scholarly journals Expression of Semliki Forest virus proteins from cloned complementary DNA. II. The membrane-spanning glycoprotein E2 is transported to the cell surface without its normal cytoplasmic domain.

1983 ◽  
Vol 97 (3) ◽  
pp. 652-658 ◽  
Author(s):  
H Garoff ◽  
C Kondor-Koch ◽  
R Pettersson ◽  
B Burke
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Cell Surface ◽  
Rough Endoplasmic Reticulum ◽  
Semliki Forest Virus ◽  
Protein Molecule ◽  
Cytoplasmic Domain ◽  
Amino Acid Residues ◽  
E2 Protein ◽  
Mutant Proteins

The E2 protein (422 amino acid residues long) of Semliki Forest virus is a spanning membrane protein which is made in the rough endoplasmic reticulum of the infected cell and transported to the cell surface. The cytoplasmic domain of this protein comprises 31 amino acid residues. We introduced deletions of various sizes into the gene region encoding this part of the protein molecule and analyzed the transport behavior of the mutant proteins. The deletions were made using exonuclease digestions of cloned cDNA encoding the E2 protein. When the mutated DNA molecules, engineered into an expression vector, were introduced into nuclei of baby hamster kidney 21 cells, membrane proteins with cytoplasmic deletions were expressed and routed to the cell surface in the same way as the wild-type protein. This suggests that the cytoplasmic domain of the E2 protein does not carry information that is needed for its transport from the rough endoplasmic reticulum to the cell surface.

scholarly journals Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products.

1984 ◽  
Vol 99 (6) ◽  
pp. 2011-2023 ◽  
Author(s):  
J W Wills ◽  
R V Srinivas ◽  
E Hunter
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Nuclear Membrane ◽  
Rous Sarcoma Virus ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Rous Sarcoma ◽  
Sarcoma Virus

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.

scholarly journals Mutants of the membrane-binding region of Semliki Forest virus E2 protein. I. Cell surface transport and fusogenic activity.

1986 ◽  
Vol 102 (3) ◽  
pp. 889-901 ◽  
Author(s):  
D F Cutler ◽  
H Garoff
Keyword(s):  
Cell Surface ◽  
Semliki Forest Virus ◽  
Membrane Binding ◽  
Binding Region ◽  
Hydrophobic Region ◽  
E2 Protein ◽  
Mutant Proteins ◽  
Surface Transport ◽  
Charged Amino Acids ◽  
Charge Cluster

Three mutations of the membrane-binding region of the Semliki Forest virus (SFV) p62 polypeptide (the precursor for virion E3 and E2) have been made by oligonucleotide-directed mutagenesis of a cDNA clone encoding the SFV structural proteins. One of the mutations (A2) substitutes a Glu for an Ala in the middle of the hydrophobic stretch which spans the bilayer. A1 and A3 alter the two basic charged amino acids in the cytoplasmic domain next to the hydrophobic region. The wild-type charge cluster of Arg-Ser-Lys (+2) has been changed to Gly-Ser-Met (0;A3) or to Gly-Ser-Glu (-1;A1). The mutant p62 proteins have been analyzed both in the presence and the absence of E1, the other half of the heterodimer spike complex of SFV. The mutant proteins expressed in COS-7 cells are glycosylated and are of the expected sizes. When co-expressed with E1, all three mutants are cleaved to yield the E2 protein and transported to the surface of COS-7 cells. When expressed in the absence of E1, the mutant p62 proteins remain uncleaved but still reach the cell surface. Once at the cell surface, all three mutants, when co-expressed with E1, can promote low pH-triggered cell-cell fusion. These results show that the three mutant p62/E2 proteins are still membrane associated in a functionally unaltered way.

scholarly journals Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

1993 ◽  
Vol 13 (4) ◽  
pp. 2554-2563 ◽  
Author(s):  
D Wojciechowicz ◽  
C F Lu ◽  
J Kurjan ◽  
P N Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Adhesion ◽  
Amino Acid ◽  
Cell Surface ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Immunoglobulin Superfamily ◽  
Cell Contact ◽  
Amino Acid Residues ◽  
Adhesion Proteins

alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells. Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.

scholarly journals Regulation of AE2-mediated Cl− Transport by Intracellular or by Extracellular pH Requires Highly Conserved Amino Acid Residues of the AE2 NH2-terminal Cytoplasmic Domain

2002 ◽  
Vol 120 (5) ◽  
pp. 707-722 ◽  
Author(s):  
A.K. Stewart ◽  
M.N. Chernova ◽  
B.E. Shmukler ◽  
S. Wilhelm ◽  
S.L. Alper
Keyword(s):  
Amino Acid ◽  
Anion Exchange ◽  
Cytoplasmic Domain ◽  
Weak Acid ◽  
Extracellular Ph ◽  
Individual Amino Acid ◽  
Amino Acid Residues ◽  
General Importance ◽  
Alanine Scan ◽  
Intracellular Alkalinization

We reported recently that regulation by intracellular pH (pHi) of the murine Cl−/HCO3− exchanger AE2 requires amino acid residues 310–347 of the polypeptide's NH2-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl− efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pHo) with unclamped or clamped pHi, or during variation of pHi at constant pHo. Wild-type AE2–mediated 36Cl− efflux was profoundly inhibited by acid pHo, with a value of pHo(50) = 6.87 ± 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312–347 identified the greatest acid shift in pHo(50) value, ∼0.8 pH units in the mutant (A)6342–347, but only a modest acid-shift in the mutant (A)6336–341. Two of the six (A)6 mutants retained normal pHi sensitivity of 36Cl− efflux, whereas the (A)6 mutants 318–323, 336–341, and 342–347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336–347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pHo and to pHi were found independently and in concert. The E346A mutation acid-shifted the pHo(50) value to the same extent whether pHi was unclamped or held constant during variation of pHo. Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pHo as well as pHi.

scholarly journals Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule

2009 ◽  
Vol 90 (7) ◽  
pp. 1730-1733 ◽  
Author(s):  
Natalia L. Varich ◽  
Konstantin S. Kochergin-Nikitsky ◽  
Evgeny V. Usachev ◽  
Olga V. Usacheva ◽  
Alexei G. Prilipov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Antigenic Variation ◽  
Strain Differences ◽  
Antigenic Structure ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Antigenic Sites

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.

Altered labelling of the cell surface and intracellular organelles with [3H]mannose in enucleated amoebae

1984 ◽  
Vol 68 (1) ◽  
pp. 83-94
Author(s):  
C.J. Flickinger
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Cell Organelles ◽  
Intracellular Organelles ◽  
And Control ◽  
Kinetics Of ◽  
Heavy Labelling

The production, transport, and disposition of material labelled with [3H]mannose were studied in microsurgically enucleated and control amoebae. Cells were injected with the precursor and samples were prepared for electron-microscope radioautography at intervals, up to 24 h later. Control cells showed heavy labelling of the rough endoplasmic reticulum and the Golgi apparatus at early intervals after injection. Later, labelling of groups of small vesicles increased, and the percentage of grains over the cell surface peaked 12 h after administration of the precursor. Two major changes were detected in enucleate amoebae. First, the kinetics of labelling of cell organelles with [3H]mannose were altered in the absence of the nucleus. The Golgi apparatus and cell surface both displayed maximal labelling at later intervals in enucleates, and the percentage of grains over the rough endoplasmic reticulum varied less with time in enucleated than in control cells. Second, the distribution of radioactivity was altered. A greater percentage of grains was associated with lysosomes in enucleates than in control cells. The change in the kinetics of labelling of the endoplasmic reticulum, Golgi apparatus and cell surface indicates that intracellular transport of surface material was slower in the absence of the nucleus. It is suggested that this is related to the decreased motility of enucleate cells.

scholarly journals Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding.

1986 ◽  
Vol 102 (3) ◽  
pp. 902-910 ◽  
Author(s):  
D F Cutler ◽  
P Melancon ◽  
H Garoff
Keyword(s):  
Cell Membranes ◽  
Semliki Forest Virus ◽  
Transmembrane Domain ◽  
Membrane Binding ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Hydrophobic Domain ◽  
E2 Protein ◽  
Amino Terminal ◽  
Charge Cluster

The p62/E2 protein of Semliki Forest virus (SFV) is a typical transmembrane glycoprotein, with an amino-terminal lumenal domain, a transmembrane (hydrophobic) domain, and a carboxy-terminal cytoplasmic domain (or tail). Our hypothesis has been that the membrane-binding polypeptide region (membrane anchor) of this protein consists of both the transmembrane domain and the adjacent positively charged peptide, Arg-Ser-Lys, which is part of the cytoplasmic domain. We have investigated three anchor mutants of the p62 protein with respect to both their disposition and their stability in cell membranes. The construction of the three mutants has been described (Cutler, D.F., and H. Garoff, J. Cell Biol., 102:889-901). They are as follows: A1, changing the basic charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Glu(-1); A2, replacing an Ala in the middle of the hydrophobic stretch with a Glu; A3, changing the charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Met(0). All three mutants retain the transmembrane configuration of the wild-type p62. In a cell homogenate they have a cytoplasmic domain that is accessible to protease. In living cells an anti-peptide antibody specific for the cytoplasmic tail of p62 reacts with the tails of both wild-type and mutant p62s following its introduction into the cytoplasm. All three mutant proteins have Triton X-114 binding properties similar to the wild-type p62. However, when the membranes of cells expressing the three mutants or the wild-type p62 protein are washed with sodium carbonate, pH 11.5, three to four times as much mutant protein as wild-type p62 is released from the membranes. Thus the stability in cell membranes of the three mutant p62 proteins is significantly reduced.

scholarly journals Amino Acid Residues in the Cytoplasmic Domain of the Mason-Pfizer Monkey Virus Glycoprotein Critical for Its Incorporation into Virions

2005 ◽  
Vol 79 (18) ◽  
pp. 11559-11568 ◽  
Author(s):  
Chisu Song ◽  
Keith Micoli ◽  
Helena Bauerova ◽  
Iva Pichova ◽  
Eric Hunter
Keyword(s):  
Amino Acid ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Cytoplasmic Domain ◽  
Virus Infectivity ◽  
Amino Acid Residues ◽  
Glycoprotein Complex ◽  
Uptake Studies ◽  
Domain Block ◽  
And Function

ABSTRACT Assembly of an infectious retrovirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer monkey virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions.

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