Examining rhodopsin folding and assembly through expression of polypeptide fragments.
Previous work on the expression of bovine opsin fragments separated in the cytoplasmic region has allowed the identification of specific polypeptide segments that contain sufficient information to fold independently, insert into a membrane, and assemble to form a functional photoreceptor. To further examine the contributions of these and other polypeptide segments to the mechanism of opsin folding and assembly, we have constructed 20 additional opsin gene fragments where the points of separation occur in the intradiscal, transmembrane, and cytoplasmic regions. Nineteen of the fragments were stably expressed in COS-1 cells. A five-helix fragment was stably produced only after coexpression with its complementary two-helix fragment. Two fragments composed of the amino-terminal region and the first transmembrane helix were not N-glycosylated and were only partially membrane integrated. One of the singly expressed fragments, which is truncated after the retinal attachment site, bound 11-cis-retinal. Of the coexpressed complementary fragments, only those separated in the second intradiscal and third cytoplasmic regions formed noncovalently linked rhodopsin. Both of the pigments showed reduced transducin activation. Therefore, while many opsin fragments contain enough information to fold and insert into a membrane, only those separated at specific locations assemble to a retinal-binding opsin.
Ridge
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Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, Maryland 20850, USA.
Lee
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Abdulaev
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D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D012243:Rhodopsin / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D017299:Rod Opsins / Q000096:biosynthesis* / Q000737:chemistry*; D014162:Transfection
D010446:Peptide Fragments; D011994:Recombinant Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D019204:GTP-Binding Proteins