Open Access
Liu, Jingjing
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
December 29, 2005
Committee Members:
  • Hui Ling Chiang, Committee Chair
  • David A Antonetti, Committee Member
  • C Randell Brown, Committee Member
  • Christopher J Lynch, Committee Member
  • John Warren Wills, Committee Member
  • FBPase
  • Protein degradation
  • V-ATPase
  • membrane fusion
  • intracellular trafficking
  • Vid vesicles
Abstract A unique protein degradation pathway has been studied using Saccharomyces cerevisiae as a model system. The gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase are induced when cells are grown in medium containing low glucose. However, they are rapidly degraded when cells are shifted to medium containing fresh glucose. Several VID (vacuole import and degradation) genes were identified in genetic screens and are required for the degradation of these cargo proteins. FBPase has been used as a marker in my study. FBPase is initially imported into a novel type of vesicle called Vid vesicles, which then deliver FBPase to the vacuole for degradation. Although the origin of the Vid vesicle is not known, ubiquitin conjugation is necessary for Vid vesicle formation. Many proteins such as the heat shock protein Ssa2p, and the immunophillin family member Cpr1p, regulate the FBPase import into Vid vesicles. The molecules that regulate Vid vesicle and vacuole fusion were largely unknown. A Vid vesicle-specific molecule, Vid24p, has been reported to be required for this process. However, the mechanism of Vid24p in the regulation of Vid vesicle and vacuole fusion was poorly understood. Membrane fusion is a thermodynamically unfavorable process. Fusion requires multiple steps including the targeting of donor vesicles to acceptor membranes, the tethering, and subsequent fusion of vesicles with the target membranes. Homotypic vacuole fusion is the most studied membrane fusion event in vivo and in vitro. Two vacuoles isolated from various genetic backgrounds can fuse in vitro with purified components. Homotypic vacuole fusion utilizes proteins that are also shared by other trafficking pathways, such as the cytosol to vacuole transport (CVT) pathway and the vacuolar protein sorting (VPS) pathway. As the Vid pathway requires Vid vesicle fusion with vacuoles to deliver of FBPase into the vacuole, it is possible that the fusion of Vid vesicles with vacuoles also utilizes these common vacuole fusion proteins. However, the molecular mechanisms of these fusion proteins in the Vid vesicle fusion may be different from those in homotypic vacuole fusion. In addition, this fusion may use molecules that are unique to the Vid pathway, and distinct signaling pathways may regulate these different fusions. The goal of my project is to characterize the fusion molecules and signaling pathways that regulate the Vid vesicle to vacuole fusion event. The work described in this dissertation provides a glimpse of the fusion molecules involved in the Vid vesicle to vacuole trafficking and fusion. The data described here implicate a role for the commonly used fusion molecules as well as unique molecules for the Vid vesicle to vacuole trafficking event. To ask whether the Vid vesicle to vacuole trafficking step requires the molecules essential for other vesicles fusion with vacuole, I studied the common fusion proteins such as SNARE and GTPase. To test this hypothesis, the GTPase Ypt7p, the HOPS complex and v- and t-SNAREs were examined for their roles in FBPase degradation. They were all essential for FBPase degradation and they functioned in the Vid vesicle trafficking to vacuole step. Furthermore, Ypt7p was identified as Vid-vesicle-essential components. Likewise, a number of v-SNAREs (Ykt6p, Nyv1p and Vti1p) and the HOPS (homotypic fusion vacuole protein sorting complex) family members Vps39p and Vps41p, were also required for the proper function of Vid vesicles. By contrast, the t-SNARE Vam3p was only necessary for vacuole, while another t-SNARE, Vam7p, functions at both vacuole and vesicles. The study of these fusion molecules suggested that Vid vesicle-vacuole trafficking exhibits characteristics similar to heterotypic membrane fusion events. It was also hypothesized that the Vid vesicle to vacuole trafficking event requires unique molecules in addition to the above-mentioned common fusion molecules. To identify novel proteins that regulate Vid vesicle trafficking, FBPase degradation mutants were screened by a colony blotting procedure using a yeast genomic gene deletion library. Via this approach, subunits of the vacuolar H+ ATPase (V-ATPase) were identified. The V-ATPase has established roles in endocytosis, VPS sorting and homotypic vacuole fusion. However, V-ATPase subunits have distinct functions in homotypic vacuole fusion. For example, V0 domain subunits are required for homotypic vacuole fusion, but the V1 domain subunits are not. In my studies, most V1 subunits and all the V0 subunits (including the a-subunit Stv1p or Vph1p) were needed for FBPase degradation. They were required for both Vid vesicle and vacuole functions, as determined by an in vitro fusion assay. However, STV1 was only required for the proper function of Vid vesicles. Moreover, STV1 and VPH1 have distinct function in the FBPase pathway. FBPase was sensitive to proteinase K digestion in the absence of STV1, while FBPase was resistant to proteinase K digestion in the absence of VPH1. The assembly of V0 and V1 domains was increased when cells were shifted from low glucose to high glucose. However, the assembly of V0 and V1 domains was independent of RAV genes. The distribution of V1 proteins is independent of V0 genes. The V1 subunits Vma2p and Vma5p were still detected on Vid vesicles and vacuoles in cells lacking V0 genes. However, missing one V0 gene usually caused other V0 subunits to be absent in the Vid vesicles and vacuoles fractions. Furthermore, the assembly of the V0 complex in the ER is necessary for FBPase trafficking, since mutants that block the assembly and transport of V0 out of the ER were defective in FBPase degradation. FBPase can be degraded in the proteasome or the vacuole depending on the length of glucose starvation. For short-termed starved cells, FBPase is degraded in the proteasome. By contrast, when long-termed starved cells are shifted to glucose, FBPase is degraded in the vacuole. How does FBPase know which pathway to go to? My hypothesis is that FBPase degradation pathway is subjected to distinct cell signaling regulation depending on the growth conditions. To test this hypothesis, the roles of glucose signaling pathways and the cAMP pathway were investigated. I found that the cAMP signaling pathway and hexokinases were needed for the vacuolar-dependent degradation of FBPase. Hexokinases was also required for FBPase degradation in the proteasome-dependent pathway. By contrast, the cAMP-dependent signaling pathway was only involved in the vacuolar FBPase degradation. Further investigation indicated that the cAMP pathway regulates the Vid vesicle to vacuole trafficking/fusion step and plays an important role on both Vid vesicles and vacuoles. In summary, these studies indicated that the FBPase Vid trafficking pathway ustilizes common fusion molecules. However, protein molecules unique to the Vid trafficking pathway, such as Stv1p, are also needed for this step of the targeting pathway. Distinct signaling pathways regulate FBPase degradation in the proteasome or the vacuole. Although the signaling pathway leading to the degradation of FBPase in the proteasome is currently unknown, the cAMP pathway is necessary for the vacuolar pathway and it plays important role in the Vid vesicles and vacuole fusion event.