The Membrane Topology of the Carboxyl-terminal Third of the Neurospora Plasma Membrane H+-ATPase

Abstract

To localize transmembrane segments in the carboxyl-terminal third of the Neurospora plasma membrane H(+)-ATPase, we constructed fusion proteins on the cDNA level. These contained DNA fragments encoding hydrophilic residues of the amino and carboxyl termini of the H(+)-ATPase with a DNA fragment encoding the putative transmembrane segment. To report translocation into microsomes, a DNA fragment encoding three consensus N-linked glycosylation sites was engineered carboxyl-terminal to the putative transmembrane segment. Fusion proteins were synthesized in a Neurospora in vitro translation system supplemented with homologous microsomes. By the criteria of glycosylation of fusion proteins by microsomes, sedimentation of products with microsomes after alkaline extraction, and analysis of protected fragments generated from proteinase K digestion of integrated products, we localized six transmembrane segments in the carboxyl-terminal third of the H(+)-ATPase. These results support a 10-segment model of the Neurospora H(+)-ATPase.