title: Direct evidence for the cytoplasmic location of the NH2- and COOH-terminal ends of the Neurospora crassa plasma membrane H+-ATPase.
abstract: Reconstituted proteoliposomes containing Neurospora plasma membrane H+-ATPase molecules oriented predominantly with their cytoplasmic portion facing outward have been used to determine the location of the NH2 and COOH termini of the H+-ATPase relative to the lipid bilayer. Treatment of the proteoliposomes with trypsin in the presence of the H+-ATPase ligands Mg2+, ATP, and vanadate produces approximately 97-, 95-, and 88-kDa truncated forms of the H+-ATPase similar to those already known to result from cleavage at Lys24, Lys36, and Arg73 at the NH2-terminal end of the molecule. These results establish that the NH2-terminal end of the H+-ATPase polypeptide chain is located on the cytoplasmic side of the membrane. Treatment of the same proteoliposome preparation with trypsin in the absence of ligands releases approximately 50 water-soluble peptides from the proteoliposomes. Separation of the released peptides by high performance liquid chromatography and spectral analysis of the purified peptides identified only a few peptides with the properties expected of a COOH-terminal, tryptic undecapeptide with the sequence SLEDFVVSLQR, and NH2-terminal amino acid sequence analysis identified this peptide among the possible candidates. Quantitative considerations indicate that this peptide must have come from H+-ATPase molecules oriented with their cytoplasmic portion facing outward, and could not have originated from a minor population of H+-ATPase molecules of reverse orientation. These results directly establish that the COOH-terminal end of the H+-ATPase is also located on the cytoplasmic side of the membrane. These findings are important for elucidating the topography of the membrane-bound H+-ATPase and are possibly relevant to the topography of other aspartyl-phosphoryl-enzyme intermediate ATPases as well.
authors: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599.
authors: J P
authors: JP
authors: Hennessey
authors: G A
authors: GA
authors: Scarborough
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D008081:Liposomes / Q000032:analysis / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000032:analysis* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
chemical_list: D008081:Liposomes; D010446:Peptide Fragments; D014638:Vanadates; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; D008274:Magnesium