Regulatory assembly of the vacuolar proton pump VOV1-ATPase in yeast cells by FLIM-FRET

TL;DR

This study uses FLIM-FRET imaging to investigate the reversible assembly and disassembly of the VOV1-ATPase proton pump in yeast cells, revealing insights into its regulation during cellular starvation and glucose addition.

Contribution

It introduces a live-cell imaging method to monitor VOV1-ATPase assembly dynamics using fluorescent tagging and FRET-FLIM, providing new understanding of its regulatory mechanisms.

Findings

Disassembly occurs during starvation

Reassembly is triggered by glucose addition

FRET-FLIM effectively tracks subunit interactions

Abstract

We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for transport processes as well as for pH and Ca2+ homoeostasis in the cell. Activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.