EXPRESSION, STABILITY, AND MEMBRANE INTEGRATION OF TRUNCATION MUTANTSOF BOVINE RHODOPSIN

Premature termination of protein synthesis by nonsense mutations is at the molecular origin of a number of inherited disorders in the family of G protein coupled seven-helix receptor proteins, To understand how such truncated polypeptides are processed by the cell, we have carrie d out COS-1 cell expression studies of mutants of bovine rhodopsin tru ncated at the first 1, 1.5, 2, 3, or 5 transmembrane segments (TMS) of the seven present in wild-type opsin, Our experiments show that succe ssful completion of different stages in the cellular processing of the protein [membrane insertion, N-linked glycosylation, stability to pro teolytic degradation, and transport from the endoplasmic reticulum (ER ) membrane] requires progressively longer lengths of the polypeptide c hain. Thus, none of the truncations affected the ability of the polype ptides to be integral membrane proteins, C-terminal truncations that g enerated polypeptides with fewer than two TMS resulted in misorientati on and prevented glycosylation at the N terminus, whereas truncations that generated polypeptides with fewer than five TMS greatly destabili zed the protein, However, all of the truncations prevented exit of the polypeptide from the ER. We conclude that during the biogenesis of rh odopsin, proper integration into the ER membrane occurs only after the synthesis of at least two TMS is completed, Synthesis of the next thr ee TMS confers a gradual increase in stability, whereas the presence o f more than five TMS is necessary for exit from the ER.