Vac8 and Atg24 Are Essential for Efficient Degradation of FAS.

To determine whether the autophagic machinery participates in FAS degradation, we monitored the delivery of FAS to the vacuole in the S. cerevisiae atg8 strain by immunoblotting and fluorescence microscopy. Fas2-GFP in the atg8 strain failed to reach the vacuole and its localization remained cytosolic (Fig. 2 A and B). This result indicated that FAS degradation depends on the core autophagy protein Atg8 and implied that autophagy degrades FAS. To further characterize FAS degradation, we investigated the steady-state levels of endogenous Fas1 at different time points of nitrogen starvation by using Fas1 antibody. After 4 h of nitrogen starvation Fas1 had dropped rapidly in WT cells, and was hardly detectable on Western blot analysis after starvation for 8 h (Fig. 2C). In contrast, Fas1 levels remained relatively stable in the pep4 strain and in the autophagy mutant strain atg8 (Fig. 2C).

The rapid decrease in FAS levels under nitrogen starvation and the need of Atg8 for this process prompted us to examine which autophagy proteins are responsible for its degradation. We did this by monitoring Fas1 levels in rich conditions and under nitrogen starvation in different autophagy-related knockout strains (Fig. S2). Fas1 degradation was inhibited upon deletion of core autophagic genes, but persisted upon deletion of most of the selective autophagy genes, including atg11 (Fig. S2). In contrast, deletion of the selective autophagy-related genes vac8 and atg24 greatly decreased the rate of FAS degradation (Fig. 3).

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Fig. 3.

Vac8 and Atg24 are essential for efficient degradation of FAS. (A) WT (BY4741) and vac8 (TOS028) S. cerevisiae cells were grown to midlog phase and shifted to SD-N medium for the indicated time periods. Cell lysates were subjected to SDS/PAGE, followed by Western blot analysis using anti-Fas1, anti-Ape1, and anti-Pgk1 antibodies. Quantifications of three individual experiments are presented on the right. Error bars represent the SDs of three independent experiments. ***P < 0.001 (Student’s t test) (Right). (B) WT (BY4741) and atg24 (TOS012) cells were grown as in A. Cell lysates were subjected to SDS/PAGE, followed by Western blot analysis using anti-Fas1 and anti-Pgk1 antibodies. Quantifications of three individual experiments are presented on the right. Error bars represent the SDs of three independent experiments. **P < 0.01, ***P < 0.001 (Student’s t test) (Right). (C) pho8::pho860 pho13 (TN124) and pho8::pho860 pho13 vac8 (TOS027) cells were grown as in A and shifted to SD-N medium for the indicated time periods. Autophagic activity was measured using the quantitative Pho860 assay. Error bars represent the SDs of three independent experiments. n.s., not significant; **P < 0.01, ***P < 0.001 (Student’s t test). (D) pho8::pho860 pho13 (TN124) and pho8::pho860 pho13 atg24 (TOS026) cells were grown as in A and shifted to SD-N medium for the indicated time periods. Autophagic activity was measured using the ALP assay. Error bars represent the SDs of three independent experiments. n.s., not significant (Student’s t test).

Vac8 is a vacuolar membrane protein required for efficient vacuolar fusion and for several selective autophagy processes, including the cytoplasm-to-vacuole targeting (CVT) pathway (11, 12). We found that vac8-knockout strains failed to efficiently degrade FAS upon nitrogen starvation (Fig. 3A and Fig. S3A). Whereas FAS degradation was inhibited, autophagy was active as indicated by delivery of the vacuolar aminopeptidase ApeI to the vacuole (Fig. 3A and Fig. S3 A and B) (12). To determine the effect of vac8 deletion on bulk autophagy, we used the Pho860 assay (14). Pho860, a truncated variant of the vacuolar alkaline phosphate Pho8, lacks the N-terminal transmembrane domain. In the absence of this domain the normal translocation of Pho8 into the endoplasmic reticulum is prevented. Pho860 accumulates in the cytosol and is delivered to the vacuole for maturation and activation only by nonselective autophagy. Although bulk autophagy was still active in vac8-knockout strains, the activity of Pho860 was lower in the vac8 strain than in the WT, as previously reported (Fig. 3C) (12). Thus, we cannot definitively conclude whether the inhibition of FAS degradation in vac8 strains is the outcome of a general decrease in the autophagic flux or a specific effect of Vac8.

The sorting nexin Atg24 reportedly participates in the CVT pathway but not in autophagy (9, 10). We found that the atg24 strain failed to efficiently degrade FAS (Fig. 3B) but maintained its ability to deliver Pho860 to the vacuole (Fig. 3D) (9, 10). Moreover, a plasmid expressing Atg24-GFP under the promoter of Atg24 was able to rescue the delivery of FAS to the vacuole in the atg24 knockout strain (Fig. S3C). Of note, the maturation rate of Pho860 to the vacuole in WT strains was significantly lower than the degradation rate of FAS (Fig. S3D). Taken together, these results suggest that FAS is selectively delivered to the vacuole upon nitrogen starvation.

FAS Degradation Depends on the Interaction with Atg8.

Atg8 is a key factor in the selective delivery of cargo proteins for degradation. The N-terminal region of Atg8 has been shown to participate in adaptor recruitment and protein recognition (15, 16). In searching for potential cargo or machinery proteins that interact with Atg8, we performed immunoprecipitation experiments on Atg8-null cells transformed with GFP-Atg8 or with GFP-Atg8 lacking the first (GFP-Atg8N8) or both (GFP-Atg8N24) N-terminal α-helices. As shown in Fig. 4A, a specific high-molecular band that coimmunoprecipitates with the full-length Atg8, but not with its two truncated forms, was identified. Mass spectrometric analysis of the resulting band recognized it exclusively as Fas1 and Fas2 (Fig. S4A). The identity of FAS was confirmed via Western blot analysis with an antibody directed against Fas1 (Fig. 4B) and by reciprocal coimmunoprecipitation using protein A-tagged Fas1 and Fas2 as bait and anti-Atg8 antibody (Fig. 4C). As shown in Fig. 4B and Fig. S4B, FAS was detected only when precipitated with the full-length Atg8, and only the full-length Atg8 precipitated with protein A-tagged FAS (Fig. 4C). It has been previously reported that the unassembled Fas2 subunit is degraded in the proteasome, whereas the unassembled Fas1 subunit is degraded by the vacuole (8). To examine the possibility that Atg8 directly interacts with the Fas1 subunit, recombinant Fas1 fused at its N-terminal to Flag tag was bound to anti-Flag beads and monitored for its ability to pull down recombinant Atg8. As depicted in Fig. 4D, Atg8 from Escherichia coli lysate as well as purified Atg8 interact with recombinant Fas1, suggesting that these proteins interact directly.

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Fig. 4.

FAS coimmunoprecipitates with Atg8 in an N-terminal-dependent manner. (A) atg8 (TOS006) S. cerevisiae cells expressing pYES-GFP-Atg8, pYES-GFP-Atg8 N8 or pYES-GFP-Atg8 N24 were grown to midlog phase. Cell lysates were subjected to immunoprecipitation using anti-GFP antibodies. The immunoprecipitates were subjected to SDS/PAGE followed by silver staining. Total is 5% of the lysate. (B) atg8 (TOS006) cells expressing pYES-GFP-Atg8, pYES-GFP-Atg8 N8 or pYES-GFP-Atg8 N24 were grown to midlog phase and shifted to SD-N medium for 2 h. Cell lysates were immunoprecipitated using anti-GFP antibodies. Immunoprecipitates were subjected to SDS/PAGE, followed by Western blot analysis using anti-Fas1 and anti-GFP antibodies. Total is 5% of the lysate. (C) Fas1-proteinA (TOS024) and Fas2-proteinA (TOS025) cells were grown to midlog phase. Cell lysates were immunoprecipitated using either IgG or protein A beads. Immunoprecipitates were subjected to SDS/PAGE, followed by Western blot analysis using anti-Fas1 and anti-Atg8 antibodies. Total is 5% of the lysate. (D) Anti-Flag beads were incubated with bacterial lysate (−) or equal amounts of lysate expressing recombinant Flag-Fas1 (+) for 1 h, washed and then incubated with bacterial lysate expressing recombinant Atg8-His for 1 h (Left) or with 4 µg purified Atg8 (Right). Beads were washed and incubated with 75 µg/mL Flag peptide for 2 h. Supernatant was collected (Elution) and subjected to SDS/PAGE, followed by Western blot analysis using anti-Flag, anti-His (Left) and anti-Atg8 (Right) antibodies. Total is 2.5% of the lysate. (E) atg8 (TOS006) cells expressing pRS316-GFP-Atg8 or pRS316-GFP-Atg8 N8 were grown to midlog phase and shifted to SD-N medium for 4 h. Cell lysates were subjected to SDS/PAGE, followed by Western blot analysis using anti-GFP antibodies. Error bars represent the SDs of three independent experiments. n.s., not significant (Student’s t test) (Right). (F) FAS2-GFP atg8 (TOS023) cells expressing pRS316-Atg8 or pRS316-Atg8 N8 were grown to midlog phase and shifted to SD-N medium for 4 h. Cell lysates were subjected to SDS/PAGE, followed by Western blot analysis using anti-GFP antibodies. Error bars represent the SDs of three independent experiments. ***P < 0.001 (Student’s t test) (Right). (G) FAS2-GFP atg8 (TOS023) cells expressing pRS316-Atg8 or pRS316-Atg8 N8 were grown to midlog phase and shifted to SD-N medium. Fas2-GFP localization was monitored after 16 h in SD-N medium. FM4-64 was used to detect vacuolar membranes. (Scale bar, 10 µm.)

To investigate the role of this interaction in FAS degradation, we monitored the GFP processing of FAS in atg8 S. cerevisiae strains expressing either full-length Atg8 or Atg8N8 under nitrogen starvation. As a control we used atg8 strains expressing GFP-Atg8 or GFP-Atg8N8. As depicted in Fig. 4E, the truncated form of GFP-Atg8 was readily processed and delivered to the vacuole. In contrast, degradation of FAS-GFP was notably inhibited in strains expressing the truncated form of Atg8, as indicated by GFP cleavage (Fig. 4F) and by fluorescence microscopy (Fig. 4G). Taken together, our results indicate that the interaction between FAS and Atg8 is important for FAS degradation.

Yeast Strains and Media.

The yeast (S. cerevisiae) strains used in this study are listed in Table S1. Yeast transformation of S. cerevisiae was performed by the lithium acetate method (27). Standard techniques for sporulation, tetrad analysis, and gene disruption in yeast were used as described previously (28). Yeast strains were grown in complete medium [YPD: 1% yeast extract, 2% peptone (wt/vol), 2% dextrose (wt/vol)], or in synthetic defined (SD) minimal medium (0.67% yeast nitrogen base lacking amino acids and nutrients, 2% dextrose (wt/vol) supplemented with amino acids depending on the strain selection), or in nitrogen starvation medium [SD-N: 2% glucose (wt/vol), 0.67% yeast nitrogen base without amino acids or ammonium sulfate].

Assays for Autophagy.

The quantitative Pho860 assay for the measurement of nonselective autophagy was performed as previously described (14). To determine FAS levels and for the GFP-processing assay, cells were grown overnight, diluted to OD 0.2−0.4, and grown to OD 0.8−1. Cells were then washed twice and shifted to nitrogen starvation medium. Cell lysis was performed as described previously (29).

FAS Activity.

Fas activity was assayed as previously described (30). Yeasts were grown to midlog phase, washed twice, and shifted to SD-N medium. At the indicated time points, 10 OD units were harvested (1,500 × g, 5 min) and washed with double distilled water. The pellet was resuspended in ice-cold 0.1 M potassium phosphate buffer pH 7.4 containing protease inhibitors and PMSF. Lysis was performed with glass beads (seven cycles of 1-min vortexing and 1 min cooling on ice). The lysate was centrifuged at 8,000 × g for 20 min at 4 °C. The assay was carried out at room temperature in a mixture containing 0.1 M potassium phosphate pH 6.5, 2 mM EDTA, 10 mM cysteine, 0.2 mM NADPH, 0.05 mM acetyl CoA, 0.3 mg BSA, 20‒40 µg total protein extract, and 0.08 mM malonyl-CoA in a total volume of 1 mL. The decrease in amount of NADPH was measured spectrophotometrically in a quartz cuvette at 340 nm. The blank rate for NADPH oxidation was measured before the addition of malonyl-CoA and was subtracted from the total rate of NADPH oxidation in the presence of malonyl-CoA. Next, 1 mU of FAS is defined as the consumption of 1 µmole of malonyl-CoA/min. FAS activity was measured as mU/mg protein and is presented as a percentage.

Cell Viability.

Cell viability was assayed by phloxine B staining as previously described (31). More than 300 cells were counted for each experiment. The average of three independent experiments is presented.

Plasmid Construction.

Yeast Atg8 was cloned into the pET28-TevH expression vector (32) fused to the C-terminal 6×His-tag or to pGEX-5X-1. Yeast Fas1 was cloned into a derivative of the expression vector pACYCDUet-1 (Novagen), harboring an N-terminal Flag-tag. Cloning of both plasmids was performed using the Restriction Free (RF) cloning strategy (33, 34).

Protein Expression and Partial Purification.

Expression of Atg8 and Fas1 in E. coli BL21(DE3) cells was induced by 200 mM IPTG (isopropyl β-d-1-thiogalactopyranoside) at 15 °C for about 18 h (32). Fas1 clone was purified using anti-DYKDDDDK G1 affinity resin (GenScript, Catalog no. L00432), according to the manufacturer’s instructions. Following the binding of Fas1 to anti-DYKDDDDK G1 affinity resin, the enriched resin was used for detection of Fas1 and Atg8 interaction. GST-Atg8 was purified with glutathione agarose and eluted with Xa factor, as previously described (35).

Z.E. is the incumbent of the Harold Korda Chair of Biology. This study was supported in part by the Israeli Science Foundation (Grant 535/11), the German-Israeli Foundation (Grant 1129/157), the Legacy Heritage Fund (Grant 1309/13), and the Conseil Pasteur-Weizmann.