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Vol. 15, Issue 5, 2189-2204, May 2004

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Early Stages of the Secretory Pathway, but Not Endosomes, Are Required for Cvt Vesicle and Autophagosome Assembly in Saccharomyces cerevisiae

Fulvio Reggiori ^* , Chao-Wen Wang ^* , Usha Nair , Takahiro Shintani ^* , Hagai Abeliovich  , and Daniel J. Klionsky ^*  

^* Life Sciences Institute and Departments of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109; Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109

Submitted July 8, 2003; Revised January 6, 2004; Accepted February 13, 2004
Monitoring Editor: Vivek Malhotra

The Cvt pathway is a biosynthetic transport route for a distinct subset of resident yeast vacuolar hydrolases, whereas macroautophagy is a nonspecific degradative mechanism that allows cell survival during starvation. Yet, these two vacuolar trafficking pathways share a number of identical molecular components and are morphologically very similar. For example, one of the hallmarks of both pathways is the formation of double-membrane cytosolic vesicles that sequester cargo before vacuolar delivery. The origin of the vesicle membrane has been controversial and various lines of evidence have implicated essentially all compartments of the endomembrane system. Despite the analogies between the Cvt pathway and autophagy, earlier work has suggested that the origin of the engulfing vesicle membranes is different; the endoplasmic reticulum is proposed to be required only for autophagy. In contrast, in this study we demonstrate that the endoplasmic reticulum and/or Golgi complex, but not endosomal compartments, play an important role for both yeast transport routes. Along these lines, we demonstrate that Berkeley bodies, a structure generated from the Golgi complex in sec7 cells, are immunolabeled with Atg8, a structural component of autophagosomes. Finally, we also show that none of the yeast t-SNAREs are located at the preautophagosomal structure, the presumed site of double-membrane vesicle formation. Based on our results, we propose two models for Cvt vesicle biogenesis.

Abbreviations used: Ape1, aminopeptidase I; CFP, cyan fluorescent protein; Cvt, cytoplasm to vacuole targeting; ER, endoplasmic reticulum; GFP, green fluorescent protein; PA, protein A; PAS, pre-autophagosomal structure; prApe1, precursor Ape1; PtdIns(3)P, phosphatidylinositol-3-phosphate; SNARE, N-ethylmaleimide-sensitive factor attachment protein receptor; t-SNARE, target-N-ethylmaleimide-sensitive factor attachment protein receptor; ts, temperature-sensitive; VFT, Vps-fifty-three; vps, vacuolar protein sorting; YFP, yellow fluorescent protein.

Present address: Hebrew University, Department of Biochemistry and Food Science, Faculty of Agriculture, Rehovot 76100, Israel.

Corresponding author. E-mail address: klionsky{at}umich.edu.

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