ABSTRACT

The Neurospora plasma membrane H-ATPase belongs to a family of cation-motive porters called P-type ATPases. Putative transmembrane segments of these enzymes contain one or more charged residues. Conditions were determined by which a transmembrane segment with charged residues is integrated into its cognate membrane. We constructed fusion proteins flanked by the hydrophilic domains of the amino and carboxyl termini of the H-ATPase that contained either one or two transmembrane segments. Neurospora in vitro translation system supplemented with homologous microsomes was programmed with RNA transcripts of these constructs. When transmembrane segment number one (M1) or number two (M2) of the H-ATPase was engineered into the construct, the resultant protein did not integrate into microsomes. When M1 and M2 were placed in tandem, the resultant protein integrated into microsomes as judged by the criteria of resistance to extraction at pH 11.5 and protection from protease digestion. The integration event depended on ATP and GTP and on microsomal protein(s). We posited that membrane topology of the amino-terminal third of the H-ATPase, and perhaps of other P-type ATPases is achieved by inserting transmembrane segments into membrane in pairs.

FOOTNOTES

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The abbreviations used are: H-ATPase, plasma membrane electrogenic, proton-translocating ATPase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; SRP, signal recognition particle; bp, base pair(s).

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