Saccharomyces cerevisiae Lacking Btn1p Modulate Vacuolar ATPase Activity to Regulate pH Imbalance in the Vacuole

Sergio Padilla-López,David A Pearce

doi:10.1074/jbc.m510625200

Sergio Padilla-López, David A Pearce

Open Access

https://doi.org/10.1074/jbc.m510625200

Copy DOI

Abstract

The vacuolar H(+)-ATPase (V-ATPase) along with ion channels and transporters maintains vacuolar pH. V-ATPase ATP hydrolysis is coupled with proton transport and establishes an electrochemical gradient between the cytosol and vacuolar lumen for coupled transport of metabolites. Btn1p, the yeast homolog to human CLN3 that is defective in Batten disease, localizes to the vacuole. We previously reported that Btn1p is required for vacuolar pH maintenance and ATP-dependent vacuolar arginine transport. We report that extracellular pH alters both V-ATPase activity and proton transport into the vacuole of wild-type Saccharomyces cerevisiae. V-ATPase activity is modulated through the assembly and disassembly of the V(0) and V(1) V-ATPase subunits located in the vacuolar membrane and on the cytosolic side of the vacuolar membrane, respectively. V-ATPase assembly is increased in yeast cells grown in high extracellular pH. In addition, at elevated extracellular pH, S. cerevisiae lacking BTN1 (btn1-Delta), have decreased V-ATPase activity while proton transport into the vacuole remains similar to that for wild type. Thus, coupling of V-ATPase activity and proton transport in btn1-Delta is altered. We show that down-regulation of V-ATPase activity compensates the vacuolar pH imbalance for btn1-Delta at early growth phases. We therefore propose that Btn1p is required for tight regulation of vacuolar pH to maintain the vacuolar luminal content and optimal activity of this organelle and that disruption in Btn1p function leads to a modulation of V-ATPase activity to maintain cellular pH homeostasis and vacuolar luminal content.

Highlights

We have previously reported that the Saccharomyces cerevisiae BTN1 gene product has high sequence similarity with the human CLN3 gene product
We propose that Btn1p has active involvement in regulation of vacuolar pH and that a disruption in the function of this protein results in altered vacuolar luminal content through alterations in transport of metabolites that are usually sequestered in this organelle
Yeast cells show an incredible plasticity during a stress that challenges pH homeostasis, making it challenging to study any kind of disruption in cellular pH homeostasis

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Media—The B11718 wild-type strain (MAT␣ his3⌬1 lys2⌬0 ura3⌬0) was purchased from RESGEN. The proton transport activity into the lumen of isolated vacuoles was measured by monitoring the formation of pH gradient across the vacuolar membrane by fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) in the presence or absence of 1 ␮M concanamycin A essentially as described by Camarasa et al [32]. 25 ␮g of vacuolar membrane vesicles was added to a cuvette containing 2 ml of reaction buffer (10 mM BisTris Propene (BTP)-MES, pH 7.0, 25 mM KCl, 2 mM MgSO4, 10% (v/v) glycerol, and 2 ␮M ACMA). In Vivo Interaction between V0 and V1 Subunits of the Vacuolar ATPase Assays—Yeast cells were grown in supplemented minimal medium and harvested by centrifugation (1000 ϫ g for 5 min). The obtained blots were analyzed by densitometry using the software GelPro analyzer version 3.1

RESULTS

Findings

DISCUSSION