- Email: [email protected]
Dual roles for the cytoplasmic domain of the VSV G protein: Protein transport and virus assembly
10
DOMAIN OF THE VSV G ASSEMBLY, M. A. Whitt, R.W. Doms, Aino Ruusala, Jonne Heienius. Ari Helenius and J.K. Rose. Departments of Pathology and Cell Biology, Yale University School of Medicine, 333 Cedar St., New Haven, Ct. 06510. We have examined a series of 7 mutations deleting amino acid sequences from the cytoplasmic domain of the VSV G protein, or substituting this domain with cytopfasmic domains from other membrane proteins. In general these mutations slowed or blocked exit of the mutant G proteins from the ER without reducing the rate of trimerization of the protein. Using a new assay to measure pH stability of G protein trimers and a series of conformation sensitive monoclonai antibodies, we found that the ectodomains of these mutant proteins were apparently correctly folded whether they were retained in the ER, transported slowly from the ER, or transported normally. Because there was no correlation between folding of the ectodomain and transport for these mutant proteins, we propose that there are important interactions on the cytoplasmic side of the membrane which regulate G protein transport. In a second series of experiments we devised two assays to anatyze the role of the cytoplasmic domain of G protein in VSV assembly. We were able to rescue the ts045 mutant of VSV (which makes a defective G protein) in cells expressing wildtype G proteins from cloned cDNA and we demonstrated directly that the rescued virus particles contain the protein expressed from cloned DNA. Mutant G proteins with substantial alterations in the sequence of the cytoplasmic domain (but which are expressed on the cell surface), are unable to rescue the ts045 mutant apparentIy because they are not incorporated into the virus. These experiments suggest that critical interactions between the cytoplasmic domain of G protein, matrix protein, and budding nucleocapsid may direct assembly of this enveioped virus, DUAL ROLES FOR THE CYTOPLASMIC PROTEIN: PROTEIN TRANSPORT AND VIRUS
11 PIEHliKANE ANCHORS OF VSV
ALICE S HUANG& STEVECHEN, Harvard Medical School,
Boston,
IISA
Uild-type vesicular stomatitis virus-infected calls contain multiple carboxy-terminal of the envelope glycoprotein G with two dominant apparent lnolecular weights, 14K and C-terminal (stem).
fragments
are
immunoprecipitated
by anti-peptide
antibodies,
anti-G(COOH)
fragments
Both 9K. and anti-G
against the laat 15 amino acids at the carboxy terminus They are made, respectively, and against the first 22 amino acids of the ektodomatn adjacent to the transmembrane region of G. indicate that the larger Pulse-chase experiments in the presence and absence of tunicamycin in the rough endoplasmtc reticulum p resumably molecular weight fragment, Gal, ia generated first, Exposure of infected cells and then apparently chased into the fast migrating, stable one, Ga2. These results Ga2 is detected in purified vfrions. to radioactive palmitic acid labels Ga2. long may have the information to direct suggest that polypeptides of approximately 71 amino acids Whether this C-terminal fragment of G their transport and incorporation into budding virions. protein is transported as a complex with other viral or host cell proteins is presently unknowo.
6
DOMAIN OF THE VSV G ASSEMBLY, M. A. Whitt, R.W. Doms, Aino Ruusala, Jonne Heienius. Ari Helenius and J.K. Rose. Departments of Pathology and Cell Biology, Yale University School of Medicine, 333 Cedar St., New Haven, Ct. 06510. We have examined a series of 7 mutations deleting amino acid sequences from the cytoplasmic domain of the VSV G protein, or substituting this domain with cytopfasmic domains from other membrane proteins. In general these mutations slowed or blocked exit of the mutant G proteins from the ER without reducing the rate of trimerization of the protein. Using a new assay to measure pH stability of G protein trimers and a series of conformation sensitive monoclonai antibodies, we found that the ectodomains of these mutant proteins were apparently correctly folded whether they were retained in the ER, transported slowly from the ER, or transported normally. Because there was no correlation between folding of the ectodomain and transport for these mutant proteins, we propose that there are important interactions on the cytoplasmic side of the membrane which regulate G protein transport. In a second series of experiments we devised two assays to anatyze the role of the cytoplasmic domain of G protein in VSV assembly. We were able to rescue the ts045 mutant of VSV (which makes a defective G protein) in cells expressing wildtype G proteins from cloned cDNA and we demonstrated directly that the rescued virus particles contain the protein expressed from cloned DNA. Mutant G proteins with substantial alterations in the sequence of the cytoplasmic domain (but which are expressed on the cell surface), are unable to rescue the ts045 mutant apparentIy because they are not incorporated into the virus. These experiments suggest that critical interactions between the cytoplasmic domain of G protein, matrix protein, and budding nucleocapsid may direct assembly of this enveioped virus, DUAL ROLES FOR THE CYTOPLASMIC PROTEIN: PROTEIN TRANSPORT AND VIRUS
11 PIEHliKANE ANCHORS OF VSV
ALICE S HUANG& STEVECHEN, Harvard Medical School,
Boston,
IISA
Uild-type vesicular stomatitis virus-infected calls contain multiple carboxy-terminal of the envelope glycoprotein G with two dominant apparent lnolecular weights, 14K and C-terminal (stem).
fragments
are
immunoprecipitated
by anti-peptide
antibodies,
anti-G(COOH)
fragments
Both 9K. and anti-G
against the laat 15 amino acids at the carboxy terminus They are made, respectively, and against the first 22 amino acids of the ektodomatn adjacent to the transmembrane region of G. indicate that the larger Pulse-chase experiments in the presence and absence of tunicamycin in the rough endoplasmtc reticulum p resumably molecular weight fragment, Gal, ia generated first, Exposure of infected cells and then apparently chased into the fast migrating, stable one, Ga2. These results Ga2 is detected in purified vfrions. to radioactive palmitic acid labels Ga2. long may have the information to direct suggest that polypeptides of approximately 71 amino acids Whether this C-terminal fragment of G their transport and incorporation into budding virions. protein is transported as a complex with other viral or host cell proteins is presently unknowo.
6