Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy

Abstract

In eukaryotic cells, nutrient starvation induces the bulk degradation of cellular materials; this process is called autophagy. In the yeast Saccharomyces cerevisiae, most of the ATG (autophagy) genes are involved in not only the process of degradative autophagy, but also a biosynthetic process, the cytoplasm to vacuole (Cvt) pathway. In contrast, the ATG17 gene is required specifically in autophagy. To better understand the function of Atg17, we have performed a biochemical characterization of the Atg17 protein. We found that the atg17delta mutant under starvation condition was largely impaired in autophagosome formation and only rarely contained small autophagosomes, whose size was less than one-half of normal autophagosomes in diameter. Two-hybrid analyses and coimmunoprecipitation experiments demonstrated that Atg17 physically associates with Atg1-Atg13 complex, and this binding was enhanced under starvation conditions. Atg17-Atg1 binding was not detected in atg13delta mutant cells, suggesting that Atg17 interacts with Atg1 through Atg13. A point mutant of Atg17, Atg17(C24R), showed reduced affinity for Atg13, resulting in impaired Atg1 kinase activity and significant defects in autophagy. Taken together, these results indicate that Atg17-Atg13 complex formation plays an important role in normal autophagosome formation via binding to and activating the Atg1 kinase.

Figure 1.

ATG17 is essential for normal autophagosome formation. (A) ATG17-null mutant appears devoid of autophagic bodies in the vacuole. Wild-type (KVY55; a) and atg17Δ (YYK382) strains transformed with either vector alone (b) or the multicopy ATG17 plasmid (c) were incubated for 4 h under nitrogen and carbon starvation condition in the presence of 1 mM PMSF. DIC images are shown. Bar, 5 μm. (B) The atg17Δ strain blocks autophagy. Wild-type (KVY55), atg9Δ (YTS007), atg17Δ (YYK382), and atg11Δ (YYK475) cells in YEPD (lanes 1, 3, 5, and 7) or in SD(-N) for 4 h (lanes 2, 4, 6, and 8) were analyzed by anti-Ape1 immunoblot (top). The autophagic activity was measured by the ALP assay before (□) and after (▪) nitrogen starvation for 6 h (bottom). (C) The atg17Δ strain is sensitive to nitrogen starvation. Wild-type (KA311A, •), atg1Δ (YYK36, ○), and atg17Δ (YYK111, □) cells were cultured in nitrogen starvation medium. Aliquots were removed at the indicated times and spread onto YEPD plates. The number of colonies was determined after 2–3 d. (D) Deletion of ATG17 causes formation of aberrantly small autophagosomes. The pep4Δ atg17Δ cells (YYK381; left) grown to log phase in YEPD were incubated in SD(-N) for 4 h and subjected to electron microscopy. Small autophagosome (opened arrowhead) and autophagic body (closed arrowhead) are detected in the cytoplasm and the vacuole (V), respectively. Note that the Cvt complex is contained in both autophagosome and autophagic body (left panel). Right panel shows normal autophagic bodies of wild-type cell. Bar, 200 nm.

Figure 2.

The interaction between Atg1 and Atg13 was not affected by atg17Δ. ^HAATG1 integrated cells (wild-type [WT], YYK422; or atg17Δ [17Δ; YYK 416]) additionally expressing Atg13 via a low-copy plasmid were enzymatically converted to spheroplasts and treated with 0.2 μg/ml rapamycin for 1 h. The total lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-HA and anti-Atg13 antibodies. The asterisks show nonspecific bands in the total lysate that were recognized by anti-HA and -Atg13 antibodies. Immunoblot with anti-Pep12 antibody (Sigma) was carried out to monitor amount of cell lysate.

Figure 3.

Atg17 interacts with Atg1 in an Atg13-dependent manner. Wild-type cells (YYK422), atg1Δ (YYK424) harboring a pATG13 CEN plasmid, and atg13Δ cells (YYK 426) were treated with rapamycin as shown in Figure 2. ^HAAtg1 was immunoprecipitated with anti-HA antibody and immunoblotted with anti-HA, Atg13, and Atg17 antibodies. Asterisks indicate nonspecific bands. Pep12 indicates that the amount of cell lysate per lane is equal.

Figure 4.

Atg17 interacts with Atg13 under starvation conditions. Both wild-type (YYK422) and atg1Δ (YYK 424) cells, which harbor additionally expressed Atg13 via a low-copy plasmid, were integrated with ^FLAGAtg17. The cells were grown in YEPD medium and were treated with rapamycin. Atg13 was immunoprecipitated with anti-Atg13 antibody. Associated proteins were visualized by immunoblotting with anti-Atg13 and FLAG antibodies. The asterisks indicate nonspecific bands. Equal amount cell lysate was loaded on the gel by monitoring with Pep12.

Figure 5.

A mutation in ATG17 abolishes complex formation and defects in autophagy. (A) atg17 mutant screened by the two-hybrid system. Plasmids transformed into PJ69–4A strains are indicated. The cells were grown on SC-Leu-Trp plate (left panel), replicaplated on SC-Ade-Leu-Trp plate (right panel), and then incubated at 30°C for 3 d. (B) Atg17^C24R does not bind Atg1. Total lysates were prepared from wild-type cells (YYK422) and atg17^C24R cells (YYK 467) chromosomally integrated with either ^HAATG1 and ^FLAGATG17 (WT) or ^HAATG1 and ^FLAGatg17^C24R (CR). The cells were grown in YEPD in the absence or presence of rapamycin. The lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Atg17 and anti-HA antibodies. (C) Atg17^C24R shows reduced affinity for Atg13. Immunoprecipitation with anti-Atg13 antibody was carried out as described in Figure 4 using wild-type cells (YYK422), atg17^C24R cells (YYK 467) harboring pRS316[ATG13], or atg13Δ cells (YYK 426). After SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti-Atg13 or anti-FLAG antibody as indicated. The asterisk indicates a nonspecific band. (D) atg17^C24R mutant is defective for autophagy. KVY55 cells (pho8::pho8Δ60) or YYK382 cells (atg17Δ pho8::pho8Δ60) harboring the wild-type ATG17 or atg17^C24R mutant on 2-μ plasmids were grown to 1 OD₆₀₀/ml in YEPD medium and then transferred to SD(-N) medium. Lysates from the cells before (0 h, □) and after 6-h starvation (▪) were used for the ALP assay. The error bars indicate the SD of three independent experiments.

Figure 6.

Interaction between Atg13 and Atg17 is required for Atg1 kinase activity. (A) Chemical genetic analysis of ATG1 using YEPD-grown cells. Cells (YYK126, atg1Δ pho8Δ60) harboring the indicated ATG1 plasmids were grown in YEPD medium, and the cells were pretreated with 1-NA-PP1 (DMSO solution) at the indicated concentrations for 1 h. The control cells were treated with DMSO. The cells were then transferred to SD(-N) medium containing the same concentration of 1-NA-PP1 or they continued growing in YEPD for 4 h. The cells were collected and subjected to Ape1 blotting (top) or ALP assay (bottom). The percentage of ALP activity relative to that of starved control cells is shown. The values represent averages of four independent experiments, with the SD as error bars. (B) Chemical genetic analysis of ATG1 using SC-grown cells. Cells (TN124–1C, atg1-1 pho8Δ60) harboring the indicated ATG1 plasmids were grown in SC-Ura medium. Thecells were treated with 1-NA-PP1, incubated in SD(-N) medium, and analyzed as described in A. The values (values of the starved control cells were normalized to 100%) represent the mean ALP activities from four independent experiments. The results from YEPD-grown cells (shown in A) are also shown. (C) Autophosphorylation activity of Atg1^M102A is inhibited by 20 μM 1-NA-PP1. Cell extract of the ^HAAtg1^M102A mutant cells before (left) and after (right) nitrogen starvation for 4 h was immunoprecipitated with anti-HA antibody, and the resultant immunocomplex was assayed for autophosphorylation activity in the presence of or absence of 20 μM 1-NA-PP1. (D) Atg1 kinase activity is impaired in atg17^C24R mutant cells. Wild-type (YYK422), atg17^C24R mutant (YYK467), or atg17Δ mutant (YYK416) cells were treated with rapamycin (0.2 μg/ml, 30 min) and subjected to Atg1 kinase assay. (E) Atg1 kinase activity in phosphorylating itself is increased in starvation condition. ^HAAtg1^WT, immunoprecipitated as described in C, was subjected to autophosphorylation assay (top), to protein kinase assay using 4 μg of recombinant Atg1 protein as a substrate (middle), and to protein kinase assay using 4 μg of myelin basic protein (MBP, bottom).