Cloning and expression of vacuolar proton-pumping ATPase subunits in the follicular epithelium of the bullfrog endolymphatic sac.
In an investigation aimed at clarifying the mechanism of crystal dissolution of the calcium carbonate lattice in otoconia (the mineral particles embedded in the otolithic membrane) of the endolymphatic sac (ELS) of the bullfrog, cDNAs encoding the A- and E-subunits of bullfrog vacuolar proton-pumping ATPase (V-ATPase) were cloned and sequenced. The cDNA of the A-subunit consisted of an 11-bp 5'-untranslated region (UTR), a 1,854-bp open reading frame (ORF) encoding a protein comprising 617 amino acids with a calculated molecular mass of 68,168 Da, and a 248-bp 3'-UTR followed by a poly(A) tail. The cDNA of the E-subunit consisted of a 72-bp 5'-UTR, a 681-bp ORF encoding a protein of 226 amino acids with a calculated molecular mass of 26,020 Da, and a 799-bp 3'-UTR followed by a poly(A) tail. Western blot and immunofluorescence analyses using specific anti-peptide antisera against the V-ATPase A- and E-subunits revealed that these subunits were present in the ELS, urinary bladder, skin, testes, and kidneys. In the ELS, positive cells were scattered in the follicular epithelium which, as revealed by electron microscopy, corresponds to the location of mitochondria-rich cells. These findings suggest that V-ATPase, including the A- and E-subunits, exists in mitochondria-rich cells of the ELS, which might be involved in dissolution of the calcium carbonate crystals in the lumen of the ELS.
Yajima
Shinya
S
Department of Biology, Faculty of Science, Shizuoka University, Japan.
Kubota
Makoto
M
Nakakura
Takashi
T
Hasegawa
Takahiro
T
Katagiri
Nobuto
N
Tomura
Hideaki
H
Sasayama
Yuichi
Y
Suzuki
Masakazu
M
Tanaka
Shigeyasu
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D004712:Endolymphatic Sac / Q000201:enzymology* / Q000648:ultrastructure; D004848:Epithelium / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D011892:Rana catesbeiana / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D025262:Vacuolar Proton-Translocating ATPases