Membrane potential mediates H(+)-ATPase dependence of "degradative pathway" endosomal fusion.
In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as "degradative endocytosis." The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for "degradative endocytosis." These endosomal pathways also have a unique distribution of their H(+)-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H(+)-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H(+)-ATPase dependent in baby hamster kidney cells, and H(+)-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H(+)-ATPase dependent, we inhibited v-type H(+)-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H(+)-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H(+)-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H(+)-ATPase in mammalian homotypic endosomal fusion.
Hammond
T G
TG
Tulane University School of Medicine, Tulane Environmental Astrobiology Center, New Orleans, Louisiana, 70112, USA.
Goda
F O
FO
Navar
G L
GL
Campbell
W C
WC
Majewski
R R
RR
Galvan
D L
DL
Pontillon
F
F
Kaysen
J H
JH
Goodwin
T J
TJ
Paddock
S W
SW
Verroust
P J
PJ
D000818:Animals; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D007425:Intracellular Membranes / Q000378:metabolism*; D007672:Kidney Cortex / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D008561:Membrane Fusion; D008564:Membrane Potentials / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014634:Valinomycin / Q000494:pharmacology; D015017:Yolk Sac
D014634:Valinomycin; D006180:Proton-Translocating ATPases; D011188:Potassium