Identification of lipid-accessible sites on the nephrops 16-kDa proteolipid incorporated into a hybrid vacuolar H(+)-ATPase: site-directed labeling with N-(1-Pyrenyl)cyclohexylcarbodiimide and fluorescence quenching analysis.
Proton translocation by the vacuolar H(+)-ATPase is mediated by a multicopy transmembrane protein, the 16-kDa proteolipid. It is proposed to assemble in the membrane as a hexameric complex, with each polypeptide comprising four transmembrane helices. The fourth helix of the proteolipid contains an intramembrane acidic residue (Glu140) which is essential for proton translocation and is reactive toward N,N'-dicyclohexylcarbodiimide (DCCD). Current theoretical models of proton translocation by the vacuolar ATPase require that Glu140 should be protonated and in contact with the membrane lipid. In this study we present direct support for this hypothesis. Modification with the fluorescent DCCD analogue N-(1-pyrenyl)cyclohexylcarbodiimide, coupled to fluorescence quenching studies and bilayer depth measurements using the parallax method, was used to probe the position of Glu140 with respect to the bilayer. Glutamate residues were also introduced mutagenically as targets for the fluorescent probe in order to map additional lipid-accessible sites on the 16-kDa proteolipid. These data are consistent with a structural model of the 16-kDa proteolipid oligomer in which the key functional residue Glu140 and discrete faces of the second and third transmembrane helices of the 16-kDa proteolipid are exposed at the lipid-protein interface.
Harrison
M
M
School of Biochemistry and Molecular Biochemistry, University of Leeds, UK. M.A.Harrison@leeds.ac.uk
Powell
B
B
Finbow
M E
ME
Findlay
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D001181:Arthropods / Q000378:metabolism*; D002234:Carbodiimides / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011510:Proteolipids / Q000737:chemistry*; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D002234:Carbodiimides; D005456:Fluorescent Dyes; C411504:N-(1-pyrenyl)cyclohexylcarbodiimide; D011510:Proteolipids; D006180:Proton-Translocating ATPases