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© The Rockefeller University Press, 0021-9525/2000//465 $5.00
The Journal of Cell Biology, Volume 148, Number 3, , 2000 465-480

Original Article

Apg9p/Cvt7p Is an Integral Membrane Protein Required for Transport Vesicle Formation in the Cvt and Autophagy Pathways



Takeshi Nodaa, John Kimb, Wei-Pang Huangb, Misuzu Babac, Chikara Tokunagaa, Yoshinori Ohsumia, and Daniel J. Klionskyb

a Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
b Section of Microbiology, University of California, Davis, California 95616
c Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Tokyo 112, Japan
Section of Microbiology, University of California, Davis, CA 95616.(530) 752-9014(530) 752-0277

djklionsky{at}ucdavis.edu

In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents.

Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins. Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.

Key Words: aminopeptidase I • lysosome • protein targeting • vacuole • yeast

© 2000 The Rockefeller University Press

Takeshi Noda and John Kim contributed equally to this work.

Abbreviations used in this paper: API, aminopeptidase I; CPY, carboxypeptidase Y; Cvt, cytoplasm to vacuole targeting; endo H, endoglycosidase H; GFP, green fluorescent protein; ORF, open reading frame; PGK, phosphoglycerate kinase; prAPI, precursor API.


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