Integration of deletion mutants of bovine rhodopsin into the membrane of the endoplasmic reticulum

Newly synthesized eukaryotic membrane proteins must be integrated into the membrane of the endoplasmic reticulum with the correct topology to enable t he subsequent acquisition of the correctly folded, functional conformation. Here, an analysis is presented of N-terminal glycosylation and steady-stat e membrane orientation of a series of truncation mutants of the seven-helix protein rhodopsin expressed in COS-1 cells. Mutants containing one, three, or five N-terminal transmembrane segments of rhodopsin, as well as mutants containing only the first transmembrane segment, but with hydrophilic exte nsions at the C-terminus were studied. The findings demonstrate that the C- terminal transmembrane segments play a crucial role in determining the fina l orientation of rhodopsin, and that the commitment to the correct orientat ion occurs only after the synthesis of at least three transmembrane segment s. The experiments also suggest that the molecular machinery involved in th e integration of a newly synthesized seven-helix membrane protein into the endoplasmic reticulum membrane is sensitive to the overall hydrophobicity o f the sequence that follows the first transmembrane segment.