 99–106) of Pex17p is similar to putative Dyn2p-binding sequences found in dynein intermediate chain and Nup159p (Stelter et al., 2007; Stuchell-Brereton et al., 2011). We investigated whether V99DAQTQTE106 mediates the interaction of Pex17p with Dyn2p by constructing a series of MBP-tagged Pex17p mutants and truncations to act as potential binding partners for GST–Dyn2p and GST–Pex14p in GST pull-down assays (Fig. 6B,C). We found that mutation or deletion of the putative Dyn2p binding domain V99DAQTQTE106 of Pex17p eliminated the interaction between Pex17p and Dyn2p. This domain was not required for Pex17p to interact with Pex14p. However, deletion of the C-terminus of Pex17p eliminates the interaction of Pex17p with Pex14p, suggesting that the C-terminus of Pex17p is required for its interaction with Pex14p.

We next investigated the functionality of the mutants Pex17pΔ102–106 and Pex17pmut by evaluating their ability to complement the growth defect of pex17Δ cells on oleic-acid-containing YPBO medium (Fig. 7A). As expected, reintroduction of the PEX17 gene restored growth of the pex17Δ strain on YPBO. In contrast, introduction of Pex17pΔ102–106 failed to restore growth of the pex17Δ strain on YPBO medium, whereas introduction of Pex17pmut led to a weak restoration of growth. The increased severity of the growth defect on YPBO medium of the pex17Δ strain expressing Pex17pΔ102–106 as compared with the dyn2Δ strain (Fig. 1B) suggests that the amino acids, Q102TQTE106, play a role in Pex17p functionality in addition to their being part of the Pex17p interaction domain with Dyn2p.

We also examined genetic interactions between DYN2 and the genes coding for the matrix protein import docking complex proteins Pex13p, Pex14p and Pex17p (Fig. 7B). Overexpression of all docking complex genes strongly retarded growth of dyn2Δ cells on oleic-acid-containing medium compared with overexpression in wild-type cells. Overexpression of PEX17 in particular had the strongest effect upon the growth of dyn2Δ cells. These results indicate that Dyn2p can mitigate the adverse effects of docking complex gene overexpression.

There is increasing evidence that individual components of the microtubule motor dynein have functions not related to transport (Bharti et al., 2011; Fan et al., 1998; Navarro-Lérida et al., 2004; Stelter et al., 2007). S. cerevisiae dynein light chain, Dyn2p, was shown to localize in part to peroxisomes through its interaction with Pex14p (Stelter et al., 2007), which, together with Pex13p and Pex17p, forms the receptor docking complex for the import of matrix proteins into the peroxisome (Rucktäschel et al., 2011). The interaction of Dyn2p with Pex14p suggested a role for Dyn2p in peroxisome biogenesis, which was also supported by the observation that S. cerevisiae cells deleted for the DYN2 gene failed to grow on medium containing oleic acid as the sole carbon source (Smith et al., 2006), the metabolism of which requires functional peroxisomes. In this study, we demonstrate a role for Dyn2p in peroxisome biogenesis in the yeast Y. lipolytica and provide evidence that Dyn2p acts together with the peroxisomal matrix protein import receptor docking complex to modulate matrix protein import into peroxisomes and peroxisome maturation.

Y. lipolytica cells deleted for the DYN2 gene exhibited impaired growth on medium containing oleic acid, and abnormal peroxisome morphology, showing that Dyn2p is required for normal peroxisomal function and biogenesis. However, the impairment in peroxisomal function resulting from deletion of the DYN2 gene is less than that caused by deletion of bona fide PEX genes such as PEX14 and PEX17 encoding components of the peroxisomal matrix protein import docking complex, which are central to the matrix protein import process and are indispensable for peroxisome biogenesis. Therefore, Dyn2p apparently has an auxiliary, rather than a primary, role in peroxisome biogenesis. An auxiliary peroxisome biogenic role for Dyn2p is also supported by our observations that normal peroxisome profiles are sometimes seen in dyn2Δ cells by both confocal fluorescence microscopy and electron microscopy, and by the presence of vesicular structures with the biochemical characteristics of wild-type peroxisomes in isopycnic density centrifugation analysis of dyn2Δ cells. Dyn2p apparently functions in peroxisome biogenesis primarily under conditions of peroxisome proliferation, because differences in peroxisome structure and matrix protein import between dyn2Δ cells and wild-type cells were most evident in cells incubated in oleic-acid-containing medium, in which peroxisome proliferation occurs, and not in medium containing glucose, which does not require peroxisomal activity for its metabolism.

Subcellular fractionation demonstrated that protein targeting to the peroxisomal matrix is compromised in cells deleted for the DYN2 gene. A comparison of the differential centrifugation fractionation patterns of matrix proteins from wild-type and dyn2Δ cells showed that matrix proteins from dyn2Δ cells localize to a high-speed supernatant fraction enriched for cytosolic proteins and a high-speed pellet fraction enriched for small vesicles, including peroxisomal vesicles, to a greater degree than matrix proteins from wild-type cells. Protease protection analysis confirmed a defect in matrix protein import in dyn2Δ cells. Density gradient centrifugation also showed that the matrix protein composition in fractions of lower buoyant density, normally enriched for small peroxisomal vesicles, differed in extracts from in wild-type and dyn2Δ cells. Similarly, matrix proteins from wild-type and dyn2Δ cells were differently distributed across fractions of high buoyant density where peroxisomes fractionate. This difference in peroxisome populations between wild-type and dyn2Δ cells was also evident by fluorescence microscopy, as the localization patterns of the peroxisomal marker proteins Pot1p–mRFP and, to a lesser extent, mRFP–SKL were different in dyn2Δ and wild-type cells. Peroxisomes without their normal complement of matrix proteins might be expected to be only partially functional, which is consistent with our observation that deletion of the DYN2 gene impairs, but does not eliminate, growth of Y. lipolytica cells on fatty-acid-containing medium, the metabolism of which requires peroxisomal activity. Together, our results demonstrate that deletion of the DYN2 gene results in impaired targeting of matrix proteins to the peroxisome, leading to compromised peroxisome maturation and function during peroxisome proliferation.

The differences in overall composition of the peroxisome populations between wild-type and dyn2Δ cells could result from direct influences on the peroxisome biogenic cascade in Y. lipolytica brought about by deletion of the DYN2 gene; however, the physical and genetic interactions between Dyn2p and the components of the peroxisomal matrix protein import docking complex, Pex13p, Pex14p and Pex17p, suggest strongly that the role of Dyn2p in peroxisome biogenesis is related, to a large degree, to its functioning in matrix protein import. Dyn2p could act as a kind of molecular glue at the docking complex, stabilizing protein–protein interactions within a multi-protein complex, similar to the role proposed for S. cerevisiae Dyn2p at the nuclear pore in stabilizing the Nup82p–Nsp1p–Nup159p complex (Stelter et al., 2007). Stabilization of the peroxisomal matrix protein import docking complex by Dyn2p could facilitate the import of matrix proteins or perhaps provide a degree of temporal control to peroxisome assembly by determining what matrix proteins are imported at what time. It should also be noted that Dyn2p interacts more strongly with Pex17p than Pex14p in our in vitro pull-down assay and that association of Dyn2p with Pex14p in BiFC experiments requires Pex17p. These data suggest that Dyn2p interacts primarily with Pex17p and that its association with Pex14p is due to the close proximity of Pex14p to Pex17p in the matrix protein import docking complex. Since Pex17p functions prior to the formation of the transient import pore (Meinecke et al., 2010), its interactions with Dyn2p and its requirement for interaction with another docking complex component, Pex14p, suggest that Dyn2p may play a stabilizing function early in the formation of the docking complex. Further study will be needed to determine whether this is indeed the case.

In summary, we have shown that Y. lipolytica dynein light chain protein, Dyn2p, functions in peroxisome biogenesis. Dyn2p localizes in part to peroxisomes and influences the targeting of proteins to the peroxisomal matrix and, by extension, peroxisome maturation, probably through its interactions with components of the matrix protein import docking complex, especially Pex17p. The role of Dyn2p in peroxisome biogenesis adds to the list of non-motility-related functions that can be attributed to individual components of the dynein motor.

The yeast strains used in this study are listed in supplementary material Table S1. Strains were cultured at 30°C. Strains containing plasmid pTC3 or pINA445 were cultured in minimal medium (YNA or YNO) supplemented with leucine, uracil and lysine, each at 50 µg/ml, as required. Strains containing plasmid pUB4 were cultured in YPA, YPD or YPBO medium supplemented with hygromycin B at 125 µg/ml. Media components were as follows: YNA, 0.67% yeast nitrogen base without amino acids, 2% sodium acetate; YNO, 0.67% yeast nitrogen base without amino acids, 0.05% Tween 40, 0.1% oleic acid; YPA, 1% yeast extract, 2% peptone, 2% sodium acetate; YPD, 1% yeast extract, 2% peptone, 2% glucose; YPG, 1% yeast extract, 2% peptone, 2% glycerol; YPBO, 0.3% yeast extract, 0.5% peptone, 0.5% K₂HPO₄, 0.5% KH₂PO₄, 1% Brij 35, 1% oleic acid.

Genes were disrupted by homologous transformation using fusion PCR-based integration (Davidson et al., 2002).

pINA445 (Nuttley et al., 1993), pTC3 (Lin et al., 1999) and pUB4 (Kerscher et al., 2001) have been described. DNA encoding mRFP–SKL and flanked by promoter and terminator sequences of the POT1 gene was amplified by PCR from pTC3-mRFP-SKL (Chang et al., 2007) and inserted at the ClaI site of pINA445 and pUB4 to produce pINA445-mRFP-SKL and pUB4-mRFP-SKL. DNA encoding Pex13p, Pex14p and Pex17p, and GFP-N (amino acids 1–156 of GFP) and GFP-C (amino acids 157–238 of GFP) used for bimolecular fluorescence complementation (split GFP) analysis, was amplified by PCR. The chimeric genes encoding Pot1p–mRFP, Dyn2p–mCherry, Dyn2p–GFP-N and Pex14p–GFP-C, and the genes encoding Pex17pmut and Pex17pΔ102–106, were constructed by fusion PCR. PCR products were inserted into the EcoRI site of pTC3 to make pTC3-POT1-mRFP, pTC3-GFP-N, pTC3-GFP-C, pTC3-DYN2, pTC3-DYN2-mCherry, pTC3-DYN2-GFP-N, pTC3-PEX14-GFP-C, pTC3-PEX13, pTC3-PEX14, pTC3-PEX17, pTC3-PEX17mut and pTC3-PEX17Δ102–106. pUB4-POT1-mRFP, pUB4-GFP-C, pUB4-DYN2, pUB4-DYN2-mCherry, pUB4-DYN2-GFP-N and pUB4-PEX14-GFP-C were made similarly to pUB4-mRFP-SKL. pINA445-PEX13, pINA445-PEX14 and pINA445-PEX17 were made similarly to pINA445-mRFP-SKL.

Fluorescence images were captured with a LCI Plan-Neofluar 63×/1.3 NA objective on an Axiovert 200 microscope equipped with a LSM510 META confocal scanner (Carl Zeiss). Stacks of 3 (Fig. 2) or 21 (Fig. 5) optical sections each spaced 1 or 0.25 µm apart were captured. Acquired images were processed as described previously (Tower et al., 2011). To remove blur, experimentally generated three-dimensional (3D) data sets were deconvolved through an iterative classic maximum likelihood estimation algorithm and an experimentally derived point spread function using Huygens Professional software (Scientific Volume Imaging, Hilversum, The Netherlands). Imaris 7.3 software (Bitplane) was used to prepare maximum intensity, or ‘Blend-view’, projections of deconvolved 3D data sets. Projections were used to generate single images. Transmission images were treated with a Gaussian filter and made white in Imaris. Collections of images were assembled into figures using Adobe Photoshop CS4 and Adobe Illustrator CS4 (Adobe Systems). Electron microscopy of whole yeast cells was performed as described previously (Eitzen et al., 1997).

Wild-type and dyn2Δ cells were cultured in YPD or YPBO medium. dyn2Δ cells transformed with pUB4-DYN2-mCherry were cultured in YPD medium supplemented with hygromycin B. Cell fractionation was performed essentially as described previously (Szilard et al., 1995). Homogenized spheroplasts were subjected to differential centrifugation at 1000 g for 10 minutes at 4°C in a JS13.1 rotor (Beckman) to yield a postnuclear supernatant (PNS) fraction. The PNS fraction was subjected to differential centrifugation at 20,000 g for 30 minutes at 4°C to yield a pellet (20KgP) fraction enriched for peroxisomes and mitochondria, and a supernatant (20KgS) fraction enriched for cytosol and high-speed pelletable organelles. The 20KgS fraction was further subjected to differential centrifugation at 200,000 g for 1 hour at 4°C in a TLA120.2 rotor (Beckman) to yield a pellet (200KgP) fraction enriched for high-speed pelletable organelles including small peroxisomal vesicles, and a supernatant (200KgS) fraction enriched for cytosol.

Peroxisomes were purified from the 20KgP fraction by isopycnic centrifugation on a discontinuous Nycodenz gradient at 100,000 g for 90 minutes in a VTi50 rotor (Beckman) (Titorenko et al., 1996). 20KgP and 200KgP fractions were treated with dilute alkali Tris buffer and separated into fractions enriched for matrix and membrane proteins by ultracentrifugation as described previously (Vizeacoumar et al., 2003).

Fusions of GST to Dyn2p, Pex14p and Pex17p were constructed using pGEX4T-1 (GE Healthcare). Recombinant expression and immobilization of GST, GST–Dyn2p, GST–Pex14p and GST–Pex17p on glutathione–Sepharose resin were performed according to the manufacturer's instructions. MBP fusions to Dyn2p, Pex14p, Pex17p and Pex17p mutations were constructed using pMAL-c2 (New England Biolabs) and expressed in E. coli strain BL21 (Invitrogen). 250 µg of purified GST, GST–Dyn2p, GST–Pex14p or GST–Pex17p immobilized on glutathione–Sepharose resin were incubated with 250 µg of E. coli lysate containing MBP or MBP fusion protein (50 µg for MBP–Pex17p and MBP–Pex17p mutations) in PBS buffer containing 0.5% Triton X-100 and 2× complete protease inhibitor (Roche) for 3 hours at 4°C on a rocking platform. After settling, the resin was washed five times with PBS buffer containing 0.5% Triton X-100. Immobilized proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE and immunoblotting.

Antibodies to Pot1p, Sdh2p, Aox1p, Aox3p, Aox5p, MLS, SKL, ICL, Pex2p, Pex3Bp, Pex19p, DsRed and GST have been described previously (Chang et al., 2009; Lambkin and Rachubinski, 2001; Titorenko et al., 2000; 2002). Mouse anti-MBP monoclonal antibody was from New England Biolabs. For immunoblot analysis, HRP-conjugated donkey anti-rabbit IgG, HRP-conjugated goat anti-guinea pig IgG, and HRP-conjugated sheep anti-mouse IgG secondary antibodies were used to detect primary antibodies, and antigen–antibody complexes were detected by enhanced chemiluminescence (GE Healthcare).

We thank Richard Poirier, Elena Savidov, Hanna Kroliczak and Dwayne Weber for expert technical assistance and members of the Rachubinski laboratory for helpful discussions. R.A.R. is an International Research Scholar of the Howard Hughes Medical Institute.