Identification and reconstitution of an isoform of the 116-kDa subunit of the vacuolar proton translocating ATPase.
We have identified a cDNA encoding an isoform of the 116-kDa subunit of the bovine vacuolar proton translocating ATPase. The predicted protein sequence of the new isoform, designated a2, consists of 854 amino acids with a calculated molecular mass of 98,010 Da; it has approximately 50% identity to the original isoform (a1) we described (Peng, S.-B., Crider, B. P., Xie, X.-S., and Stone, D.K. (1994) J. Biol. Chem. 269, 17262-17266). Sequence comparison indicates that the a2 isoform is the bovine homologue of a 116-kDa polypeptide identified in mouse as an immune regulatory factor (Lee, C.-K., Ghoshal, K., and Beaman, K.D. (1990) Mol. Immunol. 27, 1137-1144). The bovine a1 and a2 isoforms share strikingly similar structures with hydrophilic amino-terminal halves that are composed of more than 30% charged residues and hydrophobic carboxyl-terminal halves that contain 6-8 transmembrane regions. Northern blot analysis demonstrates that isoform a2 is highly expressed in lung, kidney, and spleen. To determine the possible role of the a2 isoform in vacuolar proton pump function, we purified from bovine lung a vacuolar pump proton channel (VO) containing isoform a2. This VO conducts bafilomycin-sensitive proton flow after reconstitution and acid activation, and supports proton pumping activity after assembly with the catalytic sector (V1) of vacuolar-type proton translocating ATPase (V-ATPase) and sub-58-kDa doublet, a 50-57-kDa polypeptide heterodimer required for V-ATPase function. These data indicate that the a2 isoform of the 116-kDa polypeptide functions as part of the proton channel of V-ATPases.
Peng
S B
SB
Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Li
X
X
Crider
B P
BP
Zhou
Z
Z
Andersen
P
P
Tsai
S J
SJ
Xie
X S
XS
Stone
D K
DK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011522:Protons; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011522:Protons; D012333:RNA, Messenger; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases