In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes.

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Abstract
Translation in vitro of the mRNA coding for the vesicular stomatitis virus membrane glycoprotein G in a membrane-free ribosomal extract from [human cervical carcinoma] HeLa cells allowed the synthesis of only the unglycosylated protein G1 (MW 63,000). Addition of stripped crude microsomal membranes from HeLa cells resulted in the conversion of G1 to the glycosylated protein G2 (MW 67,000). The G2 protein synthesized by the reconstructed microsomal membrane/ribosome system was segregated inside the microsomal membrane vesicles and was thus protected from the proteolytic action of trypsin and chymotrypsin. Stripped membranes were required at an early stage of protein synthesis for the synthesized protein to be inserted into the membrane vesicles and to be glycosylated. The segregated protein G2 was not completely protected from proteolytic digestion, showing that a portion of the polypeptide chain of about 3000 daltons was present on the cytoplasmic side of the membrane vesicle. Unlike the secretory proteins, the membrane glycoproteins are not completely discharged across the microsomal membranes.