Vacuolar localization of oligomeric alpha-mannosidase requires the cytoplasm to vacuole targeting and autophagy pathway components in Saccharomyces cerevisiae

One challenge facing eukaryotic cells is the posttranslational import of pr oteins into organelles. This problem is exacerbated when the proteins assem ble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this ol igomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. alpha -Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pat hway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defec tive in the Cvt and autophagy pathways. We have found that Ams1 forms an ol igomer in the cytoplasm. The oligomeric form of Ams1 is also detected in su bvacuolar vesicles in strains that are blocked in vesicle breakdown, indica ting that it retains its oligomeric form during the import process. These r esults identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.