  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.
