| The yeast vacuole is the primary site of protein and organelle degradation when nutrient conditions demand recycling by autophagocytosis for survival. Saccharomyces cerevisiae mutants blocked in autophagocytosis overlap with another set of mutants defective in the biosynthetic import of a resident vacuolar protease, aminopeptidase I (API), suggesting the pathways utilize a common set of machinery. The studies presented herein increase our understanding of the molecular mechanisms of autophagy and API import through functional and genetic analyses of the proteins that are involved. Genetic, biochemical, and microscopy methods were used to demonstrate that Apg5p functions, in the context of a newly discovered protein conjugation system, to sequester cytoplasmic cargo into double membrane vesicles. Site-directed mutagenesis techniques were utilized to identify a 10-amino acid sequence within API necessary for its oligomerization and localization to the vacuole. Finally, scanning confocal fluorescence microscopy was used to demonstrate, in vivo, the requirement of Cvt18p in peroxisome-specific autophagy. |
