Probing the structure of the Neurospora crassa plasma membrane H(+)-ATPase.
The structure of the Neurospora crassa plasma membrane H(+)-ATPase has been investigated using a variety of chemical and physiochemical techniques. The transmembrane topography of the H(+)-ATPase has been elucidated by a direct, protein chemical approach. Reconstituted proteoliposomes containing purified H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin, and the numerous peptides released were purified by HPLC and subjected to amino acid sequence analysis. In this way, seventeen released peptides were unequivocally identified as located on the cytoplasmic side of the membrane, and numerous intervening segments could be inferred to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return between sequences established to be cytoplasmically located. Additionally, three large membrane-embedded segments of the H(+)-ATPase were isolated using our recently developed methods for purifying hydrophobic peptides, and identified by amino acid sequence analysis. This information established the topographical location of virtually all of the 919 residues in the H(+)-ATPase molecule, allowing the formulation of a reasonably detailed model for the transmembrane topography of the H(+)-ATPase polypeptide chain. Separate studies of the cysteine chemistry of the H(+)-ATPase have demonstrated the existence of a single disulfide bridge in the molecule, linking the NH2- and COOH-terminal membrane-embedded domains. And, analyses of the circular dichroism and infrared spectra of the purified H(+)-ATPase have elucidated the secondary structure composition of the molecule. A first-generation model for the tertiary structure of the H(+)-ATPase based on this information and other considerations is presented.
Scarborough
G A
GA
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003545:Cysteine / Q000737:chemistry; D005656:Fungal Proteins / Q000648:ultrastructure*; D009492:Neurospora crassa; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000648:ultrastructure*
D005656:Fungal Proteins; D006180:Proton-Translocating ATPases; D003545:Cysteine