The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles in Paramecium.
The vacuolar proton-ATPase (V-ATPase) is a multisubunit enzyme complex that is able to transfer protons over membranes against an electrochemical potential under ATP hydrolysis. The enzyme consists of two subcomplexes: V0, which is membrane embedded; and V1, which is cytosolic. V0 was also reported to be involved in fusion of vacuoles in yeast. We identified six genes encoding c-subunits (proteolipids) of V0 and two genes encoding F-subunits of V1 and studied the role of the V-ATPase in trafficking in Paramecium. Green fluorescent protein (GFP) fusion proteins allowed a clear subcellular localization of c- and F-subunits in the contractile vacuole complex of the osmoregulatory system and in food vacuoles. Several other organelles were also detected, in particular dense core secretory granules (trichocysts). The functional significance of the V-ATPase in Paramecium was investigated by RNA interference (RNAi), using a recently developed feeding method. A novel strategy was used to block the expression of all six c- or both F-subunits simultaneously. The V-ATPase was found to be crucial for osmoregulation, the phagocytotic pathway and the biogenesis of dense core secretory granules. No evidence was found supporting participation of V0 in membrane fusion.
Wassmer
Thomas
T
Centre National de la Recherche Scientifique, Centre de Génétique Moleculaire, Avenue de la Terasse, Bâtiment 26, F-91198 Gif-sur-Yvette cedex, France.
Froissard
Marine
M
Plattner
Helmut
H
Kissmehl
Roland
R
Cohen
Jean
J
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D020868:Gene Silencing / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000502:physiology / Q000648:ultrastructure; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015388:Organelles / Q000502:physiology* / Q000648:ultrastructure; D010247:Paramecium / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D034622:RNA Interference / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000502:physiology; D016415:Sequence Alignment; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014617:Vacuoles / Q000502:physiology* / Q000648:ultrastructure
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D025262:Vacuolar Proton-Translocating ATPases