We investigate the reversible disassembly of V_OV₁-ATPase in life yeast cells by time resolved confocal FRET imaging. V_OV₁-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. V_OV₁-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca²⁺ homoeostasis in the cell. The activity of the V_OV₁-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of V_OV₁-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.

Date Published: 26 February 2010
PDF: 8 pages
Proc. SPIE 7569, Multiphoton Microscopy in the Biomedical Sciences X, 75690W (26 February 2010); doi: 10.1117/12.841169

Published in SPIE Proceedings Vol. 7569:
Multiphoton Microscopy in the Biomedical Sciences X
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)