scholarly journals Steady-State Plasma Membrane Expression of Human Cytomegalovirus gB Is Determined by the Phosphorylation State of Ser900

1998 ◽  
Vol 72 (8) ◽  
pp. 6657-6664 ◽  
Author(s):  
Kenneth N. Fish ◽  
Cecilia Soderberg-Naucler ◽  
Jay A. Nelson
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Amino Acid Type ◽  
Type I ◽  
Intracellular Compartments ◽  
U373 Cells

ABSTRACT Human cytomegalovirus (HCMV) infection of an astrocytoma cell line (U373) or human fibroblast (HF) cells results in a differential cell distribution of the major envelope glycoprotein gB (UL55). This 906-amino-acid type I glycoprotein contains an extracellular domain with a signal sequence, a transmembrane domain, and a 135-amino-acid cytoplasmic tail with a consensus casein kinase II (CKII) site located at Ser900. Since phosphorylation of proteins in the secretory pathway is an important determinant of intracellular trafficking, the state of gB phosphorylation in U373 and HF cells was examined. Analysis of cells expressing wild-type gB and gB with site-specific mutations indicated that the glycoprotein was equally phosphorylated at a single site, Ser900, in both U373 and HF cells. To assess the effect of charge on gB surface expression in U373 cells, Ser900 was replaced with an aspartate (Asp) or alanine (Ala) residue to mimic the phosphorylated and nonphosphorylated states, respectively. Expression of the Asp but not the Ala gB mutation resulted in an increase in the steady-state expression of gB at the plasma membrane (PM) in U373 cells. In addition, treatment of U373 cells with the phosphatase inhibitor tautomycin resulted in the accumulation of gB at the PM. Interestingly, the addition of a charge at Ser900 trapped gB in a low-level cycling pathway at the PM, preventing trafficking of the protein to thetrans-Golgi network or other intracellular compartments. Therefore, these results suggest that a tautomycin-sensitive phosphatase regulates cell-specific PM retrieval of gB to intracellular compartments.

scholarly journals Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 580-586 ◽  
Author(s):  
EA Rutledge ◽  
BJ Root ◽  
JJ Lucas ◽  
CA Enns
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Transferrin Receptor ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Soluble Form ◽  
Protein Sequence Analysis ◽  
Wild Type ◽  
Human Transferrin Receptor ◽  
Intracellular Compartments

The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron- transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

scholarly journals Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 580-586 ◽  
Author(s):  
EA Rutledge ◽  
BJ Root ◽  
JJ Lucas ◽  
CA Enns
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Transferrin Receptor ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Soluble Form ◽  
Protein Sequence Analysis ◽  
Wild Type ◽  
Human Transferrin Receptor ◽  
Intracellular Compartments

Abstract The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron- transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

scholarly journals Charged Residues Flanking the Transmembrane Domain of Two Related Toxin–Antitoxin System Toxins Affect Host Response

Toxins ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 329
Author(s):  
Andrew Holmes ◽  
Jessie Sadlon ◽  
Keith Weaver
Keyword(s):  
Amino Acid ◽  
Enterococcus Faecalis ◽  
Host Response ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
The Other ◽  
Type I ◽  
Transcriptomic Response ◽  
Specific Amino Acid ◽  
Transporter Protein

A majority of toxins produced by type I toxin–antitoxin (TA-1) systems are small membrane-localized proteins that were initially proposed to kill cells by forming non-specific pores in the cytoplasmic membrane. The examination of the effects of numerous TA-1 systems indicates that this is not the mechanism of action of many of these proteins. Enterococcus faecalis produces two toxins of the Fst/Ldr family, one encoded on pheromone-responsive conjugative plasmids (FstpAD1) and the other on the chromosome, FstEF0409. Previous results demonstrated that overexpression of the toxins produced a differential transcriptomic response in E. faecalis cells. In this report, we identify the specific amino acid differences between the two toxins responsible for the differential response of a gene highly induced by FstpAD1 but not FstEF0409. In addition, we demonstrate that a transporter protein that is genetically linked to the chromosomal version of the TA-1 system functions to limit the toxicity of the protein.

scholarly journals Role of the Varicella-Zoster Virus gB Cytoplasmic Domain in gB Transport and Viral Egress

2002 ◽  
Vol 76 (2) ◽  
pp. 591-599 ◽  
Author(s):  
Thomas C. Heineman ◽  
Susan L. Hall
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Varicella Zoster Virus ◽  
Cultured Cells ◽  
Signal Sequence ◽  
Cytoplasmic Domain ◽  
Varicella Zoster ◽  
Golgi Transport ◽  
Viral Egress

ABSTRACT To study the function of the varicella-zoster virus (VZV) gB cytoplasmic domain during viral infection, we produced a VZV recombinant virus that expresses a truncated form of gB lacking the C-terminal 36 amino acids of its cytoplasmic domain (VZV gB-36). VZV gB-36 replicates in noncomplementing cells and grows at a rate similar to that of native VZV. However, cells infected with VZVgB-36 form extensive syncytia compared to the relatively small syncytia formed during native VZV infection. In addition, electron microscopy shows that very little virus is present on the surfaces of cells infected with VZV gB-36, while cells infected with native VZV exhibit abundant virions on the cell surface. The C-terminal 36 amino acids of the gB cytoplasmic domain have been shown in transfection-based experiments to contain both an endoplasmic reticulum-to-Golgi transport signal (the C-terminal 17 amino acids) and a consensus YXXφ (where Y is tyrosine, X is any amino acid, and φ is any bulky hydrophobic amino acid) signal sequence (YSRV) that mediates the internalization of gB from the plasma membrane. As predicted based on these data, gB-36 expressed during the infection of cultured cells is transported inefficiently to the Golgi. Despite lacking the YSRV signal sequence, gB-36 is internalized from the plasma membrane; however, in contrast to native gB, it fails to localize to the Golgi. Therefore, the C-terminal 36 amino acids of the VZV gB cytoplasmic domain are required for normal viral egress and for both the pre- and post-Golgi transport of gB.

Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

2001 ◽  
Vol 281 (1) ◽  
pp. C215-C223 ◽  
Author(s):  
Robert T. Watson ◽  
Jeffrey E. Pessin
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Glucose Transporter ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Snare Protein ◽  
Glut 4 ◽  
Golgi Localization ◽  
Syntaxin 3

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.

scholarly journals Regulation of type I collagen mRNA in lung fibroblasts by cystine availability

1998 ◽  
Vol 331 (2) ◽  
pp. 417-422 ◽  
Author(s):  
David C. RISHIKOF ◽  
Ping-Ping KUANG ◽  
Christine POLIKS ◽  
Ronald H. GOLDSTEIN
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Type I Collagen ◽  
State Level ◽  
Lung Fibroblasts ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Mrna ◽  
Amino Acid Availability

The steady-state level of α1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases α1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of α1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of α1(I) collagen mRNA. We found that re-expression of α1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of α1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased α1(I) collagen mRNA levels. The combination of glutamine and cystine increased α1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase α1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of α1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of γ-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of α1(I) collagen mRNA.

scholarly journals Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II

1999 ◽  
Vol 13 (4) ◽  
pp. 578-586 ◽  
Author(s):  
Stéphane A. Laporte ◽  
Antony A. Boucard ◽  
Guy Servant ◽  
Gaétan Guillemette ◽  
Richard Leduc ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Transmembrane Domain ◽  
At1 Receptor ◽  
High Yield ◽  
Type I ◽  
Terminal Amino Acid ◽  
Contact Points ◽  
Terminal Amino

Abstract To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-l-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the[ Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166–199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of[ Sar1, Bpa8]AngII, whereas the N-terminal amino acid of[ Bpa1]AngII interacts with the second extracellular loop of the AT1 receptor.

scholarly journals AMIGO, a transmembrane protein implicated in axon tract development, defines a novel protein family with leucine-rich repeats

2003 ◽  
Vol 160 (6) ◽  
pp. 963-973 ◽  
Author(s):  
Juha Kuja-Panula ◽  
Marjaana Kiiltomäki ◽  
Takashi Yamashiro ◽  
Ari Rouhiainen ◽  
Heikki Rauvala
Keyword(s):  
Hippocampal Neurons ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Transmembrane Protein ◽  
Protein Family ◽  
Type I ◽  
Fiber Tracts ◽  
Neuronal Tissues ◽  
Immunoglobulin Domain ◽  
Leucine Rich Repeats

Ordered differential display identified a novel sequence induced in neurons by the neurite-promoting protein amphoterin. We named this gene amphoterin-induced gene and ORF (AMIGO), and also cloned two other novel genes homologous to AMIGO (AMIGO2 and AMIGO3). Together, these three AMIGOs form a novel family of genes coding for type I transmembrane proteins which contain a signal sequence for secretion and a transmembrane domain. The deduced extracellular parts of the AMIGOs contain six leucine-rich repeats (LRRs) flanked by cysteine-rich LRR NH2- and COOH-terminal domains and by one immunoglobulin domain close to the transmembrane region. A substrate-bound form of the recombinant AMIGO ectodomain promoted prominent neurite extension in hippocampal neurons, and in solution, the same AMIGO ectodomain inhibited fasciculation of neurites. A homophilic and heterophilic binding mechanism is shown between the members of the AMIGO family. Our results suggest that the members of the AMIGO protein family are novel cell adhesion molecules among which AMIGO is specifically expressed on fiber tracts of neuronal tissues and participates in their formation.

scholarly journals Human cytomegalovirus US9 protein contains an N-terminal signal sequence and a C-terminal mitochondrial localization domain, and does not alter cellular sensitivity to apoptosis

2009 ◽  
Vol 90 (5) ◽  
pp. 1172-1182 ◽  
Author(s):  
Lana Mandic ◽  
Matthew S. Miller ◽  
Corinne Coulter ◽  
Brian Munshaw ◽  
Laura Hertel
Keyword(s):  
Human Cytomegalovirus ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Brefeldin A ◽  
Genomic Region ◽  
Hygromycin B ◽  
Mitochondrial Localization ◽  
Dual Localization ◽  
Carbonyl Cyanide ◽  
Necessary And Sufficient

The human cytomegalovirus (CMV) US2–US11 genomic region contains a cluster of genes whose products interfere with antigen presentation by the major histocompatibility complex (MHC) proteins. Although included in this cluster, the US9 gene encodes a glycoprotein that does not affect MHC activity and whose function is still largely uncharacterized. An in silico analysis of the US9 amino-acid sequence uncovered the presence of an N-terminal signal sequence (SS) and a C-terminal transmembrane domain containing the specific hallmarks of known mitochondrial localization sequences (MLS). Expression of full-length US9 and of US9 deletion mutants fused to GFP revealed that the N-terminal SS mediates US9 targeting to the endoplasmic reticulum (ER) and that the C-terminal MLS is both necessary and sufficient to direct US9 to mitochondria in the absence of a functional SS. This dual localization suggested a possible role for US9 in protection from apoptosis triggered by ER-to-mitochondria signalling. Fibroblasts infected with the US2–US11 deletion mutant virus RV798 or with the parental strain AD169varATCC were equally susceptible to death triggered by exposure to tumour necrosis factor (TNF)-α, tunicamycin, thapsigargin, brefeldin A, lonidamine and carbonyl cyanide m-chloro phenyl hydrazone, but were 1.6-fold more sensitive to apoptosis induced by hygromycin B. Expression of US9 in human embryonic kidney 293T cells or in fibroblasts, however, did not protect cells from hygromycin B-mediated death. Together, these results classify US9 as the first CMV-encoded protein to contain an N-terminal SS and a C-terminal MLS, and suggest a completely novel role for this protein during infection.

scholarly journals Ponticulin is an atypical membrane protein.

1994 ◽  
Vol 126 (6) ◽  
pp. 1421-1431 ◽  
Author(s):  
A L Hitt ◽  
T H Lu ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Metabolic Labeling ◽  
Cortical Actin ◽  
Intact Cells ◽  
Membrane Spanning

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

Sign in / Sign up

Export Citation Format

Share Document