Messenger RNA of opsin from bovine retina: Isolation and partial sequence of the in vitro translation product

Abstract

Opsin, the apoprotein of the visual pigment rhodopsin, is synthesized on membranes of the rough endoplasmic reticulum and subsequently passes through the Golgi apparatus to the rod outer segment. This pathway parallels the early stages of biosynthesis of some secretory proteins and viral membrane glycoproteins. Most of these proteins are initially synthesized as precursor molecules with a short-lived hydrophobic extra peptide segment at the NH2 terminus. Therefore we investigated whether or not the immediate translation product of opsin mRNA contains a similar short-lived NH2-terminal extra peptide. The mRNA coding for opsin was isolated from bovine retina polysomes precipitated by antibodies to opsin. The mRNA directed the cell-free synthesis of a protein comparable in size to opsin that was specifically precipitated by anti-opsin antibodies. Sequence analyses of the immunoprecipitated protein labeled with six radioactive amino acids (Met, Asn, Pro, Phe, Tyr, Val) provided the following result: MATERIALS AND METHODS Anti-Opsin Antibodies. Bovine opsin was isolated electrophoretically from sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gels as described (24). In the presence of 0.1% NaDodSO4 the protein was mixed with an equal volume of complete Freund's adjuvant and injected at 7-day intervals into a goat. Antibody content of sera was tested by precipitin analysis. The initial opsin solution that contained NaDodSO4 inhibited precipitin formation. Therefore the solution of NaDodSO4-solubilized opsin was dialyzed against 1000 vol of 0.15 M NaCl at 20°C. During dialysis a precipitate occasionally formed. The centrifuged supernatant (3000 X g for 10 min) was cooled to 4°C and any additional precipitated material (presumably NaDodSO4 and aggregated protein) was removed by further centrifugation. Over 80% of the opsin was recovered in the clear supernatant, and it formed specific precipitates with anti-opsin antibodies. The dialyzed opsin solution (1 mg/ml) could also be coupled directly to CNBr-activated Sepharose in 0.1 M NaHCO3 (25) Haven, CT 06510. 2654The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci. USA 76 (1979) 2655 135SMet-tRNAMet. The two [35SSMet-tRNAMet species that transfer methionine to the NH2-terminal (initiator MettRNAIN'tt) and internal positio'Siin proteins were prepared from wheat germ as described (27

The four cytoplasmically made subunits of yeast mitochondrial cytochrome c oxidase are synthesized individually and not as a polyprotein.

“…There are now several precedents for uncleaved signal sequences. These have been detected so far among the signal sequences addressed either to the cotranslational translocation system in the rough endoplasmic reticulum (33)(34)(35) or to the cotranslational translocation system in the prokaryotic plasma membrane (36) or to the two posttranslational translocation systems in the mitochondrial membranes (37,38).…”

Studies on the Biosynthesis of Microsomal Membrane Proteins

“…Although it may be speculated that the presence of uncleaved amino-terminal insertion signals may be a characteristic feature of microsomal membrane proteins, which could play a role in maintaining their segregation in the endoplasmic reticulum membrane domain, it should be noted that other membrane proteins, such as retinal opsin [68] and erythrocyte band 3 [69], are also synthesized in bound ribosomes and are not cleaved proteolytically. These proteins, however, differ from the microsomal polypeptides in that their amino-terminal segments do not remain membrane associated.…”

Assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the E1 glycoprotein of coronavirus mouse hepatitis virus A59.

“…Other examples of spanning-membrane proteins without a cleavable signal sequence are rhodopsin (37) and band III protein (38 2 and 3 with lanes 7 and 6). Presumably, NH2-terminal methionine normally is cleaved from the protein before insertion into the membrane.…”