The peripheral complex of the tobacco hornworm V-ATPase contains a novel 13-kDa subunit G.
A prominent 16-kDa protein copurifies with the V-ATPase isolated from both posterior midgut and Malpighian tubules of Manduca sexta larvae and thus was believed to represent a V-ATPase subunit. [14C]N,N'-dicyclohexylcarbodiimide labeling and its position on SDS-electrophoresis gels revealed that this protein was different from the 17-kDa proteolipid. A cDNA clone encoding a highly hydrophilic protein with a calculated molecular mass of 13,692 Da was obtained by immunoscreening. Monospecific antibodies, affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16-kDa protein of the purified V-ATPase, confirming that a cDNA encoding this protein had been cloned. In vitro translation of the cRNA showed that the cloned 13-kDa subunit behaved like a 16-kDa protein on SDS-electrophoresis gels. The cloned protein showed 37% amino acid sequence identity to the 13-kDa V-ATPase subunit Vma10p recently cloned from yeast and some similarity to subunit b of bacterial F-ATPases. In contrast to the Vma10p protein, which behaved like a V0 subunit, the M. sexta 13-kDa protein behaved like a V1 subunit, since it could be stripped from the membrane by treatment with the chaotropic salt KI and by cold inactivation. When KI dissociated V-ATPase subunits were reassociated by dialysis that removed the KI, a soluble, 450-kDa complex of the M. sexta V-ATPase could be purified by gel chromatography. This V1 complex consisted of subunits A, B, E, and the 13-kDa subunit, confirming that the cloned protein is a new V-ATPase subunit and a member of the peripheral V1 complex of the V-ATPase. We designate this new V1 component subunit G.
Lepier
A
A
Zoological Institute, University of Munich, D-80021 Munich, Germany.
Gräf
R
R
Azuma
M
M
Merzendorfer
H
H
Harvey
W R
WR
Wieczorek
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003429:Cross Reactions; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology; D046911:Macromolecular Substances; D018402:Manduca / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases