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- Fig. 1.
Identification of the Atg2-interaction region in Atg18. (A) Atg18, which comprises seven WD-40 domains, is able to interact with Atg2 by the Y2H assay. Atg2 and Atg18 were fused to the activation domain (AD) and/or the DNA-binding domain (BD) of the transcription factor Gal4. Plasmids were transformed into the PJ69-4A strain and colonies were spotted on medium lacking uracil, tryptophan and histidine. Growth on these plates indicates that the tested proteins interact. The empty pGAD-C1 plasmid was used as a control. (B) Overview of the amino acid sequence of Atg18. The seven β-sheets forming the blades of Atg18 β-propeller are underlined and the loops connecting them are indicated with boxes. Charged and polar amino acids present in each loop that were substituted with alanines are indicated in bold. (C) Mutations in a single loop do not disrupt the binding between Atg2 and Atg18. AD fusions of the different Atg18 mutants were tested for their ability to interact with the BD-Atg2 chimera using the Y2H assay as in A. (D) Loops 1 and 2 of Atg18 are essential for binding with Atg2. Combinations of several mutated Atg18 loops were cloned in the pGAD-C1 vector and tested for interaction with BD-Atg2 as in A.
- Fig. 2.
Amino acids in loop2 of the Atg18 β-propeller are essential for the Atg2-Atg18 interaction in vivo. (A) The identified Atg18 loop 2 and 1,2 mutants do not bind to Atg2. Cell lysates from the atg18Δ (JGY3) and atg18Δ ATG2-PA (FRY387) strains transformed with plasmids expressing 13×myc-tagged Atg18 loop mutants were subjected to pull-down experiments. Affinity isolates were resolved by SDS-PAGE and analyzed by western blotting. On each lane of the SDS-PAGE gel, 1% of cell lysate or 20% of the affinity isolate was loaded. Although there is a small amount of Atg18(L1) in the affinity eluate of the negative control (lane 8), this fusion protein is highly enriched in the sample containing Atg2-PA (lane 3), indicating that it still binds to Atg2-PA. (B) Atg2-binding mutants of Atg18 still bind phosphoinositides. To determine the phosphoinositide-binding capacity of the various Atg18 constructs, the atg18Δ strain transformed with plasmids expressing the 13×myc-tagged Atg18 loop mutants under the control of the GAL1 promoter were grown on galactose overnight to induce protein overexpression (∼70 fold, not shown) before incubating native cell extracts on phospholipid strips. The lipids present on the membranes are indicated next to the panels.
- Fig. 3.
Atg18-Atg2 interaction is essential for autophagy. (A) Mutations in the Atg18 loops 1, 2 and 5 cause an autophagy impairment. Wild type (SEY6210) and atg18Δ (JGY3) cells carrying both the pCuGFPATG8414 construct and one of the plasmids expressing the untagged Atg18 loop mutants were grown in rich medium and transferred to starvation medium to induce autophagy. Cell aliquots were taken at 0, 1, 2 and 4 h, before analyzing the cell extracts by western blotting. The detected bands were quantified using the Odyssey software and the percentages of GFP-Atg8 were plotted in a graph. Data represent the average of three experiments±s.e.m. (B) Mutations in Atg18 loop2 do not affect the function of Atg18 at the vacuole. The atg18Δ (atg18Δ) cells carrying an empty vector (pRS415) or one of the plasmids expressing untagged wild type Atg18, Atg18(L2) or Atg18(L5) were grown in rich medium to an early log phase and labeled with FM4-64 to visualize the vacuole. Representative fields are shown. Scale bars: 5 µm.
- Fig. 4.
The Atg2-Atg18 interaction is unnecessary for PAS formation. (A) PAS formation was assessed using CFP-tagged Atg8. The atg18Δ strain with the integrated CFP-Atg8 fusion and carrying no construct (ERY068), 13×myc-tagged ATG18 (ERY070) or one of the 13×myc-tagged ATG18 loop mutants (ERY072 and ERY074) were grown in rich medium before being nitrogen starved for 3 h to induce autophagy. Cells were imaged by fluorescence microscopy before and after nitrogen starvation. For clarity, the cyan-blue fluorescence signal was converted into yellow. DIC, differential interference contrast. Scale bars: 5 µm. (B) Quantification of the percentage of cells with a single CFP-Atg8-positive punctum presented in A. Data represent the average of two independent experiments±s.e.m., and asterisks indicate a significant difference compared with the wild type (WT) (two-tailed t-test: P<0.05).
- Fig. 5.
Atg2 association with the PAS does not require Atg18 and/or Atg21. (A) Atg2 is recruited in an Atg18- and Atg21-independent manner to the PAS. The atg18Δ strain expressing endogenous Atg2-GFP and carrying either no other constructs (ERY087), integrated 13×myc-tagged ATG18 (ERY094) or a 13×myc-tagged ATG18 loop mutant (ERY095 and ERY097), or the atg18Δ atg21Δ strain expressing only endogenous Atg2-GFP (ERY103), were processed as in Fig. 4A. (B) Quantification of the percentage of cells with a single Atg2-GFP-positive dot presented in A. The data represent the average of two experiments±s.e.m.
- Fig. 6.
Atg18 binding to Atg2 is essential for its recruitment to the PAS. (A) Atg2-binding mutants of Atg18 do not localize to the PAS. The atg18Δ strain carrying the mCheV5-Atg8 fusion and genomically integrated GFP-tagged ATG18 (ERY090) or the GFP-tagged ATG18 loop mutants 2 or 5 (ERY091 and ERY093), and the atg18Δ atg2Δ strain carrying GFP-tagged ATG18 (ERY102) were analyzed as in Fig. 4A. White arrows indicate colocalization of the fluorescence signals. (B) Quantification of the percentage of cells with colocalizing puncta presented in A and supplementary material Fig. S3. All data represent the average of two independent experiments±s.e.m. Asterisks indicate a significant difference compared with the wild type (WT) (two-tailed t-test: P<0.05).
- Fig. 7.
Atg2-Atg18 association at the PAS depends on both the Atg2- and phosphoinositide-binding motifs of Atg18. (A) Atg2-Atg18 interaction at the PAS was visualized using the BiFC system. Wild type (WT), atg3Δ or atg13Δ cells expressing endogenous Atg2-VN and/or Atg18-VC (ERY117, ERY118 and ERY119) were grown in rich medium before being nitrogen starved for 3 h. Fluorescence images were taken before and after nitrogen starvation. Arrows highlight the BiFC signals. DIC, differential interference contrast. Scale bars: 5 µm. (B) Quantification of the percentage of cells analyzed in A that are positive for a perivacuolar BiFC punctum. The data represent the average of two experiments±s.e.m., and asterisks indicate a significant difference compared with the WT (two-tailed t-test: P<0.05). (C) Wild type cells or atg14Δ cells expressing endogenous Atg2-VN and Atg18-VC, Atg18(L2)-VC or Atg18(L5)-VC (ERY132, ERY133, ERY137 and ERY146) were analysed as in A. (D) Quantification of the percentage of cells positive for a single perivacuolar BiFC punctum analyzed in C and carried out as in B. (E) The Atg18-Atg2 interaction is severely impaired when these two proteins cannot be recruited to the PAS. Cell lysates from atg18Δ (JGY3), atg18Δ ATG2-PA (FRY387) and atg18Δ atg14Δ ATG2-PA (ERY145) strains transformed with plasmids expressing 13×myc-tagged wild type Atg18 or Atg18(L2) were subjected to pull-down experiments as in Fig. 2A.
- Fig. 8.
Model for Atg18 β-propeller function in autophagy. (A) Putative structure of the Atg18 β-propeller generated with PyMol software. On the left, a cartoon view of the predicted structure of the Atg18 β-propeller from the top and side is shown. The blades are colored in yellow, loop 1 in marine blue, loop 2 in dark blue and loop 5 in red. In the middle, the molecular surface of the Atg18 β-propeller is presented with the same colors. On the right, the β-propeller is displayed in line view with the mutated residues in loops 2 and 5 highlighted in red. The FRRG sequence is located in loop 5, whereas the residues important for Atg2 binding are situated in loop 2. (B) Alignment of the amino acid sequence around loop 2 of the β-propeller of Atg18, Atg21 and Hsv2. Part of the amino acid sequences of Atg18, Atg21 and Hsv2 from S. cerevisiae were aligned using ClustalW2 software. Blades 2 and 3 of the Atg18 β-propeller are underlined and highlighted in grey. Loop 2 is bordered by a box and the mutated residues are in bold. (C) Alignment of the amino acid sequence around loop 2 of the β-propeller of Atg18 and WIPI4 from various organisms. The amino acid sequences of Atg18, EPG-6 from C. elegans, and WIPI4 from H. sapiens, M. musculus and D. rerio, have been aligned and presented as in B. (D) Model for Atg18 recruitment to the PAS. At an early stage of PAS formation, PtdIns is converted into PtdIns3P by the PtdIns 3-kinase complex I. This lipid is essential for the subsequent association of Atg2 to this structure. Presence of PtdIns3P and Atg2 on the autophagosomal membrane triggers the recruitment of Atg18 through its β-propeller. It is presently unclear whether these events occur on the phagophore or on another precursor membrane.
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