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.