Identification of the major cytoplasmic regions of the Neurospora crassa plasma membrane H(+)-ATPase using protein chemical techniques.
The transmembrane topography of the Neurospora crassa plasma membrane H(+)-ATPase has been investigated using purified, reconstituted components and direct protein chemical techniques. Reconstituted proteoliposomes containing H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin to liberate peptides present on the cytoplasmic surface of the H(+)-ATPase as recently described (Hennessey, J.P., Jr., and Scarborough, G. (1990) J. Biol. Chem. 265, 532-537. The released peptides were then separated from the proteoliposomes by gel filtration chromatography and further purified by high performance liquid chromatography. Fourteen such peptides were identified by NH2-terminal amino acid sequence analysis, directly defining these parts of the molecule as present on the cytoplasmic surface of the membrane. Moreover, this information identified several additional flanking stretches as likely to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return. These results and the results of other recent experiments establish 417 residues of the 919 present in the ATPase molecule, at positions 2-100, 186-256, 441-663, and 897-920, as cytoplasmically located. Taken together with the results of our preliminary investigations of the membrane embedded sectors of the ATPase, this information allows the formulation of a reasonably detailed model for the transmembrane topography of the ATPase polypeptide chain.
Scarborough
G A
GA
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599.
Hennessey
J P
JP
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000201:enzymology; D008081:Liposomes; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011510:Proteolipids / Q000302:isolation & purification; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000302:isolation & purification
D008081:Liposomes; D010446:Peptide Fragments; D011510:Proteolipids; C020485:proteoliposomes; D006180:Proton-Translocating ATPases