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Synthesis and Assembly of Membrane Glycoproteins

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Synthesis and Assembly of Membrane Glycoproteins Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 570-574. February 1979 Biochemistry Synthesis and assembly of membrane glycoproteins: Presence of leader peptide in nonglycosylated precursor of membrane glycoprotein of vesicular stomatitis virus (NH2-terminal sequence/processing in vitro/insertion into membranes in vitro/glycosylation in vitro/radiosequencing) ROBERT A. IRVING*, FRANCES TONEGUZZO*t, SUNG H. RHEEt, THEO HOFMANNf, AND HARA P. GHOSH* *Department of Biochemistry, McMaster University, Hamilton, Ontario L8S 4J9; and *Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada Communicated by George E. Palade, October 13,1978 ABSTRACT Translation of mRNA encoding vesicular sto- this ribosome-membrane junction results in the passage of the matitis virus envelope glycoprotein G by a membrane-free ri- bosomal extract obtained from HeLa cells yielded a nongly- nascent polypeptide chain through the membrane, the dis- cosylated protein G1 (Mr 63,000). In the presence of added mi- charge of the completed protein into the lumen of the endo- crosomal -membranes, G1 was converted to the glycosylated plasmic reticulum, and the proteolytic cleavage of the signal protein G2 (Mr 67,000) which is inserted in the membrane ves- sequence (7-9). The vectorially discharged and processed icles as a transmembrane protein. Labeling with methionine protein is then transported through the cell and secreted (7). donated by wheat germ initiator tRNAIet showed that GI but The existence, at the NH2 terminus, of a signal or leader se- not G2 contains methionine in the NH2-terminal position. De- termination of the NH2-terminal sequence of G1, G2, and G quence containing 15-30 amino acid residues has been dem- showed that a leader peptide of 16 amino acids is present in G1 onstrated recently for a number of secretory proteins- (9-16). but absent from the glycosylated proteins G2 and G. This leader Because of our interest in the synthesis and assembly of peptide contains at least 62% hydrophobic amino acids and is membrane glycoproteins, we looked for the presence of a leader removed presumably during insertion of G1 into the mem- sequence in the nonglycosylated precursor of the transmem- brane. brane glycoprotein G of VSV. In this communication we pro- An important aspect of membrane biogenesis is the mechanism vide direct evidence that the nonglycosylated precursor protein by which integral membrane proteins are transported from the GI contains a leader peptide of 16 amino acids that, in the site of synthesis and are assembled in the membrane. The en- presence of membranes, is cleaved during the.conversion of G1 veloped viruses that serve as models to study membrane to G2.. A preliminary report of this work has been presented structure and function also are excellent systems for study-of (17). membrane biogenesis (1). A study of the mechanisms of syn- thesis and glycosylation of the viral membrane glycoproteins MATERIALS AND METHODS and of their insertion into the membrane, transport along cel- Plaque-purified VSV (Indiana HR-LT) and HeLa S3 cells were lular membrane systems, and ultimate migration to the plasma grown as described (3, 4). Radioactive amino acids were ob- membrane of the infected cell can help to elucidate the se- tained from New England Nuclear or Amersham/Searle. Hen quence of events involved in membrane biogenesis. Because egg-white lysozyme was from Worthington. cell surface glycoprotein molecules are believed to be involved Preparation. of Formylated and Nonformylated in cellular recognition and information exchange processes Methionyl-tRNAmet. Initiator tRNAMet was purified from wheat occurring on the cell membrane (2), insight into these processes germ as described (18) and was free from tRNAm" species may be gained from studies on membrane glycoprotein syn- which donates methionine into internal positions of a poly- thesis and assembly. Studies from this (3, 4) and other labora- peptide chain (18). [35S]Met-tRNAmlet was obtained by charging tories (5, 6) have shown that translation in vitro of the. mRNA tRNAjlet with methionine, using Escherichia coli methionyl- coding for the membrane glycoprotein G (Mr 69,000) of the tRNA synthetase which can aminoacylate only tRNAIMe, (18). enveloped vesicular stomatitis virus (VSV) in the absence of The charged [a5S]Met-tRNAmei was recovered (18) and chem- microsomal membranes results in the synthesis of the nongly- ically formylated as described (19). cosylated protein G1 (Mr 63,000). In the presence of added Synthesis of VSV-Specific Proteins. Ribosomal systems, microsomal membrane vesicles, however, G1 is converted to containing either membranes (S-4 extract) or free from mem- the glycosylated protein G2 (Mr 67,000) which is inserted into branes (S-27 extract), as well as stripped microsomal membranes the vesicle membrane. It was also shown that insertion into the were isolated from uninfected HeLa cells as described (3, 4). membrane and glycosylation are cotranslational events. Unlike The coupled transcription-translation system containing ribo- secretory proteins, which are also synthesized by membrane- somes from HeLa cells and purified ribonucleoprotein particles bound ribosomes and are discharged vectorially across the from VSV-infected L cells has been described (3, 4). VSV- microsomal membrane (7), glycoprotein G2 is not completely specific mRNA was prepared from VSV-infected HeLa cells discharged but spans the membrane (4-6). by extraction with phenol/CHCl3/sodium dodecyl sulfate It has been postulated that the transport of secretory proteins followed by precipitation with LiCl/ethanol as described (20). across membranes is initiated by the association of a signal or The isolated RNA was used to direct VSV-specific protein leader sequence at the NH2 terminus of the nascent polypeptide chain with the endoplasmic reticulum (8, 9). The formation of Abbreviations: VSV, vesicular stomatitis virus; protein G, fully glyco- sylated virion glycoprotein of Mr 69,000; protein G2, nonsialated The publication costs of this article were defrayed in part by page glycoprotein (Mr 67,000) synthesized in vitro; protein G1, nongly- charge payment. This article must therefore be hereby marked "ad- cosylated precursor protein (Mr 63,000) synthesized in vitro. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate t Present address: Department of Zoology, University of California, this fact. Berkeley, CA 94720. 570 Downloaded by guest on September 28, 2021 Biochemistry: Irving et al. Proc. Natl. Acad. Sci. USA 76 (1979) 571 a b c d e f g h j a b c d e f g h L _ I sw L_ ia.J PI g--G .. ***'........... G2 _--0 "-*-' :-r# * G2- Gi r. NS . .n N . MI .. mmww IWONUM ap4m G 0M- IWI, Y NS _wn mgj - N_ W - aLtm W M _ _ _ _W FIG. 1. Autoradiogram of proteins synthesized in vitro by direct - 1Ii;E translation of VSV mRNA and by the coupled system. The reaction mixtures for protein synthesis by the direct translation system con- FIG. 2. Autoradiograms of proteins synthesized in vitro and tained 40 mM Hepes (pH 7.6), 90 mM KCl, 3.2 mM Mg acetate, 1 mM containing formyl[35Sjmethionine and [35S]methionine transferred ATP, 0.2 mM GTP, 1 mM dithiothreitol, 10 mM creatine phosphate, from formyl[35SJMetAtRNAMet and [35SJMetAtRNAMet, respectively. 40,Mg of creatinekinase per ml, 80 gM spermine, 19 unlabeled amino The conditions of the reaction mixtures were the same as in the direct acids each at 20 gM, 40 gg of unfractionated VSV mRNA per ml, 200 translation system in Fig. 1 with the following modifications: reaction MCi of [35Slmethionine per ml, and 300 ,l of the preincubated HeLa mixtures containing charged tRNAMet also contained 200 MM nonla- extract (3, 4). The coupled system contained the same components beled methionine and the amounts of formyl[35S]Met-tRNAjet and except for the following modification: 75 mM KCl, 4.5 mM Mg acetate, [35S]Met-tRNAMet used were 1 X 106 cpm/ml and 1.2 X 106 cpm/ml, 0.8 mM each of CTP, UTP, and GTP, 2 mM ATP, and 0.7 mg of the respectively. Lanes: a and e, VS-virion proteins; b-d, proteins syn- purified ribonucleoprotein particles from VSV-infected L cells instead thesized in the presence of membranes and in reaction mixtures ofthe VSV mRNA (3,4). All incubations were at 30'C for 90 min. The containing 35S]methionine, formyl[35SJMet-tRNAMet, and [35S]- [35Sjmethionine-labeled proteins were electrophoresed on 10% Met-tRNA, et, respectively; f-h, proteins synthesized in the absence polyacrylamide slab gel containing 0.1% sodium dodecyl sulfate (3, of membranes and in reaction mixtures containing [35S]methionine, 4). Lanes: a and j, VS-virion proteins; b and c, proteins synthesized formyl[35S]MetAtRNAMet, and [35S]Met-tRNAMet, respectively. The in the absence of membranes in a direct translation and in a coupled presence of additional peptides migrating between N and M in the reaction mixture, respectively; d and e, proteins synthesized in the reaction mixtures containing membranes (lanes f, g, and h) could be presence of membranes in a direct translation and in a coupled re- due to premature termination or proteolysis of the synthesized pro- action mixture, respectively; f-i, proteolytic digestion of the proteins teins. synthesized in reaction mixtures shown in b-e, respectively. zyme was used as a carrier protein in place of apomyoglobin. synthesis in HeLa cell ribosomal systems in the absence and in The proteins were sequentially degraded for 12 or 24 cycles in the presence of microsomal membranes. The reaction condi- a Beckman 890C sequencer with a volatile buffer containing tions were identical to those described for the coupled system N-dimethylaminobenzylamine (22). The phenylthiohydantoin (3, 4) except for the following changes: UTP and CTP were derivative of norleucine was added as an internal standard to omitted and the concentrations of KC1, magnesium acetate, each tube in the fraction collector.
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