Activation of H+-ATPase of the Plasma Membrane of Saccharomyces cerevisiae by Glucose: The Role of Sphingolipid and Lateral Enzyme Mobility

Sergey Permyakov,Nataliya Suzina,Airat Valiakhmetov,Hendrik W Van Veen

doi:10.1371/journal.pone.0030966

Abstract

Activation of the plasma membrane H+-ATPase of the yeast Saccharomyces cerevisiae by glucose is a complex process that has not yet been completely elucidated. This study aimed to shed light on the role of lipids and the lateral mobility of the enzyme complex during its activation by glucose. The significance of H+-ATPase oligomerization for the activation of H+-ATPase by glucose was shown using the strains lcb1-100 and erg6, with the disturbed synthesis of sphyngolipid and ergosterol, respectively. Experiments with GFP-fused H+-ATPase showed a decrease in fluorescence anisotropy during the course of glucose activation, suggesting structural reorganization of the molecular domains. An immunogold assay showed that the incubation with glucose results in the spatial redistribution of ATPase complexes in the plasma membrane. The data suggest that (1) to be activated by glucose, H+-ATPase is supposed to be in an oligomeric state, and (2) glucose activation is accompanied by the spatial movements of H+-ATPase clusters in the PM.

Highlights

H+-ATPase, hereafter, Pma1, of the yeast plasma membrane (PM), one of the major structural proteins of the PM, belongs to a P-type ATPase [1,2] and is encoded by the PMA1 gene [3]
The regulation of Pma1 activity was shown for the first time by Serrano, who discovered that incubation with glucose resulted in a reversible many-fold enhancement of the enzyme’s activity, a decrease in Km, and an increase in Vmax [6]
Little is known about the role of lipids in glucose activation of Pma1

Summary

Introduction

H+-ATPase, hereafter, Pma, of the yeast plasma membrane (PM), one of the major structural proteins of the PM, belongs to a P-type ATPase [1,2] and is encoded by the PMA1 gene [3]. The enzyme hydrolyzes ATP and forms an electrochemical proton gradient on the PM and drives the transport of basic nutrients across the PM [4]. As has been shown previously, Pma consumes up to 20% of cellular ATP and, not surprisingly, its activity is under strict control [5]. The regulation of Pma activity was shown for the first time by Serrano, who discovered that incubation with glucose resulted in a reversible many-fold enhancement of the enzyme’s activity, a decrease in Km, and an increase in Vmax [6]. The sugars utilized via the glycolytic pathway (fructose and mannose) were shown to lead to the enhancement of the enzyme’s activity [6]. Sugars (D-xylose and galactose) metabolized through other pathways, as well as nonmetabolized glucose analogs (3-Omethylglucose and deoxyglucose), did not result in any enhancement

Objectives

Methods

Results