AMINOPEPTIDASE-I IS TARGETED TO THE VACUOLE BY A NONCLASSICAL VESICULAR MECHANISM

The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassic al targeting mechanism. Recent genetic studies indicate that the biosy nthetic pathway that transports API uses many of the same molecular co mponents as the degradative autophagy pathway. This overlap coupled wi th both in vitro and in vivo analysis of API import suggested that, li ke autophagy, API transport is vesicular. Subcellular fractionation ex periments demonstrate that API precursor (prAPI) initially enters a no nvacuolar cytosolic compartment. In addition, subvacuolar vesicles con taining prAPI were purified from a mutant strain defective in breakdow n of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to c ontain vacuolar marker proteins. Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation. These data suggest that cyto solic vesicles containing prAPI fuse with the vacuole to release a mem brane-bounded intermediate compartment that is subsequently broken dow n, allowing API maturation.