Biomedical Sciences ETDs

Publication Date

Spring 5-13-2017

Abstract

We studied two signals that regulate the yeast vacuolar proton pump, V-ATPase, glucose and PI(3,5)P2.  V-ATPase regulation by glucose is linked to the glycolytic enzyme PFK-1. Chapter one investigates the relationship between V-ATPase and glycolysis in cells overexpressing the PFK-1 subunit Pfk2p.  We measured: (i) PFK-1 activity to determine the effect on PFK-1 function; and (ii) NADH autofluorescence to determine the effect on glycolysis. PFK2 overexpression decreased PFK-1 catalytic activity. However, the NADH synthesis rate increased by 1.4 – 2.6-fold, suggesting that excess Pfk2p enhanced the apparent glycolytic capacity of the cells. Vacuolar acidification was measured after the glucose concentration was increased from 2% to 4% in Pfk2p overexpressing cells, although NADH synthesis was not different at 2% and 4% glucose. Thus, failure to re-activate re-assembled V-ATPase pumps in 2% glucose was not attributed to glycolytic defects or reduced glycolytic capacity. We propose that re-addition of 4% glucose stimulates other signals that reactivate V-ATPase proton transport.

V-ATPase activity also can be controlled by the vacuolar membrane lipid PI(3,5)P2. PI(3,5)P2 interacts with V-ATPase and other proteins including TORC1,  a master kinase that responds to nutrients availability. Chapter two studies the interplay with V-ATPase, TORC1, and PI(3,5)P2. The results showed that constitutively high levels of PI(3,5)P2  (i) cannot rescue the Vma- growth phenotype of cells with catalytically inactive V-ATPase; and (ii) cannot activate TORC1 if V-ATPase is inactive. Because excess PI(3,5)P2 altered vacuolar pH and morphology, it remains elusive whether PI(3,5)P2 integrates V-ATPase and TORC1 functions.

Keywords

V-ATPase, Proton pump, Vacuole, pH, Glycolysis, Phosphofructokinase-1, NADH, ATP, Phosphatidylinositol, PI(3, 5)P2

Document Type

Thesis

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Masters

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Dr. Karlett J. Parra

Second Committee Member

Dr. Summer Raines Hayek

Third Committee Member

Dr. Meilian Liu

Download

Share

COinS