When glucose is added to yeast cells that are starved for 3 days, fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase 2 are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. In this study, we examined the distribution of FBPase at the ultrastructural level. FBPase was observed in areas close to the plasma membrane and in cytoplasmic structures that are heterogeneous in size and density. We have isolated these intracellular structures that contain FBPase, the Vid vesicle marker Vid24p, and the endosomal marker Pep12p. They appeared irregular in size and shape. In yeast, actin polymerization plays an important role in early steps of endocytosis. Mutants that affect actin polymerization inhibited FBPase degradation, suggesting that actin polymerization is important for FBPase degradation. Both FBPase and malate dehydrogenase 2 were associated with actin patches. Vid vesicle proteins such as Vid24p or Sec28p were also at actin patches, although they dissociated from these structures at later time points. We propose that Vid24p and Sec28p are present at actin patches during glucose starvation. Cargo proteins arrive at these sites following the addition of glucose, and the endocytic vesicles then pinch off from the plasma membrane. Following the fusion of endosomes with the vacuole, cargo proteins are then degraded in the vacuole.Protein synthesis and degradation play important roles in determining the levels of proteins in cells. For protein degradation, two major pathways have been characterized (1-6). These are the ubiquitin/proteasome pathway and the lysosomal targeting pathway (1-6). Lysosomes degrade intracellular proteins as well as proteins that are internalized from the plasma membrane. The lysosomal pathway can be regulated by nutrients or by hormones in the media (4 -7). During starvation, a macroautophagy pathway is induced that degrades proteins or organelles non-selectively (8 -10). Another chaperone-mediated autophagy pathway degrades proteins based on the presence of a particular KFERQ sequence (11)(12)(13)(14).The yeast vacuole is homologous to mammalian lysosomes (15)(16)(17). A number of protein trafficking pathways lead to the yeast vacuole. For example, the Vps pathway targets carboxypeptidase Y from the endoplasmic reticulum to the Golgi, and then to the vacuole for maturation (18,19). The Cvt pathway targets aminopeptidase I from the cytosol to the vacuole for processing (20,21). During nitrogen starvation, a non-selective autophagy pathway is induced, and ATG genes play important roles in this pathway (20 -23). Interestingly, many of these ATG genes are utilized by the Cvt pathway (20 -23). ATG genes are also needed for micro-or macro-pexophagy pathways that degrade peroxisomes when yeast cells are transferred from methanol or ethanol to glucose (24, 25). The macroautophagy pathway is inhibited by Tor1 and is stimulated by rapamycin. The induction of autophagy by rapamycin has been used to clear large aggregates that cannot be cleared by the proteasome path...

No abstract

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.