ONLY THE FIRST AND THE LAST HYDROPHOBIC SEGMENTS IN THE COOH-TERMINAL3RD OF NA,K-ATPASE ALPHA-SUBUNIT INITIATE AND HALT, RESPECTIVELY, MEMBRANE TRANSLOCATION OF THE NEWLY SYNTHESIZED POLYPEPTIDE - IMPLICATIONS FOR THE MEMBRANE TOPOLOGY

We studied the topogenic properties of five hydrophobic segments (H5-H 9) in the COOH-terminal third of Na,K-ATPase alpha subunit using in vi tro insertion of fusion proteins into endoplasmic reticulum membranes, These fusion proteins consisted of several different lengths of trunc ated alpha subunit starting at Met(729) and a reporter protein, chlora mphenicol acetyltransferase, that was linked in frame after each hydro phobic segment. We found that membrane insertion of the newly synthesi zed COOH-terminal third was initiated by H5 and terminated by H9, indi cating that here only H5 and H9 have topogenic function. The other thr ee, H6-H8, did not have topogenic function in the native context and w ere translocated into the endoplasmic reticulum lumen. These results w ere in striking contrast to the previous models in which four or six h ydrophobic segments were proposed to cross the membrane. Furthermore, the findings suggest a novel mechanism for achieving the final membran e topology of the COOH-terminal third of the alpha subunit.