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 the subsequent acquisition of the correctly folded, functional conformation. Here, an analysis is presented of N-terminal glycosylation and steady-state 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 extensions at the C-terminus were studied. The findings demonstrate that the C-terminal transmembrane segments play a crucial role in determining the final orientation of rhodopsin, and that the commitment to the correct orientation occurs only after the synthesis of at least three transmembrane segments. The experiments also suggest that the molecular machinery involved in the integration of a newly synthesized seven-helix membrane protein into the endoplasmic reticulum membrane is sensitive to the overall hydrophobicity of the sequence that follows the first transmembrane segment.
Heymann
J A
JA
National Cancer Institute, NIH, Laboratory of Biochemistry, Bethesda, MD 20892, USA. heymannj@mail.nih.gov
Subramaniam
S
S
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002254:Carbonates / Q000378:metabolism; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017353:Gene Deletion; D006031:Glycosylation; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D012243:Rhodopsin / Q000096:biosynthesis* / Q000235:genetics*; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D002254:Carbonates; D012243:Rhodopsin; D014357:Trypsin