Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit.
An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H(+)ATPase (V-ATPase), located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6v1B1 (B1), cause distal renal tubular acidosis in humans but surprisingly, B1(-/-) mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1(-/-) mice compared with B1(+/+). Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1(-/-) mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1(-/-) mice as in B1(+/+) (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1(-/-) mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1(-/-) mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility.
Da Silva
Nicolas
N
Massachusetts General Hospital, Harvard Medical School, Program in Membrane Biology, Nephrology Div., 185 Cambridge St., CPZN 8150, Boston, MA 02114-2790, USA. ndasilva@partners.org
Shum
Winnie W C
WW
El-Annan
Jaafar
J
Păunescu
Teodor G
TG
McKee
Mary
M
Smith
Peter J S
PJ
Brown
Dennis
D
Breton
Sylvie
S
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology*; D016764:Cell Polarity; D004822:Epididymis / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development; D004847:Epithelial Cells / Q000201:enzymology*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007834:Lasers; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D042282:Microdissection / Q000379:methods; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008875:Middle Aged; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; C480628:Atp6v1b1 protein, mouse; D025262:Vacuolar Proton-Translocating ATPases