Vid28 protein is required for the association of vacuole import and degradation (Vid) vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction.

Gluconeogenic enzymes are induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. The Vid pathway is linked to the nonclassical secretory and internalizing pathways. In prolonged starved cells, substantial amounts of the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) are in the extracellular fraction (periplasm). However, when glucose is added to glucose-starved cells, levels of extracellular FBPase decrease rapidly. Ultrastructural studies indicate that FBPase is in Vid/endosomes following glucose addition, suggesting that FBPase is internalized in response to glucose refeeding. Under the same conditions, the majority of Vid vesicle proteins are in the intracellular fraction. In yeast, actin polymerization is involved in endocytosis. Vid vesicles associate with actin patches initially, and they dissociate later. Here, we show that VID28 plays a critical role in the association of Vid vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction. Vid28p was distributed to Vid vesicles and interacted with other Vid vesicle proteins. Vid28p contains an Armadillo (ARM) domain required for FBPase degradation. When VID28 was deleted or when the ARM domain was mutated, Vid vesicles failed to co-localize with actin patches, and Vid vesicle proteins appeared in the extracellular fraction. We suggest that the ARM domain is required for the association of Vid vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction.

Vps34p is required for the decline of extracellular fructose-1,6-bisphosphatase in the vacuole import and degradation pathway.

When Saccharomyces cerevisiae are starved of glucose for a prolonged period of time, gluconeogenic enzymes such as fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase are induced. However, when glucose is added to prolonged-starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. The Vid pathway merges with the endocytic pathway to remove intracellular and extracellular proteins simultaneously. Ultrastructural and cell extraction studies indicate that substantial amounts of FBPase were in the extracellular fraction (periplasm) during glucose starvation. FBPase levels in the extracellular fraction decreased after glucose re-feeding in wild-type cells. The decline of FBPase in the extracellular fraction was dependent on the SLA1 and ARC18 genes involved in actin polymerization and endocytosis. Moreover, the reduction of extracellular FBPase was also dependent on the VPS34 gene. VPS34 encodes the PI3 kinase and is also required for the Vid pathway. Vps34p co-localized with actin patches in prolonged-starved cells. In the absence of this gene, FBPase and the Vid vesicle protein Vid24p associated with actin patches before and after the addition of glucose. Furthermore, high levels of FBPase remained in the extracellular fraction in the Δvps34 mutant during glucose re-feeding. When the Asn-736 residue of Vps34p was mutated and when the C-terminal 11 amino acids were deleted, mutant proteins failed to co-localize with actin patches, and FBPase in the extracellular fraction did not decrease as rapidly. We suggest that VPS34 plays a critical role in the decline of extracellular FBPase in response to glucose.

Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway.

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.

The TOR complex 1 is distributed in endosomes and in retrograde vesicles that form from the vacuole membrane and plays an important role in the vacuole import and degradation pathway.

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.

The vacuole import and degradation pathway utilizes early steps of endocytosis and actin polymerization to deliver cargo proteins to the vacuole for degradation.

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.