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

Abstract

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. INTRODUCTIONThe lysosome/vacuole is the major cellular center for degradation, recycling, and storage of biological constituents. Several well-characterized transport routes are implicated in protein delivery to this organelle during normal growth conditions. These pathways are conserved among eukaryotic organisms, but work in the yeast Saccharomyces cerevisiae has had a major impact on their characterization and in the identification of the molecular machinery (Conibear and Stevens, 1998). The route called the alkaline phosphatase pathway provides a direct transport connection between the Golgi complex and the vacuole Piper et al., 1997). A second itinerary from the Golgi complex to the vacuole passes through the late endosome and is known as the carboxypeptidase Y pathway (Piper et al., 1995;Babst et al., 1998;Conibear and Stevens, 1998). These two pathways are required for the biogenesis and maintenance of vacuoles, whereas a third route, endocytosis, has a catabolic function. Endocytosis mediates the downregulation of plasma membrane proteins by delivering them via the late endosome to the vacuole for degradation (Hicke, 1999;Katzmann et al., 2002). Before reaching the late endosome, these proteins pass through the early endosome where some components are recycled back to the cell surface (Hicke et al., 1997;Prescianotto-Baschong and Riezman, 1998;Lewis et al., 2000). The carboxypeptidase Y pathway and endocytosis converge at the endosome where another route, the multivesicular body pathway, delivers both resident enzymes and substrates to the vac...

Atg27 Is Required for Autophagy-dependent Cycling of Atg9

“…These characteristics of Atg27 lead us to suggest that Atg27 may also mark the membrane source that is donated to the forming vesicles. The localization of Atg27 to the Golgi complex fits with previous studies implicating this organelle as another source of membrane for the forming vesicles (Reggiori et al, 2004b).…”

“…In pep4⌬ cells, which lack vacuolar proteinase A activity, the breakdown of autophagic bodies is blocked, allowing them to be visualized by electron microscopy. To eliminate the background vesicles that are delivered into the vacuole through the multivesicular body pathway, we deleted the VPS4 gene (Reggiori et al, 2004b). Wild-type, atg1⌬, and atg27⌬ strains additionally harboring pep4⌬ vps4⌬ double mutations were grown in YPD to early log phase and then shifted to SD-N for 5 h and prepared for electron microscopy as described in Materials and Methods.…”

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

“…34 Accordingly, we next decided to examine the requirement for phosphorylation at S122 in selective types of autophagy. Aminopeptidase I (Ape1), a vacuole resident hydrolase, is initially synthesized as a cytosolic precursor (prApe1), and is delivered to the vacuole through either nonselective autophagy or the cytoplasm-to-vacuole targeting (Cvt) pathway, depending on nutrient conditions; [35][36][37][38] in either case delivery is a selective process depending on a receptor protein, Atg19. 5,39,40 Upon delivery to the vacuole, the propeptide of prApe1 is enzymatically removed generating the mature hydrolase.…”

Mitochondrial DNA deletions inhibit proteasomal activity and stimulate an autophagic transcript

“…Induction of SNARE proteins is essential for the process of autophagy [53,[73][74][75]. Thus it is possible that the subsarcolemmal proliferation of mitochondria observed in KSS/CPEO and revealed by the Gomori trichrome stain in raggedred fibers is in fact the result of increased autophagic activity.…”