The vacuolar-atpase of Paramecium multimicronucleatum: gene structure of the B subunit and the dynamics of the V-ATPase-rich osmoregulatory membranes.
Previous studies have shown that the vacuolar-ATPase (V-ATPase) of the contractile vacuole complexes (CVCs) in Paramecium multimicronucleatum is necessary for fluid segregation and osmoregulation. In the current study, immunofluorescence showed that the development of a new CVC begins with the formation of a new pore around which the collecting canals form. The decorated membranes are then deposited around the newly formed collecting canals. Quick-freeze deep-etch techniques reveal that six 10-nm-wide V-ATPase V, sectors, tightly packed into a 20 x 30-nm rectangle, form two rows of these compacted sectors that helically wrap around the cytosolic side of decorated membrane tubules. During new CVC formation, packing of decorated tubules around mature CVCs was temporarily disrupted so that some of these decorated tubules became transformed into decorated vesicles. Freeze-fracturing of these decorated vesicles revealed a highly pitted E-face and a particulate P-face. The V-ATPase was purified for the first time in any ciliated protozoan and shown to contain, as in other cells, the V1 subunits A to E, and four 14-20 kDa polypeptides. The B subunit was cloned and found to be encoded by one gene containing four short introns. This subunit has 510 amino acid residues with a predicted molecular weight of 56.8 kDa, a value similar to B subunits of other organisms. Except for the N- and C-termini, it has a 75% sequence identity with other B subunits, suggesting that the B subunits in Paramecium, like other species, have been conserved and that the entire surface of this subunit may be important in interacting with other subunits.
Biology Program, University of Hawaii, Honolulu 96822, USA. fok@hawaii.edu
Agnes K
AK
Fok
Kiyoshi
K
Yamauchi
Akinori
A
Ishihara
Marilynn S
MS
Aihara
Masaki
M
Ishida
Richard D
RD
Allen
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D015723:Gene Library; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010247:Paramecium / Q000201:enzymology* / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics*; D014617:Vacuoles / Q000201:enzymology / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D014882:Water-Electrolyte Balance / Q000235:genetics / Q000502:physiology
D025262:Vacuolar Proton-Translocating ATPases