Characterization of the Functions of VID30 and VPS34 in the Vacuole Import and Degradation Pathway

Open Access
Author:
Alibhoy, Abbas Abizar
Graduate Program:
Genetics
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 18, 2012
Committee Members:
  • Hui Ling Chiang, Dissertation Advisor
  • Sarah Bronson, Committee Member
  • Jin Ming Yang, Committee Member
  • Christopher Martin Yengo, Committee Member
  • Vincent Chau, Committee Member
Keywords:
  • Autophagy
  • Gluconeogenesis
  • Vacuole Import and Degradation
  • Actin
  • VPS34
  • VID30
Abstract:
Glucose deprivation induces the synthesis of gluconeogenic enzymes such as fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), phosphoenolpyruvate carboxykinase (Pck1p) and isocitrate lyase (Icl1p) in Saccharomyces cerevisiae. Following glucose replenishment, these gluconeogenic enzymes are inactivated and degraded. The mechanisms by which these enzymes are degraded in response to glucose are dependent on the duration of starvation. Glucose replenishment of cells starved for one day results in these proteins being degraded in the proteasome. In contrast, replenishment of glucose to cells starved for three days leads to these proteins being degraded in the vacuole via the Vid (vacuole import and degradation) pathway. For the Vid pathway, the proteins are sequestered in specialized vesicles termed Vid vesicles. The Vid vesicles converge with the endocytic pathway and deliver their cargo to the vacuole for degradation. Recent studies have shown that internalization, mediated by actin polymerization, is essential for delivery of cargo proteins to the vacuole for degradation. The objective of this dissertation was to characterize the functions of two proteins, Vid30p and Vps34p, in the Vid pathway. The VID30 and VPS34 genes were identified in a transposon library screen for mutants that were defective in FBPase degradation. VPS34 encodes a class III phosphatidylinositol (PtdIns) 3-kinase (PI3K). It was demonstrated that VID30 and VPS34 play a critical role in the association and dissociation of Vid vesicles and actin patches respectively. In cells lacking the VID30 gene, FBPase and the Vid vesicle protein Vid24p were not localized to actin patches, suggesting that Vid30p is important for the association of Vid vesicles and actin patches. Vid30p contains two protein domains, a LisH (lissencephaly type 1-like homology) and a CTLH (C-terminal to the LisH) domain. The LIS1 gene is mutated in Miller-Dieker lissencephaly. The CTLH domain has been implicated in microtubule function. Both domains are required for FBPase degradation. Surprisingly, ultra-structural and cell extraction studies indicated that 40-70% of FBPase was in the extracellular fraction (periplasm) during glucose starvation. FBPase levels in the extracellular fraction (periplasm) decreased following glucose re-feeding in wild-type cells. The decline of FBPase in the extracellular fraction was dependent on the SLA1 and ARC18 involved in actin polymerization and endocytosis. Moreover, the decline of extracellular FBPase was also dependent on the VPS34 gene. Vps34p is co-localized with actin patches in three-day starved cells. In the absence of VPS34, FBPase and Vid vesicle protein Vid24p associated with actin patches before and after the addition of glucose. When the N736 residue of Vps34p was mutated to K (which inhibits kinase activity) and when the C-terminal 11 amino acids were deleted, mutant proteins failed to localize to actin patches and FBPase in the extracellular fraction did not decrease as rapidly. This suggests that VPS34 plays a critical role in the internalization of extracellular FBPase in response to glucose. This dissertation defines the roles of two proteins not previously associated with the Vid pathway and implicates trafficking through the periplasm and actin polymerization mediated endocytosis in the turnover of gluconeogenic enzymes in response to glucose replenishment after prolonged starvation.