J. Cell Biol.
© The Rockefeller University Press
0021-9525/97/07/37/08 $2.00
Volume 138, Number 1, July 14, 1997 37-44

Aminopeptidase I Is Targeted to the Vacuole by a Nonclassical Vesicular Mechanism

Sidney V. Scott,^* Misuzu Baba, Yoshinori Ohsumi,^§ and Daniel J. Klionsky^*

^* Section of Microbiology, University of California, Davis, California 95616;  Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Tokyo 112, Japan; and ^§ Department of Cell Biology, National Institute for Basic Biology, Okazaki 444, Japan

The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism. Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway. This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular. Subcellular fractionation experiments demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment. In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to contain 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 cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation.

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