MEMBRANE INTEGRATION OF NA,K-ATPASE ALPHA-SUBUNITS AND BETA-SUBUNIT ASSEMBLY

The control of membrane insertion of polytopic proteins is still poorl y understood, We carried out in vivo translation/insertion experiments in Xenopus oocytes with combined wild type or mutant membrane segment s of the alpha-subunit of the heterodimeric Na,K-ATPase linked to a gl ycosylation reporter sequence. We confirm that the four N-terminaI hyd rophobic segments of the alpha-subunit behave as alternating signal an chor/stop transfer motifs necessary for two lipid-inserted membrane pa irs, For the six C-terminal membrane segments, however, proper packing depends on specific sequence information and association with the bet a-subunit, M5 is a very inefficient signal anchor sequence due to the presence of prolines and polar amino acids. Its correct membrane inser tion is probably mediated by posttranslational hairpin formation with M6, which is favored by a proline pair in the connecting loop, M7 has partial signal anchor function, which may be mediated by the presence of glycine and glutamine residues, The formation of a transmembrane M7 /M8 pair requires the association of the beta-subunit, which induces a conformational change in the connecting extracytoplasmic loop that fa vors M7/M8 packing, The formation of the M9/M10 pair appears to be pre dominantly mediated by the efficient stop transfer function of M10. Mu tations that provide signal anchor function to M5, M7, and M9 abolish or impede the transport activity of the enzyme, These data illustrate the importance of specific amino acids near or within hydrophobic regi ons as well as of subunit oligomerization for correct topographical al ignment that is necessary for proper folding and/or activity of oligom eric membrane proteins.