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 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 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 Interaction of wild-type signal sequences and their charged variants with model and natural membranes

1993 ◽  
Vol 293 (1) ◽  
pp. 43-49 ◽  
Author(s):  
N M Rao ◽  
R Nagaraj
Keyword(s):  
Amino Acids ◽  
Synthetic Peptides ◽  
Model Membranes ◽  
Signal Peptides ◽  
Wild Type ◽  
Signal Sequences ◽  
Hydrophobic Region ◽  
Phospholipases A2 ◽  
Charged Amino Acids

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

scholarly journals Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

2010 ◽  
Vol 84 (18) ◽  
pp. 9019-9026 ◽  
Author(s):  
Andrea Schuessler ◽  
Kerstin Laib Sampaio ◽  
Laura Scrivano ◽  
Christian Sinzger
Keyword(s):  
Endothelial Cell ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Protein Domain ◽  
Cell Infection ◽  
Peptide Motifs ◽  
Charged Amino Acids ◽  
C Terminus ◽  
Charge Cluster ◽  
A Charge

ABSTRACT The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.

scholarly journals Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction.

1992 ◽  
Vol 118 (5) ◽  
pp. 1057-1070 ◽  
Author(s):  
B A Lee ◽  
D J Donoghue
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Immunofluorescent Localization ◽  
Receptor Ligand ◽  
Surface Transport ◽  
Established Cell ◽  
Autocrine Transformation ◽  
Nih 3T3 ◽  
Retention Signal

An important question regarding autocrine transformation by v-sis is whether intracellularly activated PDGF receptors are sufficient to transform cells or whether activated receptor-ligand complexes are required at the cell surface. We have addressed this question by inhibiting cell surface transport of a membrane-anchored v-sis protein utilizing the ER retention signal of the adenoviral transmembrane protein E3/19K. A v-sis fusion protein containing this signal was retained within the cell and not transported to the cell surface as confirmed by immunofluorescent localization experiments. Also, proteolytic maturation of this protein was suppressed, indicating inefficient transport to post-Golgi compartments of the secretory pathway. When compared with v-sis proteins lacking a functional retention signal, the ER-retained protein showed a diminished ability to transform NIH 3T3 cells, as measured by the number and size of foci formed. In newly established cell lines, the ER-retained protein did not down-regulate PDGF receptors. However, continued passage of these cells selected for a fully transformed phenotype exhibiting downregulated PDGF receptors and proteolytically processed v-sis protein. These results indicate that productive autocrine interactions occur in a post-ER compartment of the secretory pathway. Transport of v-sis protein beyond the Golgi correlated with acquisition of the transformed phenotype. Furthermore, suramin treatment reversed transformation and upregulated the expression of cell surface PDGF receptors, suggesting an important role for receptor-ligand complexes localized to the cell surface.

scholarly journals ADP-Ribosylation Factors Modulate the Cell Surface Transport of G Protein-Coupled Receptors

2010 ◽  
Vol 333 (1) ◽  
pp. 174-183 ◽  
Author(s):  
Chunmin Dong ◽  
Xiaoping Zhang ◽  
Fuguo Zhou ◽  
Huijuan Dou ◽  
Matthew T. Duvernay ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Surface Transport ◽  
Adp Ribosylation ◽  
G Protein Coupled

scholarly journals Reduced Affinity to and Inhibition by DKK1 Form a Common Mechanism by Which High Bone Mass-Associated Missense Mutations in LRP5 Affect Canonical Wnt Signaling

2005 ◽  
Vol 25 (12) ◽  
pp. 4946-4955 ◽  
Author(s):  
Minrong Ai ◽  
Sheri L. Holmen ◽  
Wim Van Hul ◽  
Bart O. Williams ◽  
Matthew L. Warman
Keyword(s):  
Cell Surface ◽  
Bone Mass ◽  
Wnt Signaling ◽  
Common Mechanism ◽  
Missense Mutations ◽  
Canonical Wnt Signaling ◽  
High Bone Mass ◽  
Mutant Proteins ◽  
High Bone ◽  
Canonical Wnt

ABSTRACT The low-density-lipoprotein receptor-related protein 5 (LRP5), a coreceptor in the canonical Wnt signaling pathway, has been implicated in human disorders of low and high bone mass. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma syndrome, and heterozygous missense mutations in families segregating autosomal dominant high bone mass (HBM) phenotypes have been identified. We expressed seven different HBM-LRP5 missense mutations to delineate the mechanism by which they alter Wnt signaling. None of the mutations caused activation of the receptor in the absence of ligand. Each mutant receptor was able to reach the cell surface, albeit at differing amounts, and transduce exogenously supplied Wnt1 and Wnt3a signal. All HBM mutant proteins had reduced physical interaction with and reduced inhibition by DKK1. These data suggest that HBM mutant proteins can transit to the cell surface in sufficient quantity to transduce Wnt signal and that the likely mechanism for the HBM mutations' physiologic effects is via reduced affinity to and inhibition by DKK1.

scholarly journals Release from Endoplasmic Reticulum Matrix Proteins Controls Cell Surface Transport of MHC Class I Molecules

Traffic ◽  
2015 ◽  
Vol 16 (6) ◽  
pp. 591-603 ◽  
Author(s):  
Susanne Fritzsche ◽  
Esam T. Abualrous ◽  
Britta Borchert ◽  
Frank Momburg ◽  
Sebastian Springer
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Mhc Class I ◽  
Matrix Proteins ◽  
Class I ◽  
Surface Transport ◽  
Mhc Class I Molecules
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