Autophagy and the cvt pathway both depend on AUT9

Abstract

In growing cells of the yeast Saccharomyces cerevisiae, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen for aut(-) mutants and isolated aut9-1 cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid aut9-1 mutant cells with a genomic library, in this study we identified and characterized the AUT9 gene, which is allelic with CVT7. aut9-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.

FIG. 1

(A) Genomic fragments isolated after complementation of the sporulation defect of homocygous aut9Δ cells. (B) Hydrophobicity analysis of the Aut9p indicates five potential transmembrane domains. (C) Aut9p is the representative of a protein family of unknown function. Sequences were aligned by the Clustal method. Homologous residues (1 distance unit) are shaded.

FIG. 1

(A) Genomic fragments isolated after complementation of the sporulation defect of homocygous aut9Δ cells. (B) Hydrophobicity analysis of the Aut9p indicates five potential transmembrane domains. (C) Aut9p is the representative of a protein family of unknown function. Sequences were aligned by the Clustal method. Homologous residues (1 distance unit) are shaded.

FIG. 2

aut9Δ cells (B to E) show a defect in the accumulation of autophagic vesicles in the vacuole during starvation in the presence of PMSF. As a control, we also checked cells with defects in the major vacuolar endoproteinases (pep4Δ prb1Δ) (A and F to I). Freeze etching nicely illustrates the accumulation of autophagic vesicles in starved pep4Δ prb1Δ cells (I). Cells were taken from the logarithmic growth phase (C and F), starved for 5 h (D and G), or starved for 24 h (E, H, and I). (A and B) Nomarski optics (bar, 20 μm). (C to H) Thin-section electron microscopy; I, freeze etching electron microscopy (bars, 1 μm).

FIG. 2

aut9Δ cells (B to E) show a defect in the accumulation of autophagic vesicles in the vacuole during starvation in the presence of PMSF. As a control, we also checked cells with defects in the major vacuolar endoproteinases (pep4Δ prb1Δ) (A and F to I). Freeze etching nicely illustrates the accumulation of autophagic vesicles in starved pep4Δ prb1Δ cells (I). Cells were taken from the logarithmic growth phase (C and F), starved for 5 h (D and G), or starved for 24 h (E, H, and I). (A and B) Nomarski optics (bar, 20 μm). (C to H) Thin-section electron microscopy; I, freeze etching electron microscopy (bars, 1 μm).

FIG. 3

aut9Δ cells, like pep4Δ cells, show a reduced survival rate during starvation compared to wild-type cells. Cells were starved in 1% potassium acetate solution, and aliquots were plated out to determine the proportion of surviving cells.

FIG. 4

(A) Maturation of proaminopeptidase I is impaired in aut9Δ cells. (B) cvt7-1 cells are allelic with aut9Δ cells. (C) Fusion proteins of GFP with the amino and carboxy termini of Aut9p, respectively, are biologically active.

FIG. 5

Total protein turnover during starvation for nitrogen was measured in cvt⁻ mutants by labeling all proteins with [³⁵S]methionine and determining the amount of acid-soluble small peptides generated by proteolysis. Data shown are the average from several independent experiments. Some cvt⁻ mutants such as cvt9-1 cells show wild-type-like or partially reduced protein turnover, suggesting that these mutants are affected predominantly in the cvt pathway rather than in autophagy.

FIG. 6

(A) Accumulation of quinacrine in the vacuoles of growing and starved aut9Δ cells suggests wild-type-like acidification. Starved cells were incubated for 4 h in 1% potassium acetate. Bar, 20 μm. (B) In growing and in starved aut9Δ cells, only mature CPY is detectable in immunoblots. (C) Growth of aut9Δ cells is wild-type-like on YP medium containing ethanol as a carbon source. As a control, mss51Δ cells, known to exhibit a petite phenotype (5), are included.

FIG. 7

(A) Biologically active GFP-Aut9 fusion protein in aut9Δ cells grown to an OD of 2.2, visualized at punctate structures in the cytosol; (B) DAPI staining; (C) Nomarski optics; (D) overlay of panels A to C. Bar, 20 μm.