Identification of the membrane-embedded regions of the Neurospora crassa plasma membrane H(+)-ATPase

Abstract

Reconstituted proteoliposomes containing functional Neurospora crassa plasma membrane H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface exposed were treated with trypsin and then subjected to Sepharose CL-6B column chromatography to remove the liberated peptides. The peptides remaining associated with the liposomes were then separated from the phospholipid by Sephadex LH-60 column chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six H(+)-ATPase peptides with approximate molecular masses of 7, 7.5, 8, 10, 14, and 21 kDa were found to be tightly associated with the liposomal membrane. Amino acid sequencing of the 7-, 7.5-, and 21-kDa peptides in the LH-60 eluate identified them as H(+)-ATPase fragments beginning at residues 99 or 100, 272, and 660, respectively. After further purification, the approximately 10- and 14-kDa peptides were also similarly identified as beginning at residues 272 and 660. The approximately 8-kDa fragment was purified further but could not be sequenced, presumably indicating NH2-terminal blockage. To identify which of the liposome-associated peptides are embedded in the membrane, H(+)-ATPase molecules in the proteoliposomes were labeled from the hydrophobic membrane interior with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and cleaved with trypsin, after which the membrane-associated peptides were purified and assessed for the presence of label. The results indicate that the approximately 7-, 7.5-, and 21-kDa peptides are in contact with the lipid bilayer whereas the approximately 8-kDa peptide is not. Taken together with the results of our recent analyses of the peptides released from the proteoliposomes, this information establishes the transmembrane topography of nearly all of the 919 residues in the H(+)-ATPase molecule.