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. Author manuscript; available in PMC: 2017 Jan 25.
Published in final edited form as: Curr Biol. 2016 Jan 7;26(2):150–160. doi: 10.1016/j.cub.2015.11.054

The Atg17-Atg31-Atg29 complex coordinates with Atg11 to recruit the Vam7 SNARE and mediate autophagosome-vacuole fusion

Xu Liu 1,‡, Kai Mao 1,2,‡, Angela YH Yu 3, Amin Omairi-Nasser 4, Jotham Austin II 5, Benjamin S Glick 4, Calvin K Yip 3, Daniel J Klionsky 1,*
PMCID: PMC4729596  NIHMSID: NIHMS744311  PMID: 26774783

Summary

Macroautophagy (hereafter autophagy) is an evolutionarily conserved process in which portions of the cytoplasm are engulfed, degraded and subsequently recycled. The Atg17-Atg31-Atg29 complex translocates to the phagophore assembly site (PAS), where an autophagosome forms, at a very early stage of autophagy, playing a vital role in autophagy induction. Here, we identified a novel role of this complex in a late stage of autophagy where it coordinates with Atg11 to regulate autophagy-specific fusion with the vacuole. Atg17 and Atg11 interact with the vacuolar SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) Vam7 independent of each other. Several hydrophobic residues in helix 1 and helix 4 of Atg17 and the SNARE domain of Vam7 mediate the Atg17-Vam7 interaction. An F317D mutation of Atg17, which diminished its interaction with Vam7 without affecting its interaction with Atg13 or Atg31, leads to a defect in the fusion of autophagosomes with the vacuole and decreased autophagy activity. These results provide the first demonstration that the Atg17-Atg31-Atg29 complex functions in both early and late stages of autophagy, and provides a mechanistic explanation for the coordination of autophagosome completion and fusion with the vacuole.

Keywords: autophagy, lysosome, stress, vacuole, yeast

Graphical Abstract

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Introduction

Macroautophagy (hereafter autophagy) is a highly controlled cellular degradative pathway where numerous cargoes including protein aggregates, superfluous or damaged organelles and invading pathogens are targeted to the vacuole, or the analogous organelle in higher eukaryotes, the lysosome, for degradation [1–4]. In yeast, during autophagy an initial double-membrane structure named the phagophore is formed at the PAS. The phagophore engulfs its cargoes sequentially, while expanding through lipid addition. Following closure of the phagophore, the de novo formed autophagosome fuses with the vacuole. The cargoes together with the inner membrane of the autophagosome are degraded in the lumen before they are eventually recycled. Autophagy has many physiological roles, whereas autophagic dysfunction is associated with many pathologies, including cancer, diabetes, and certain types of neurodegenerative disease [5–7].

Following the induction of autophagy, Atg17 is among the very first Atg proteins that translocate to the PAS [8, 9]. Atg17 forms a stable ternary complex with Atg31 and Atg29, with Atg31 positioned as the bridge between the other two proteins [10]. The Atg17-Atg31-Atg29 complex is indispensible for autophagy induction. First, this complex recruits other Atg proteins, including Atg1and Atg13, to the PAS [11, 12]. Second, structural data indicate that this complex forms a dimer with two crescents [12, 13]; this curved structure might enable the complex together with Atg1 and Atg13 to tether Atg9-containing vesicles, which may be membrane sources for autophagosome formation, positioning them for fusion into the expanding phagophore.

After formation of a complete autophagosome, the double-membrane vesicle fuses with the vacuole. In yeast, previous studies have indicated that the fusion machinery for other targeting pathways that terminate at the vacuole is also required for autophagosome-vacuole fusion, including the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins[14–20]. Similarly, the homotypic fusion and protein sorting (HOPS) tethering complex, RAB7 and SNARE proteins also have a role in mammalian autophagy [21–27]. However, those studies in yeast were carried out in strains where the corresponding genes were deleted. As a result, all fusion events at the vacuole, not only autophagosome-vacuole fusion, were affected. Accordingly, those data have not provided specific information on the regulation of autophagosome-vacuole fusion. Here we found that the Atg17-Atg29-Atg31complex coordinates with Atg11 to recruit the SNARE protein Vam7 to the PAS when autophagy is induced. Impaired recruitment of Vam7 resulted in a defect in autophagosome-vacuole fusion and diminished autophagy flux. The results demonstrate that distinct molecular machinery is employed for regulating autophagy-vacuole fusion. Moreover, these data reveal a role of the Atg17-Atg29-Atg31complex at a late stage of autophagy.

Results

The Atg17-Atg31-Atg29 Complex Interacts with the Vam7 SNARE Protein at the PAS

The Atg17-Atg31-Atg29 complex is a scaffold that is vital for PAS organization during autophagy induction. In a genome-wide study of protein-protein interaction via the yeast two-hybrid (Y2H) assay, Atg17 was found to bind Vam7, a vacuolar SNARE protein [28]. This interaction appears counterintuitive because the Atg17-Atg31-Atg29 complex was thought to only act at a very early stage of autophagy, whereas Vam7 is required for autophagosome fusion with the vacuole, a relatively late stage of the process. Therefore, we wanted to determine whether this interaction was valid and might reveal a novel role of the Atg17-Atg31-Atg29 complex in regulating autophagy.

First, we confirmed the previous result, interaction between Atg17 and Vam7, using the Y2H assay with the wild-type Y2H strain PJ69-4A (data not shown). Although it is a useful system, in the Y2H assay proteins are typically overexpressed and artificially directed to the nucleus. Therefore, to further validate the Atg17-Vam7 interaction, we utilized another assay, bimolecular fluorescence complementation (BiFC) [29]. In this assay, the Venus yellow fluorescent protein (vYFP) is split into two fragments, VN (N terminus of vYFP) and VC (C terminus of vYFP), and these are separately tagged to two proteins of interest. If the two proteins are able to interact, VN and VC will be brought into proximity with each other and are essentially able to reform the native three-dimensional structure of vYFP and emit a fluorescent signal.

We tested the interactions between VC-Vam7 and the respective Atg(17, 29, or 31)-VN fusion proteins. None of the three pairs produced a detectable signal under growing conditions (YPD medium, “+”). In contrast, a substantial percentage of Atg17-VN VC-Vam7 and Atg29-VN VC-Vam7, but not Atg31-VN VC-Vam7, cells displayed a vYFP signal under starvation conditions (SD-N medium, “−N”) (Figure 1A). As controls, we examined Atg17-VN VC, Atg29-VN VC and Atg31-VN VC cells, where only the VC peptide was expressed. None of the cells expressing these combinations showed a vYFP signal even under starvation conditions (Figure S1A). In addition, similar to Vam7, the Vam3 protein is a vacuolar SNARE that is needed for the fusion of autophagosomes with the vacuole. As an additional control for the specificity of the Atg17-Vam7 interaction we examined whether any of the components of the Atg17-Atg31-Atg29 complex showed an interaction with Vam3 via the BiFC assay, however, in all cases the results were negative (Figure S1B and data not shown).

Figure 1. The Atg17-Atg31-Atg29 Complex Recruits Vam7 to the PAS upon Autophagy Induction.

Figure 1

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(A) VC-VAM7 ATG17-VN (KDM1586), VC-VAM7 ATG29-VN (KDM1587), and VC-VAM7 ATG31-VN (KDM1588) cells were grown in YPD to mid-log phase (+N) and then shifted to SD-N medium (−N) for 1 h to induce autophagy. Images were captured by fluorescence microscopy and are representative single Z-sections. Scale bar, 2.5 μm. The quantification of the percentage of cells showing a vYFP signal is indicated below each panel. Error bars represent the standard deviation (SD) of three independent experiments. *** p<0.001. DIC, differential interference contrast.

(B) VC-VAM7/ATG17-VN CFP-ATG8 (405) (KDM1591) cells were grown as described in (A). FM 4–64 staining was used to visualize the vacuole limiting membrane. Cells were observed under a fluorescence microscope. Images were collected as in (A). The quantification of colocalization of vYFP and CFP signals is shown on right. The percentage is calculated from the number of cells showing colocalization divided by the total number of cells with both vYFP and CFP signals. Error bars represent the SD of three independent experiments. ***, p<0.001. Scale bar, 2.5μm.

(See also Table S1 and Figure S1.)

Next, we wanted to determine where the interaction between the ternary complex and Vam7 occurs. Under starvation conditions, approximately 60% of the Atg17-Vam7 puncta (i.e., a vYFP signal) colocalized with CFP-Atg8, a PAS marker (Figure 1B). These colocalized puncta were located next to the vacuole, suggesting that the Atg17-Atg31-Atg29 complex recruits Vam7 to the PAS in autophagy-inducing conditions.

The Atg17-Atg31-Atg29 Complex Recruits Cytosolic Vam7 to the PAS at an Early Stage of Autophagy

The next question we addressed was to examine when the Atg17-Atg31-Atg29 complex recruits Vam7 to the PAS. In particular, we wanted to determine whether this recruitment occurs at an early stage of autophagy when the ternary complex itself translocates to the PAS or only after autophagosome completion, as is the case with the mammalian protein STX17 that is recruited to completed autophagosomes to allow fusion with the lysosome [26]. To answer this question, we deleted ATG1 or ATG9 in the ATG17-VNVC -VAM7 strain. In atg1Δ or atg9Δ strains, autophagy can be initiated, but complete autophagosomes cannot be formed. A vYFP signal was still observed in the null strains after nitrogen starvation, indicating that the Atg17-Atg31-Atg29 complex is able to recruit Vam7 to the PAS at an early stage of autophagy (Figure 2A). Noticeably, the percentage of cells showing vYFP puncta was significantly higher in the atg1Δ and atg9Δ strains compared to the wild-type (WT) strain (Figure 2B). This higher level of Atg17-VN-VC-Vam7 puncta likely reflects the block in autophagy and the accumulation of autophagy-related proteins at the PAS.

Figure 2. Cytosolic Vam7 Is Recruited to the PAS at an Early Stage of Autophagy.

Figure 2

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(A)VC -VAM7 ATG17-VN WT (KDM1586), atg1Δ (XLY108), and atg9Δ (XLY111) cells were grown as described in Figure 1A. Images were captured by fluorescence microscopy and are representative single Z-sections. Scale bar, 2.5 μm.

(B) Quantification of the percentage of cells showing a vYFP signal in (A). Error bars represent the SD of three independent experiments. ***, p<0.001.

(C) VC-VAM7 ATG17-VN CFP-ATG8(405) (KDM1591) and VC-VAM7ΔPX ATG17-VN CFP-ATG8(405) (XLY107) cells were grown as described in Figure 1A. Cell lysates were prepared, subjected to SDS-PAGE and analyzed by western blot.

(D) VC-VAM7 ATG17-VN CFP-ATG8(405) (KDM1591) and VC-VAM7 PXΔATG17-VN CFP-ATG8(405) (XLY107) cells were grown as described in Figure 1A. Images were captured by fluorescence microscopy and are representative single Z-sections. Scale bar, 2.5 μm.

(E) Quantification of the percentage of cells showing a vYFP signal in (D). Error bars represent the SD of three independent experiments. **, p<0.01.

(See also Figure S2.)

There are two pools of cellular Vam7; one of these pools is cytosolic, and the other is associated with the vacuolar membrane through its phox homology (PX) domain, which binds phosphatidylinositol-3-phosphate (PtdIns3P) [30]. We constructed a strain with VC- Vam7ΔPX and Atg17-VN to determine whether membrane binding was required for the Atg17-Vam7 interaction. The truncated VC- Vam7ΔPX protein was as stable as the WT protein (Figure 2C), but a previous study demonstrated that Vam7ΔPX localizes only in the cytosol [30]. To verify the localization of the Vam7 construct lacking the PX domain we compared the phenotype of Vam7-GFP to Vam7ΔPX-GFP. In contrast to the punctate and vacuolar membrane distribution seen with Vam7-GFP, the Vam7ΔPX-GFP chimera was distributed diffusely in the cytosol (Figure S2A). In addition, we examined the subcellular distribution of the proteins by fractionation. VC-Vam7 was located primarily in the vacuole membrane-enriched P13 fraction, in addition to a cytosolic pool, whereas VC-Vam7ΔPX was recovered entirely in the cytosolic supernatant fraction (Figure S2B). Despite its strictly cytosolic localization, under starvation conditions, we were still able to observe a vYFP signal corresponding to Atg17-VN-VC-Vam7ΔPX (Figure 2D and 2E). Moreover, these BiFC dots showed good colocalization with CFP-Atg8, implying that the Atg17-Atg31-Atg29 complex recruits cytosolic Vam7 to the PAS (Figure 2D). Nonetheless, we cannot rule out the possibility that this complex is also able to recruit membrane-associated Vam7 to the PAS.

Even though the PX domain of Vam7 was not required for the interaction with Atg17, we wondered whether it was necessary for normal autophagy activity. Based on a well established quantitative autophagy assay, the Pho8Δ60 assay [31–33], cells expressing VC-Vam7ΔPX showed almost a complete block in autophagy, while the activity of VC-Vam7 cells was comparable to wild-type cells (Figure S2C). This finding suggests that the PX domain of Vam7 plays some other indispensible role(s) in autophagy.

Atg17 Directly Interacts with the SNARE Domain of Vam7

In order to examine the functional consequences of disrupting the interaction between the Atg17-Atg31-Atg29 complex and Vam7, we first asked which component(s) in the ternary complex mediates the interaction. In the BiFC assay, both Atg17-VN-VC-Vam7 and Atg29-VN-VC-Vam7 cells showed a vYFP signal (Figure 1A). To determine if one or both of these interactions was direct, we deleted ATG31 in both strains because this protein connects Atg17 with Atg29 in the complex. We found that the Atg17-VN-VC-Vam7 cells still produced BiFC dots in the absence of Atg31, and these dots colocalized with CFP-Atg8 under starvation conditions (Figure 3A and 3B). In contrast, an Atg29-VN-VC-Vam7 signal was no longer detected after deletion of ATG31 (Figure 3C and 3D).

Figure 3. Atg17 Directly Interacts with the SNARE Domain of Vam7.

Figure 3

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(A) VC-VAM7 ATG17-VN CFP-ATG8(405) (WT, KDM1591) and atg31Δ (XLY100) cells were grown as described in Figure 1A. Images were captured by fluorescence microscopy and are representative single Z-sections. Scale bar, 2.5 μm.

(B) Quantification of cells showing a vYFP signal in (A). Error bars represent the SD of three independent experiments. **, p<0.01.

(C) VC-VAM7 ATG29-VN WT (KDM1587) and atg31Δ (XLY101) cells were grown as described in Figure 1A. Images were captured by fluorescence microscopy and are representative single Z-sections. Scale bar, 2.5 μm.

(D) Quantification of cells showing a vYFP signal in (C). Error bars represent the SD of three independent experiments. ***, p<0.001.

(E) MKO Y2H (YCY149) cells were co-transformed with the indicated plasmids. These cells were grown in SMD -Ura -Leu +Met medium to mid-log phase. Dilutions as indicated were grown on non-selective and selective plates for 3 days and then imaged.

(F) MKO Y2H (YCY149) cells were transformed with the indicated plasmids, and then analyzed for growth as in (E). “−” indicates no significant growth; “+”, indicates the relative extent of growth.

(G) Interaction between Atg17 and the Vam7 SNARE domain in vitro. Affinity isolation experiments were performed by co-expressing His-tagged Atg17 with either the GST-tagged Vam7 SNARE domain or the GST tag alone as described in Materials and Methods. The Coomassie-stained gel shows the GST construct inputs. Glutathione resin was used for precipitation and the western blot was probed using anti-His antibody.

TL, total lysate; E, eluate.

We have previously constructed a multiple-knockout (MKO) Y2H strain, in which most of the known ATG genes are deleted, including ATG29 and ATG31 [10]. In this MKO Y2H strain we found that cells with AD-Atg17 and BD-Vam7 still showed robust growth on selection plates, suggesting that the interaction between Atg17 and Vam7 was independent of most other Atg proteins (Figure 3E). As expected, Atg29 was not able to bind Vam7 in the MKO Y2H strain, in agreement with our results using the wild-type Y2H strain (data not shown).

Next, we decided to map the Vam7 domain that is required for binding Atg17. Vam7 consists of the N-terminal PX domain, an intermediate region and a C-terminal SNARE domain [30, 34]. We constructed a set of BD-Vam7 constructs that contain various domains of the Vam7 protein (Figure 3F). Only cells with AD-Atg17 and BD-Vam7241–316, which contains only the SNARE domain, showed stronger growth on selective plates than the control. In agreement with this result, BD-Vam7Δ245–303, in which only the SNARE domain was deleted, was not able to interact with AD-Atg17 (Figure 3F). These results suggest that the SNARE domain of Vam7 is necessary and sufficient for interaction with Atg17.

To further validate our findings that Vam7 binds Atg17 and that the SNARE domain mediates this interaction, we carried out an in vitro affinity isolation analysis. Escherichia coli cells were transformed with plasmids expressing Atg17-hexahistidine (6xHis) and glutathione S-transferase (GST) alone or as a GST-Vam7 SNARE domain chimera (GST-SNARE). Cell lysates were loaded onto glutathione resin and after extensive washing the proteins were eluted and analyzed by western blot. GST-SNARE, but not GST alone, was able to affinity isolate Atg17-6xHis (Figure 3G). Based on these results, we conclude that Atg17 directly interacts with the SNARE domain of Vam7.

Hydrophobic Residues in Helix 1 and Helix 4 of Atg17 Mediate its Interaction with Vam7

We could not block the interaction between Atg17 and Vam7 by deleting the SNARE domain without affecting all fusion events at the vacuole. Thus, in order to determine the function of the recruitment of Vam7 by Atg17 we needed to turn instead to Atg17. Deletion of ATG17 blocks autophagy at a very early stage, which would obscure a potential role for Atg17 in regulating autophagosome-vacuole fusion. Accordingly, we needed to identify an Atg17 mutant that specifically affects the Atg17-Vam7 interaction without disrupting its interaction with Atg31-Atg29 or Atg13-Atg1. The crystal structure of a partial Atg17-Atg31-Atg29 complex has been resolved, indicating the presence of four highly organized α-helices in Atg17 [13]. To map the domains required for interaction with Vam7, a set of AD-Atg17 deletion mutants were constructed (Figure 4A and S3A). Atg17 deletion mutants that lack part of helix 1 (92–106) or helix 4 (296–312, or 313–334) showed diminished interaction with Vam7 by the Y2H assay (Figure 4A and S3A).

Figure 4. Hydrophobic Residues in Helix 1 and Helix 4 of Atg17 Mediate its Interaction with Vam7.

Figure 4

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(A) MKO Y2H (YCY149) cells were transformed with the indicated plasmids and analyzed as described in Figure 3F. “−” indicates no significant growth; “+”, indicates the relative extent of growth.

(B) MKO Y2H (YCY149) cells were co-transformed with the indicated plasmids. Cells were grown as described in Figure 3E and imaged. A, H, L and U correspond to adenine, histidine, leucine and uracil, respectively.

(C) VC-VAM7 ATG17-VN (KDM1586), VC-VAM7 ATG17L105D-VN (XLY143),VC -VAM7 ATG17F317D-VN (XLY144) andVC -VAM7 ATG17I325D-VN (XLY145) cells were grown as described in Figure 1A. Images were captured by fluorescence microscopy and are representative single Z-sections. Scale bar, 2.5 μm.

(D) Quantification of the percentage of cells showing a vYFP signal in (C). Error bars represent the SD of three independent experiments. ***, p<0.001. FD, F317D; ID, I325D; LD, L105D.

(E) MKO Y2H (YCY149) cells were co-transformed with the indicated plasmids. These cells were then grown as described in Figure 3E and imaged.

(F) Interaction between Atg17F317D and the Vam7 SNARE domain in vitro. Affinity isolation experiments were performed by co-expressing His-tagged Atg17 or Atg17F317D with the GST-tagged Vam7 SNARE domain or the GST tag alone. The Coomassie-stained gel shows the GST construct inputs. Glutathione resin was used for precipitation and the western blot was probed using anti-His antibody.

(G) Quantitative infrared western to compare binding affinities between wild-type (WT) Atg17 and Atg17F317D and the Vam7 SNARE domain. Error bars correspond to standard deviation calculated from data obtained from three independent experiments. SN, SNARE. ***, p<0.001.

(H) Wild-type (WLY176) cells with empty vector, and atg17Δ (XLY134) cells with either empty vector, pCu-Atg17 (WT), pCu-Atg17L105D (LD), pCu-Atg17F317D (FD), or pCu-Atg17I325D (ID) were grown in SMD -Ura medium to mid-log phase. The cells were then shifted to SD-N for 4 h to induce autophagy, and the autophagy activity in these samples was analyzed by the Pho8Δ60 assay. The activity of atg17Δ (XLY134) cells with pCu-Atg17 (WT) after starvation was set to 100% and other values were normalized. The graph shows the average activity obtained from three independent experiments. Error bars represent the SD. **, p<0.01; ***, p<0.001.

(See also Figure S3.)

Because we had shown that the SNARE domain of Vam7, which is also in a helix structure, mediates its interaction with Atg17, we hypothesized that the binding between Atg17 and Vam7 is due to a “helix-helix” interaction, which is usually mediated by hydrophobic residues from both sides of the interacting helices. Based on an alignment of yeast Atg17 homologs, we chose the conserved hydrophobic residues from 92–106 and 296–334, namely I98, L105, I310, L313, I314, F317, I325 and L331, for further analysis (Figure S3B).

We mutated these residues to charged hydrophilic residues by site directed mutagenesis and tested their interaction with Vam7 by the Y2H assay. Among the mutants that we tested, Atg17L105D, Atg17F317D and Atg17I325D showed diminished interaction with Vam7 (Figure 4B). As a control, we tested the stability of these mutants compared to the WT protein. The GFP-Atg17L313K,I314D, GFP-Atg17L105D and GFP-Atg17F317D mutants were somewhat less stable than the GFP-Atg17WT protein (Figure S3C). However, because the AD-Atg17L313K,I314D mutant still showed a strong interaction with BD-Vam7 (Figure 4B), the impaired interaction with Vam7 seen with the Atg17L105D, Atg17F317D and Atg17I325D mutants was unlikely to be due to a lower level of the mutant proteins.

To confirm the results seen with the Y2H assay, we analyzed the three mutants by the BiFC assay. The percentage of cells showing a vYFP signal after nitrogen starvation in the Atg17-VN strains with L105D, F317D or I325D mutated was substantially decreased (Figure 4C, D).

Next, we determined whether these mutations affected other known interactions of Atg17. Because Atg1 and Atg29 interact with Atg17 through Atg13 and Atg31, respectively, we tested the Atg17 point mutants for interaction with Atg13 and Atg31 by the Y2H assay. The Atg17F317D mutant showed an essentially normal interaction with both Atg13 and Atg31, while Atg17L105D and Atg17I325D were particularly defective in interactions with Atg13 and Atg31, respectively (Figure 4E).

Because the Atg17F317D mutant was the only one that showed diminished interaction with Vam7 without substantially affecting its binding affinity to Atg13 and Atg31, we decided to focus our analysis on this mutant. First, the defective interaction of the mutant with Vam7 was further demonstrated by an in vitro affinity isolation assay. Although the GST-Vam7 SNARE domain chimera was able to affinity isolate Atg17-6xHis (Figure 3G), it was not able to efficiently pull down Atg17F317D-6xHis (Figure 4F, G). We also examined whether the F317D mutation affects the efficiency of Atg17 movement to the PAS. During nitrogen starvation GFP- Atg17F317D displayed significant colocalization with RFP-Ape1 (a PAS marker), at a frequency similar to the wild-type protein (Figure S3D).

As the next part of the analysis we asked whether the Atg17F317D mutant was competent for autophagy based on the Pho8Δ60 assay. Contrary to our expectations, the mutant Atg17F317D did not affect the autophagy activity compared to the wild-type protein. The Atg17L105D mutant showed almost a complete block of autophagy, while the Atg17I325D mutant displayed an approximately 50% decrease in activity (Figure 4H). With either of the latter two mutants, however, we were not able to exclude the possibility that the defects in interaction with Atg13 or Atg31, rather than Vam7, led to the autophagy defects.

One possible explanation for the lack of an autophagy phenotype with the Atg17F317D mutant is that the defect in the interaction with Vam7 is not severe enough to cause a detectable block in autophagy activity. We found that cells expressing AD-Atg17F317D and BD-Vam7 still grew quite well on plates lacking histidine (a less stringent selection condition), although they were not able to grow on plates without adenine (a harsher selection condition) (Figure 4B). In this case, the residual binding affinity may be adequate for autophagy to occur at a nearly wild-type level. A second possibility is that another protein might play a partially redundant role in recruiting Vam7 to the PAS, so that the autophagy activity defect from the Atg17F317D mutant was complemented.

We tested the first possibility by isolating mutants that display more severe defects in the interaction between Vam7 than Atg17F317D. Substitutions of glycine, histidine, lysine or glutamine for Phe317, however, did not show a stronger block in binding (Figure S3E). Next, we made some double mutants including Atg17F317D,I325A, Atg17F317D,I325G, and Atg17F317G,I325G. Unfortunately, although these double mutants displayed a stronger defect in binding Vam7, they also had blocks in their interaction with either Atg13 or Atg31 (Figure S3F and S3G, and data not shown), preventing us from excluding the possibility that the F317D mutation caused only a weak phenotype.

Atg11 Recruits Vam7 to the PAS Independent of the Atg17-Atg31-Atg29 Complex

Next, we tested the second possibility by looking at an Atg protein that may have a similar role in recruiting Vam7 as that proposed for Atg17. In this regard, Atg11 stands out for three reasons. First, Atg11 is predicted to have four major α-helical coiled-coil (CC) domains, similar to Atg17, which would allow it to engage in “helix-helix” interactions with Vam7. Second, like Atg17, Atg11 plays a role in formation and organization of the PAS [12, 35]. Third, the atg17Δ strain is able to generate a small number of autophagic bodies under starvation conditions, whereas an atg11Δ atg17Δ double mutant displays an essentially complete block in autophagy [35].

To assess this possibility, a Y2H assay was conducted in the MKO strain background to examine whether Atg11 could bind directly to Vam7. Cells with AD-Atg11 and BD-Vam7, but not control cells, grew on plates lacking histidine, suggesting a potential direct Atg11-Vam7 interaction (Figure 5A). However, the observation that they cannot grow on plates without adenine (data not shown) indicates that the Atg11-Vam7 interaction is weaker than that of Atg17-Vam7. Next, we tested Atg11 mutants that lack each of the four predicted coiled-coil domains to determine which region of Atg11 is involved in mediating the interaction. AD-Vam7 was still able to show binding affinity to AD-Atg11 Δ272–321(ΔCC1) and AD -Atg11Δ627–858(ΔCC3), but not toward AD -Atg11 Δ536–576(ΔCC2) or AD -Atg11Δ859–1178(ΔCC4), suggesting that the latter two α-helical domains of the protein are required for the interaction (Figure 5A).

Figure 5. Atg11 Recruits Vam7 to the PAS Independent of the Atg17-Atg31-Atg29 Complex.

Figure 5

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(A) MKO Y2H (YCY149) cells were co-transformed with the indicated plasmids. These cells were grown in SMD -Ura -Leu +Met medium to mid-log phase. Dilutions as indicated were grown on non-selective and selective plates for 4 days and then imaged.

(B) ATG11-VN VC-VAM7 (XLY124), VN-ATG11 VC (XLY087) and VN-ATG11 VC-VAM7 (XLY128) cells were grown as described in Figure 1A. Images were captured by fluorescence microscopy and are representative of single Z-sections. Scale bar, 2.5 μm.

(C) VN-ATG11 VC-VAM7CFP-ATG8 (405) (WT, XLY128), atg17Δ (XLY130), atg29Δ (XLY131) cells were grown as described in Figure 1A. Images were captured by fluorescence microscopy and are representative of single Z-sections. Scale bar, 2.5 μm.

(D) Quantification of cells showing a vYFP signal and colocalization of vYFP and CFP signals in (C). The percentage of colocalization is calculated from number of cells showing co-localization divided by total number of cells with both vYFP and CFP signals. Error bars represent the SD of three independent experiments. NS, not significant.

To further verify the interaction between Atg11 and Vam7, a BiFC assay was performed. In ATG11-VN VC-VAM7 cells, where Atg11-VN was expressed at its endogenous level, we were not able to detect a vYFP signal (Figure 5B). In contrast, the vYFP signal was observed in more than 90% of VN-ATG11 VC-VAM7 cells, in which Atg11 was tagged at its N terminus, and the VN-Atg11 chimera was overexpressed (Figure 5B). Although Atg11 can self-interact, the vYFP signal observed in these cells was not due to overexpression of VN-Atg11; cells expressing VN-ATG11 and VC alone, where both VN-Atg11 and the VC peptide were overexpressed, did not display a vYFP signal (Figure 5B).

We next examined whether this interaction occurs at the PAS. Indeed, more than 70% of VN-Atg11-VC-Vam7 vYFP dots colocalized with CFP-Atg8 under starvation conditions (Figure 5C and 5D). Previous studies have shown that Atg11 interacts with the Atg17-Atg31-Atg29 complex through Atg29 [12]. Therefore, we decided to examine whether Atg11-dependent recruitment of Vam7 to the PAS requires the ternary complex. We deleted either ATG17 or ATG29 in the VN-ATG11 VC-VAM7 strain and carried out a BiFC assay. Similar to the WT strain, significant colocalization of vYFP and CFP-Atg8 (~70%) was still observed in both strains (Figure 5C and 5D). These data suggest that Atg11 can recruit Vam7 to the PAS independent of the Atg17-Atg31-Atg29 complex.

Impaired Recruitment of Vam7 to the PAS Results in a Defect in Autophagosome-Vacuole Fusion

We next tested whether the Atg17F317D mutant would display a defect in autophagy activity in the atg11Δ background. In the atg11Δ atg17Δ background, compared to wild-type Atg17, the Atg17F317D mutant showed an approximate 30% decrease in autophagy acitivity (Figure 6A), in contrast to the essentially wild-type activity seen in the atg17Δ background (Figure 4H). Moreover, the Atg17I325D mutant, which had approximately 50% of the wild-type activity in the atg17Δ strain background, displayed almost a complete block of autophagy in the atg11Δ atg17Δ background.

Figure 6. Impaired Recruitment of Vam7 to the PAS Leads to Defects in Autophagosome-Vacuole Fusion and Autophagy Activity.

Figure 6

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(A) atg11Δ (XLY135) cells with empty vector, and atg11Δ atg17Δ (XLY136) cells with either empty vector, pCu-Atg17 (WT), pCu-Atg17F317D, or pCu-Atg17I325D were grown in SMD -Ura to mid-log phase. The cells were then shifted to SD-N medium for 4 h and autophagy activity was analyzed by the Pho8Δ60 assay. The activity of atg11Δ atg17Δ (XLY136) cells with pCu-Atg17 (WT) after starvation was set to 100% and other values were normalized. The graph shows the average activity obtained from three independent experiments. Error bars represent the SD. **, p<0.01.

(B) A schematic model of the prApe1 protease protection assay. PK, proteinase K.

(C) vam7Δ (XLY137) cells with empty vector, and atg11Δ atg17Δ (XLY136) cells with either empty vector, pCu-Atg17 (WT) or pCu-Atg17 (F317D) were grown in SMD-Ura medium to mid-log phase. The cells were then shifted to SD-N medium for 4 h. Total cell lysates from lysed spheroplasts were centrifuged at 300 x g to pellet cell debris. Then the cell lysates were further centrifuged at 5000 x g to obtain a prApe1-enriched membrane fraction, which was split into four aliquots and subjected to different treatments: no addition, 0.2% Triton X-100 (TX-100), proteinase K (PK), or proteinase K with 0.2% Trition X-100 on ice for 30 min. After protein precipitation, samples were analyzed by western blot. S.E., short exposure; L.E., long exposure.

(D) The quantification of percentage of protected prApe1 from three independent experiments. The percentage is calculated from amount of prApe1 after PK treatment divided by total level of Ape1 without any treatment. *, p<0.05.

(E) The atg17Δ vam7Δ GFP-ATG8 (XLY142) cells with pCu-Atg17 (WT), and the atg11Δ atg17Δ GFP-ATG8 (XLY147) cells with pCu-Atg17 (F317D) were grown in SMD-Ura to mid-log phase. Cells were then shifted to SD-N medium for 4 h to induce autophagy before samples were frozen and analyzed by correlative light and electron microscopy. The top two panels show samples observed by TEM tomography, while the bottom panel shows results from STEM tomography. The bottom right corner of each electron tomographic image shows the same cell visualized by fluorescence microscopy. An overlay of the fluorescent signal on the tomographic volume of the same cell is also shown. Tomographic models of the outlined autophagic structures are shown to the right; the lower model is the same as the top one, but is rotated 90° around the x-axis. Scale bars, 300 nm.

(See also Figure S4, Movie S1, S2 and S3.)

To determine whether the autophagy defect we observed with the Atg17F317D mutant in the atg11Δ atg17Δ background was due to impaired autophagosome-vacuole fusion, we performed a protease protection assay [36]. In brief, the propeptide of the cytosolic precursor form of the vacuolar hydrolase aminopeptidase I (prApe1) is sensitive to cleavage by exogenous proteases added to lysed yeast spheroplasts; once enclosed in an autophagosome, however, the propeptide is protected from digestion (Figure 6B). In the vam7Δ cells, as a result of a block in autophagosome-vacuole fusion, approximately half of the prApe1 was protected from exogenously added proteinase K (Figure 6C). In contrast, the prApe1 was completely sensitive to proteinase K treatment in the atg11Δ atg17Δ cells, where autophagosome formation is defective. Compared to cells with wild-type Atg17, in the atg11Δ atg17Δ cells expressing the Atg17F317D mutant, a small but consistent portion of the prApe1 pool was protected from proteinase K (Figure 6C and 6D).

Next, to validate our protease protection assay data, we investigated the autophaogosome structure of the Atg17F317D mutant by correlative light and electron microscopy, combining fluorescence microscopy and tomography. GFP-Atg8 was utilized as a PAS/autophagosome marker to correlate the fluorescence with autophagosomal structures observed by EM. In both the atg17Δ vam7Δ cells expressing the Atg17WT protein, and the atg11Δ atg17Δ cells expressing the Atg17F317D mutant, we were able to observe circular double-membrane structures correlated with the GFP fluorescence signal in the sections examined by transmission electron microscopy (TEM) tomography (Figure 6E, Movie S1 and S2), suggesting that the autophagosomes in the Atg17F317D mutant might be sealed. However, by TEM tomography the sections are 0.3 μm, which are not of sufficient thickness to cover a complete autophaogosome. Accordingly, we further investigated the autophagosomes in the Atg17F317D mutant, using scanning transmission electron microscopy (STEM) tomography, which covers thicker sections (up to 2 μm) [37]. By this method, we observed fully closed autophagosomes in the Atg17F317D mutant (Figure 6E and Movie S3).

To further support the observation that autophagosome formation in the Atg17F317D mutant was not defective, we transformed either the empty vector, or a plasmid expressing wild-type ATG17 or the ATG17 (F317D) mutant into atg11Δ atg17Δ vam7Δ cells, where the VAM7 deletion enhances the accumulation of completed autophagosomes. As expected, in those cells with the empty vector in which autophagosome formation was blocked, the prApe1 was completely sensitive to proteinase K treatment (Figure S4). In contrast, in cells expressing the Atg17F317D mutant, a substantial pool of the prApe1 was insensitive to proteinase K treatment, similar to that seen in cells with wild-type Atg17. Based on these results, we conclude that the autophagy defect seen with the Atg17F317D mutant was due to defective autophagosome-vacoule fusion, but not autophagosome formation.

Discussion

The Atg17-Atg31-Atg29 complex was identified as a scaffold indispensible for PAS organization and vesicle tethering at a very early stage of autophagy [12, 13]. In this study we characterized a role of this complex in regulating autophagosome-vacuole fusion, a relatively late stage of the process. We found that the Atg17-Atg31-Atg29 complex recruits the vacuolar SNARE protein Vam7 to the PAS at an early stage of autophagy. In vitro and in vivo analyses indicate that Atg17 directly interacts with the SNARE core domain of Vam7. This interaction might block SNARE complex formation between Vam7 and other SNARE proteins, such as Vti1 and Vam3, inhibiting premature fusion of incomplete autophagosomes/phagophores with the vacuole. However, if this model is correct, it is not clear how Vam7 is released from Atg17 upon autophagosome maturation. Similar to many other Atg proteins, Atg17 will dissociate from the completed autophagosome and be recycled for the next round of autophagosome formation. A previous study has shown that the clearance of PtdIns3P by the phosphatase Ymr1 is required for disassembly of the Atg machinery from completed autophagosomes, which is a requisite for subsequent fusion with the vacuole [38]. We think it is possible that clearance of PtdIns3P promotes dissociation of Atg17 from autophagosomes, releasing Vam7 to mediate fusion between autophagosomes and the vacuole. In addition, we found that the PX domain of Vam7, which is able to bind PtdIns3P, is required for normal autophagy activity, even though it is not directly involved in mediating the Atg17-Vam7 interaction (Figure 2D and S2C). We think it is likely that the removal of PtdIns3P from the membrane normally releases the PX domain of Vam7, promoting the disassociation of the protein from Atg17.

Atg11, which also functions as a scaffold protein in autophagy, interacts with Vam7 at the PAS independent of the Atg17-Atg31-Atg29 complex. Previous data suggest that Atg11 and Atg17 are partially redundant, with Atg11 functioning in selective types of autophagy, but also in PAS organization primarily under growing conditions [12, 35, 39]. Along these lines, we observed interaction between Atg11 and Vam7 under nutrient-rich conditions by the BiFC assay (data not shown). In contrast, Atg17 did not interact with Vam7 in this condition (Figure 1A). Also, a previous study has shown that Atg17 is not required for the cytoplasm-to-vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole under growing conditions [8]. These lines of evidence suggest that Atg11 recruits Vam7 to Cvt vesicles as part of the Cvt pathway. This pool of Vam7 is enough for mediating the fusion of Cvt vesicles with the vacuole considering that the Cvt pathway essentially operates at a basal level relative to induced autophagy. Upon nitrogen starvation autophagy is highly stimulated, and the Atg17-Atg31-Atg29 complex is now needed to recruit an adequate amount of Vam7 to the PAS.

Our analysis of the interaction between Atg17 and Vam7 reveals that certain hydrophobic residues in helix 1 and helix 4 of Atg17 mediate its interaction with Vam7. Point mutants Atg17L105D, Atg17F317D and Atg17I325D are defective in interaction with Vam7, with the Atg17F317D mutant showing only a partial defect. But only the Atg17F317D mutant is still able to interact with Atg13 and Atg31, as does the wild-type protein. In addition, the Atg17F317D mutant is able to suppress the autophagy defect in the atg17Δ strain to a level similar to that of the wild-type protein. In contrast, the Atg17F317D mutant can only rescue the autophagy activity to approximately 70% in an atg11Δ atg17Δ double deletion strain compared to the wild-type protein. This implies that the binding affinity of wild-type Atg17 for Vam7 is relatively saturated for starvation-induced autophagy so that a partial defect in recruiting Vam7 does not lead to an obvious functional defect.

Previous studies on the topic of autophagosome-vacuole/lysosome fusion demonstrated a requirement for the general fusion machinery that operates at this organelle [14–22, 24–27]. A major question, however, concerned the specific regulation of autophagosome-vacuole/lysosome fusion. Autophagy presents a unique situation in this regard compared to other vesicular transport pathways. For example, in the secretory pathway vesicles do not bud off from the originating organelle until they are essentially complete. Thus, fusion with the target/acceptor organelle cannot occur prematurely. In contrast, the phagophore expands sequentially and thus must be prevented from fusing with the vacuole/lysosome prior to completion. In mammals, the functional counterpart of Atg17 is RB1CC1/FIP200 [40–43]. RB1CC1 is predicted to be largely helical. The similarity in secondary structure might imply a conserved role of RB1CC1 to recruit a SNARE protein(s) to the phagophore or autophagosomes. Recent studies in higher eukaryotes identified the autophagosomal SNARE protein STX17 that specifically mediates fusion of autophagosomes with lysosomes, together with SNAP29 and VAMP8 [26, 27]. It is not known whether RB1CC1 interacts with these SNARE proteins. During the preparation of our manuscript, it was reported that ATG14 interacts with STX17 and SNAP29 on mature autophagosomes to promote membrane tethering and the fusion of autophagosomes with lysosomes [44]. It is possible that RB1CC1 coordinates with ATG14 to recruit these SNAREs to autophagosomes in mammalian cells. Our study provides evidence that a similar specific mechanism is employed to regulate autophagosome-vacuole fusion in yeast. Overall, conserved molecular machinery might be employed to regulate autophagosome-vacuole/lysosome fusion.

Experimental Procedures

Yeast Strains, Media and Growth Conditions

Yeast strains used in this study are listed in Table S1. Under growing conditions, yeast cells were grown in YPD or SMD media [45]. To induce autophagy, cells were cultured in nitrogen starvation media SD-N [45].

Plasmids

For the Y2H assay, pGAD-Atg17 was constructed by ligation of the polymerase chain reaction-amplified ATG17 open reading frame (ORF) fragment into the pGAD-C1 vector between the ClaI and SalI sites. Plasmids pGAD-Atg11(WT, Δ272–321, Δ536–576, Δ627–858, Δ859–1178), pGAD-Atg13, pGAD-Atg31 and pGBDU-Atg17 have been described previously [8, 10]. The truncation and point mutants of Atg17 were made by site-directed deletion or mutagenesis from either pGAD-Atg17 or pGBDU-Atg17. Plasmids pGBDU-Vam7, pGBDU-Vam7(1–125), pGBDU-Vam7(119–254) and pGBDU-Vam7(241–317) were constructed by ligation of the corresponding polymerase chain reaction-amplified DNA fragments from the VAM7 ORF into the pGBDU-C1 vector between the EcoRI and BamHI sites. The deletion mutants of Vam7 were made by site-directed mutagenesis based on pGBDU-Vam7.

The ATG17 ORF fragment was ligated into the ClaI and SalI sites of pCu(416) [46] and pCu-GFP(416) vectors to construct pCu-Atg17(416) and pCu-GFP-Atg17(416). Atg17 point mutants were then made by site-directed mutagenesis.

Fluorescence Microscopy

For fluorescence microscopy, images were captured on a DeltaVision microscope with a 100x objective and a CCD camera (CoolSnap HQ; Photometrics). Twelve Z-sections with 0.3-μm spacing between two neighboring sections were taken for each picture.

Other Methods

Western blot, the Pho8Δ60 assay and the prApe1 protease protection assay were performed as described previously [36, 47, 48]. The detailed procedures for the BiFC assay, in vitro GST affinity isolation, and correlative light and electron microscopy (CLEM) tomography analysis are described in the Supplemental Experimental Procedures.

Supplementary Material

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Acknowledgments

This work was supported by grant GM053396 to DJK, an operating grant from the Canadian Institutes of Health Research (MOP-126126) and startup funds from the University of British Columbia to CKY, and by funding through the Biological Systems Science Division, Office of Biological and Environmental Research, Office of Science, U.S. Dept. of Energy, under Contract DE-AC02-06CH11357 with BSG. Fluorescence microscopy was performed at the Integrated Microscopy Core Facility, and electron microscopy was performed at the Electron Microscopy Core Facility, with support for both facilities coming from the NIH-funded Cancer Center Support Grant P30 CA014599.

Abbreviations

Atg

autophagy-related

BiFC

bimolecular fluorescence complementation

CC

coiled-coil

Cvt

cytoplasm-to-vacuole targeting

GFP

green fluorescent protein

ORF

open reading frame

PAS

phagophore assembly site

STEM

scanning TEM

TEM

transmission electron microscopy

VC

C terminus of Venus YFP

VN

N terminus of Venus YFP

WT

wild type

Y2H

yeast two-hybrid

Footnotes

Author Contributions

X.L., K.M., A.Y.H.Y., and A.O.-N. conducted the experiments, and J.A.II assisted with electron microscopy analyses. X.L., K.M., A.O.-N., B.S.G., C.K.Y. and D.J.K. designed the experiments. X.L., A.O.-N., B.S.G., C.K.Y. and D.J.K. wrote the manuscript.

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References

  • 1.Klionsky DJ, Codogno P. The mechanism and physiological function of macroautophagy. J Innate Immun. 2013;5:427–433. doi: 10.1159/000351979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature. 2011;469:323–335. doi: 10.1038/nature09782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol. 2007;9:1102–1109. doi: 10.1038/ncb1007-1102. [DOI] [PubMed] [Google Scholar]
  • 4.Yang Z, Klionsky DJ. Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol. 2010;22:124–131. doi: 10.1016/j.ceb.2009.11.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Deretic V, Levine B. Autophagy, immunity, and microbial adaptations. Cell Host Microbe. 2009;5:527–549. doi: 10.1016/j.chom.2009.05.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Lynch-Day MA, Mao K, Wang K, Zhao M, Klionsky DJ. The role of autophagy in Parkinson’s disease. Cold Spring Harb Perspect Med. 2012;2:a009357. doi: 10.1101/cshperspect.a009357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Narendra D, Tanaka A, Suen DF, Youle RJ. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol. 2008;183:795–803. doi: 10.1083/jcb.200809125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Cheong H, Yorimitsu T, Reggiori F, Legakis JE, Wang CW, Klionsky DJ. Atg17 regulates the magnitude of the autophagic response. Mol Biol Cell. 2005;16:3438–3453. doi: 10.1091/mbc.E04-10-0894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Suzuki K, Kubota Y, Sekito T, Ohsumi Y. Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes Cells. 2007;12:209–218. doi: 10.1111/j.1365-2443.2007.01050.x. [DOI] [PubMed] [Google Scholar]
  • 10.Cao Y, Nair U, Yasumura-Yorimitsu K, Klionsky DJ. A multiple ATG gene knockout strain for yeast two-hybrid analysis. Autophagy. 2009;5:699–705. doi: 10.4161/auto.5.5.8382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Kabeya Y, Kamada Y, Baba M, Takikawa H, Sasaki M, Ohsumi Y. Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy. Mol Biol Cell. 2005;16:2544–2553. doi: 10.1091/mbc.E04-08-0669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mao K, Chew LH, Inoue-Aono Y, Cheong H, Nair U, Popelka H, Yip CK, Klionsky DJ. Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation. Proc Natl Acad Sci U S A. 2013;110:E2875–2884. doi: 10.1073/pnas.1300064110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ragusa MJ, Stanley RE, Hurley JH. Architecture of the Atg17 complex as a scaffold for autophagosome biogenesis. Cell. 2012;151:1501–1512. doi: 10.1016/j.cell.2012.11.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Balderhaar HJ, Arlt H, Ostrowicz C, Brocker C, Sundermann F, Brandt R, Babst M, Ungermann C. The Rab GTPase Ypt7 is linked to retromer-mediated receptor recycling and fusion at the yeast late endosome. J Cell Sci. 2010;123:4085–4094. doi: 10.1242/jcs.071977. [DOI] [PubMed] [Google Scholar]
  • 15.Darsow T, Rieder SE, Emr SD. A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole. J Cell Biol. 1997;138:517–529. doi: 10.1083/jcb.138.3.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Fischer von Mollard G, Stevens TH. The Saccharomyces cerevisiae v-SNARE Vti1p is required for multiple membrane transport pathways to the vacuole. Mol Biol Cell. 1999;10:1719–1732. doi: 10.1091/mbc.10.6.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Harding TM, Morano KA, Scott SV, Klionsky DJ. Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway. J Cell Biol. 1995;131:591–602. doi: 10.1083/jcb.131.3.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ishihara N, Hamasaki M, Yokota S, Suzuki K, Kamada Y, Kihara A, Yoshimori T, Noda T, Ohsumi Y. Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion. Mol Biol Cell. 2001;12:3690–3702. doi: 10.1091/mbc.12.11.3690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Nordmann M, Cabrera M, Perz A, Brocker C, Ostrowicz C, Engelbrecht-Vandre S, Ungermann C. The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Current biology : CB. 2010;20:1654–1659. doi: 10.1016/j.cub.2010.08.002. [DOI] [PubMed] [Google Scholar]
  • 20.Wang CW, Stromhaug PE, Shima J, Klionsky DJ. The Ccz1-Mon1 protein complex is required for the late step of multiple vacuole delivery pathways. The Journal of biological chemistry. 2002;277:47917–47927. doi: 10.1074/jbc.M208191200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Fader CM, Sanchez DG, Mestre MB, Colombo MI. TI-VAMP/VAMP7 and VAMP3/cellubrevin: two v-SNARE proteins involved in specific steps of the autophagy/multivesicular body pathways. Biochim Biophys Acta. 2009;1793:1901–1916. doi: 10.1016/j.bbamcr.2009.09.011. [DOI] [PubMed] [Google Scholar]
  • 22.Furuta N, Fujita N, Noda T, Yoshimori T, Amano A. Combinational soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins VAMP8 and Vti1b mediate fusion of antimicrobial and canonical autophagosomes with lysosomes. Mol Biol Cell. 2010;21:1001–1010. doi: 10.1091/mbc.E09-08-0693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Gutierrez MG, Munafo DB, Beron W, Colombo MI. Rab7 is required for the normal progression of the autophagic pathway in mammalian cells. J Cell Sci. 2004;117:2687–2697. doi: 10.1242/jcs.01114. [DOI] [PubMed] [Google Scholar]
  • 24.Manil-Segalen M, Lefebvre C, Jenzer C, Trichet M, Boulogne C, Satiat-Jeunemaitre B, Legouis R. The C. elegans LC3 acts downstream of GABARAP to degrade autophagosomes by interacting with the HOPS subunit VPS39. Dev Cell. 2014;28:43–55. doi: 10.1016/j.devcel.2013.11.022. [DOI] [PubMed] [Google Scholar]
  • 25.Takats S, Pircs K, Nagy P, Varga A, Karpati M, Hegedus K, Kramer H, Kovacs AL, Sass M, Juhasz G. Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol Biol Cell. 2014;25:1338–1354. doi: 10.1091/mbc.E13-08-0449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Itakura E, Kishi-Itakura C, Mizushima N. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell. 2012;151:1256–1269. doi: 10.1016/j.cell.2012.11.001. [DOI] [PubMed] [Google Scholar]
  • 27.Takats S, Nagy P, Varga A, Pircs K, Karpati M, Varga K, Kovacs AL, Hegedus K, Juhasz G. Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila. J Cell Biol. 2013;201:531–539. doi: 10.1083/jcb.201211160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 2000;403:623–627. doi: 10.1038/35001009. [DOI] [PubMed] [Google Scholar]
  • 29.Sung MK, Huh WK. Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae. Yeast. 2007;24:767–775. doi: 10.1002/yea.1504. [DOI] [PubMed] [Google Scholar]
  • 30.Cheever ML, Sato TK, de Beer T, Kutateladze TG, Emr SD, Overduin M. Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nat Cell Biol. 2001;3:613–618. doi: 10.1038/35083000. [DOI] [PubMed] [Google Scholar]
  • 31.Klionsky DJ, Emr SD. Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. The EMBO journal. 1989;8:2241–2250. doi: 10.1002/j.1460-2075.1989.tb08348.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Klionsky DJ, Emr SD. A new class of lysosomal/vacuolar protein sorting signals. The Journal of biological chemistry. 1990;265:5349–5352. [PubMed] [Google Scholar]
  • 33.Noda T, Matsuura A, Wada Y, Ohsumi Y. Novel system for monitoring autophagy in the yeast Saccharomyces cerevisiae. Biochemical and biophysical research communications. 1995;210:126–132. doi: 10.1006/bbrc.1995.1636. [DOI] [PubMed] [Google Scholar]
  • 34.Ungermann C, Wickner W. Vam7p, a vacuolar SNAP-25 homolog, is required for SNARE complex integrity and vacuole docking and fusion. Embo J. 1998;17:3269–3276. doi: 10.1093/emboj/17.12.3269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Cheong H, Nair U, Geng J, Klionsky DJ. 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. 2008;19:668–681. doi: 10.1091/mbc.E07-08-0826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Yen WL, Klionsky DJ. Proteinase protection of prApe1 as a tool to monitor Cvt vesicle/autophagosome biogenesis. Autophagy. 2012;8:1245–1249. doi: 10.4161/auto.20916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Sousa AA, Azari AA, Zhang G, Leapman RD. Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging. J Struct Biol. 2011;174:107–114. doi: 10.1016/j.jsb.2010.10.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Cebollero E, van der Vaart A, Zhao M, Rieter E, Klionsky DJ, Helms JB, Reggiori F. Phosphatidylinositol-3-phosphate clearance plays a key role in autophagosome completion. Curr Biol. 2012;22:1545–1553. doi: 10.1016/j.cub.2012.06.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Lynch-Day MA, Klionsky DJ. The Cvt pathway as a model for selective autophagy. FEBS Lett. 2010;584:1359–1366. doi: 10.1016/j.febslet.2010.02.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Chan EY. mTORC1 phosphorylates the ULK1-mAtg13-FIP200 autophagy regulatory complex. Sci Signal. 2009;2:pe51. doi: 10.1126/scisignal.284pe51. [DOI] [PubMed] [Google Scholar]
  • 41.Ganley IG, Lam du H, Wang J, Ding X, Chen S, Jiang X. ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy. J Biol Chem. 2009;284:12297–12305. doi: 10.1074/jbc.M900573200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, Iemura S, Natsume T, Takehana K, Yamada N, et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell. 2009;20:1981–1991. doi: 10.1091/mbc.E08-12-1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, Cao J, Kundu M, Kim DH. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol Biol Cell. 2009;20:1992–2003. doi: 10.1091/mbc.E08-12-1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Diao J, Liu R, Rong Y, Zhao M, Zhang J, Lai Y, Zhou Q, Wilz LM, Li J, Vivona S, et al. ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature. 2015 doi: 10.1038/nature14147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Jin M, He D, Backues SK, Freeberg MA, Liu X, Kim JK, Klionsky DJ. Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation. Curr Biol. 2014;24:1314–1322. doi: 10.1016/j.cub.2014.04.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Labbe S, Thiele DJ. Copper ion inducible and repressible promoter systems in yeast. Methods in enzymology. 1999;306:145–153. doi: 10.1016/s0076-6879(99)06010-3. [DOI] [PubMed] [Google Scholar]
  • 47.Shintani T, Klionsky DJ. Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway. J Biol Chem. 2004;279:29889–29894. doi: 10.1074/jbc.M404399200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Noda T, Matsuura A, Wada Y, Ohsumi Y. Novel system for monitoring autophagy in the yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1995;210:126–132. doi: 10.1006/bbrc.1995.1636. [DOI] [PubMed] [Google Scholar]

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