Dissertations, Theses, and Capstone Projects

Date of Degree

9-2015

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Chang-Hui Shen

Subject Categories

Molecular Biology

Keywords

vacuole; yeast

Abstract

In eukaryotes, V-ATPases play an essential role in cellular pH homeostasis as they transport hydrogen ions into the vacuoles. This assures an acidic vacuolar interior and normal physiological processes associated with this organelle. Studies show that mutations in any of the VMA genes encoding for subunits of the V-ATPase pump result in growth inhibition in the absence of inositol, suggesting that V-ATPases may play an important role in phospholipid biosynthesis or vice versa. It is not clear how VMA genes affect phospholipid biosynthesis and how the regulators of phospholipid biosynthetic genes may affect ATPase activity. Here, we employ biochemical and genetic analyses to understand the connection. In the first part of the study, we evaluated how VMA3 an indispensable gene required for proper V-ATPase function affects phospholipid biosynthesis. Using growth analysis, we demonstrated that vma3Δ cells were sensitive to pH conditions in the absence of inositol. This result strongly suggests that VMA3 can influence phospholipid gene expression. Subsequently, we observed that HXK2 gene was down-regulated in vma3Δ cells using real-time PCR analysis. Acetic acid sensitivity assay further confirmed reduced expression of HXK2 gene in vma3Δ cells. As such, we have demonstrated that the VMA3 plays an important role in phospholipid biosynthesis through regulation of HXK2 gene expression.

Recent studies show that some phospholipid genes play important roles in vacuolar morphology and acidification. Among these genes are transcription factors, INO2 and OPI1. To determine their role on vacuolar function, we first performed CaCl2 sensitivity test which screens for genes important in vacuolar ATPase activity. Our results showed that WT, opi1Δ and ino2Δ cells exhibited similar growth sensitivity at 10 mM CaCl2, whereas at 60 mM CaCl2 both opi1Δ and ino2Δ cells exhibited moderate increase in sensitivity compared to WT. As microscopic analyses did not show any significant differences in vacuolar morphology of opi1Δ or ino2Δ compared to WT, vacuolar pH analyses showed significant increases in vacuolar acidification of opi1Δ but not ino2Δ cells compared to WT. Moreover, we observed that some of the genes that encode for V-ATPases were down-regulated significantly in both opi1Δ and ino2Δ. Furthermore, both opi1Δ and ino2Δ cells showed lower ATPase activity than WT cells, suggesting that the transcription factors affecting expression of phospholipid genes play an important role in V-ATPase pump activity and vacuolar homeostasis. It is possible that down-regulation of ATPase activity is to maintain normal H+ concentration in the vacuole and to compensate a pH imbalance caused by the lack Opi1p or Ino2p. Together, our results have provided new insights into the connection between phospholipid biosynthesis and cellular pH homeostasis.

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