A NOVEL INTEGRATION SIGNAL THAT IS COMPOSED OF 2 TRANSMEMBRANE SEGMENTS IS REQUIRED TO INTEGRATE THE NEUROSPORA PLASMA-MEMBRANE H-ATPASE INTO MICROSOMES()

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 wer e 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 o f the H+-ATPase that contained either one or two transmembrane segment s. Neurospora in vitro translation system supplemented with homologous microsomes was programmed with RNA transcripts of these constructs. W hen transmembrane segment number one (M1) or number two (M2) of the H-ATPase was engineered into the construct, the resultant protein did n ot integrate into microsomes. When M1 and M2 were placed in tandem, th e resultant protein integrated into microsomes as judged by the criter ia of resistance to extraction at pH 11.5 and protection from protease digestion. The integration event depended on ATP and GTP and on micro somal protein(s). We posited that membrane topology of the amino termi nal third of the H+-ATPase, and perhaps of other P-type ATPases is ach ieved by inserting transmembrane segments into membrane in pairs.