title: The amino and carboxyl termini of the Neurospora plasma membrane H+-ATPase are cytoplasmically located.
abstract: Based on hydropathy analysis, the P-type cation translocating ATPases are believed to have similar topological arrangements in the membrane, but little independent evidence exists for their precise pattern of transmembrane folding. As a first step toward defining the topology of the Neurospora plasma membrane H+-ATPase, we have mapped the orientation of the amino and carboxyl termini. In three different types of experiments, both termini of the H+-ATPase were shown to be exposed at the cytoplasmic surface of the plasma membrane: 1) antibodies specific for the amino and carboxyl termini bound to permeabilized but not intact cells; 2) inside-out plasma membrane vesicles were approximately 100-fold more effective than intact cells in competing for antibody binding; and 3) trypsin, which is known to proteolyze three sites at the amino terminus and one site at the carboxyl terminus of the purified Neurospora H+-ATPase (Mandala, S. M., and Slayman, C. W. (1988) J. Biol. Chem. 263, 15122-15128), was found in the present study to cleave the same sites in inside-out plasma membrane vesicles but not in intact cells. These results indicate that the ATPase polypeptide traverses the membrane an even number of times, in support of a previously published topological model (Hager, K. M., Mandala, S. M., Davenport, J. W., Speicher, D. W., Benz, E. J., Jr., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697).
authors.affiliation: Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
authors.forename: S M
authors.initials: SM
authors.lastname: Mandala
authors.forename: C W
authors.initials: CW
authors.lastname: Slayman
mesh_terms: D000936:Antigen-Antibody Complex; D002462:Cell Membrane / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D015151:Immunoblotting; D007700:Kinetics; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000378:metabolism*; D014357:Trypsin; D014638:Vanadates / Q000494:pharmacology
chemical_list: D000936:Antigen-Antibody Complex; D007106:Immune Sera; D010446:Peptide Fragments; D014638:Vanadates; D014357:Trypsin; D006180:Proton-Translocating ATPases