THE MEMBRANE TOPOLOGY OF THE CARBOXYL-TERMINAL 3RD OF THE NEUROSPORA PLASMA-MEMBRANE H(+)ATPASE

To localize transmembrane segments in the carboxyl-terminal third of t he Neurospora plasma membrane H+-ATPase, we constructed fusion protein s on the cDNA level. These contained DNA fragments encoding hydrophili c residues of the amino and carboxyl termini of the H+-ATPase with a D NA fragment encoding the putative transmembrane segment. To report tra nslocation into microsomes, a DNA fragment encoding three consensus N- linked glycosylation sites was engineered carboxyl-terminal to the put ative transmembrane segment. Fusion proteins were synthesized in a Neu rospora in vitro translation system supplemented with homologous micro somes. By the criteria of glycosylation of fusion proteins by microsom es, sedimentation of products with microsomes after alkaline extractio n, and analysis of protected fragments generated from proteinase K dig estion of integrated products, we localized six transmembrane segments in the carboxyl-terminal third of the H+-ATPase. These results suppor t a 10-segment model of the Neurospora H+-ATPase.