Research Interests: Biology, Cell Cycle, Enzyme Inhibitors, Cell Biology, Neuronal cell death, and 15 moreCell Division, Cellular Signalling, Mitosis, Cell line, Humans, Mice, Animals, Microtubules, Histones, HeLa cells, Neurodegenerative Disease, Cell Proliferation, MAP Kinase, Biochemistry and cell biology, and Mitotic Spindle
Golgin-160 is a member of the coiled-coil family of golgin proteins, which are proposed to regulate the structure of the Golgi complex. The C-terminal two-thirds of golgin-160 is predicted to form a coiled-coil domain and the N-terminal... more
Golgin-160 is a member of the coiled-coil family of golgin proteins, which are proposed to regulate the structure of the Golgi complex. The C-terminal two-thirds of golgin-160 is predicted to form a coiled-coil domain and the N-terminal head domain contains several putative binding domains, regulatory motifs and phosphorylation sites. Recently, it has been demonstrated that caspase-dependent cleavage of the golgin-160 head domain occurs rapidly after induction of apoptosis. The role of golgin-160 phosphorylation and the functional implications for Golgi structure have not been defined. In this study, we investigated the kinase(s) responsible for phosphorylation of golgin-160. Signaling through the small G-protein Rac and mixed-lineage-kinase-3 (MLK3) resulted in increased phosphorylation of golgin-160. The intracellular distribution of MLK3 overlapped with that of golgin-160 and the two proteins could be co-immunoprecipitated. In vitro kinase assays demonstrated that MLK3 directly p...
Research Interests: Biology, Membrane Proteins, Medicine, Signal Transduction, Biological Sciences, and 15 moreHumans, Animals, Cell, Phosphorylation, HeLa cells, Substrate Specificity, Golgi Apparatus, MAP Kinase, Protein Binding, Intracellular Space, Protein Transport, Autoantigens, CHO cells, Cricetinae, and Medical and Health Sciences
To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a... more
To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a panel of well characterized mutant G proteins and immunoprecipitation with anti-BiP antibodies to determine if BiP interacted with newly synthesized G protein and/or mutant G proteins retained in the endoplasmic reticulum. We made three major observations: 1) BiP bound transiently to folding intermediates of wild-type G protein which were incompletely disulfide-bonded; 2) BiP did not bind stably to all mutant G proteins which remain in the endoplasmic reticulum; and 3) BiP bound stably only to mutant G proteins which do not form correct intrachain disulfide bonds.
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We isolated forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself... more
We isolated forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself in the membranes of the cis-Golgi network. A fraction of these virions then acquires a second envelope by enwrapping trans-Golgi network membranes to form the intracellular enveloped virus. Lipids were analyzed by high performance thin layer chromatography and digital densitometry to establish a steady-state lipid profile of viral membranes, which should reflect the compositions of the cis-Golgi network and trans-Golgi network. Phosphatidyl-inositol was slightly enriched in the cis-Golgi network of HeLa cells, whereas the trans-Golgi network showed a minor increase in phosphatidylserine and sphingomyelin. Similarly, cholesterol was only slightly more abundant in the trans-Golgi compared to the cis-Golgi. An unusual lipid, semilysobisphosphatidic ac...
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We have developed a monoclonal antibody with HLA-DR5 serologic specificity. The antibody, SFR3-DR5, binds specifically to DR5-positive lymphoblastoid cell lines, and immunoprecipitates alpha- and beta-chains characteristic of DR antigens... more
We have developed a monoclonal antibody with HLA-DR5 serologic specificity. The antibody, SFR3-DR5, binds specifically to DR5-positive lymphoblastoid cell lines, and immunoprecipitates alpha- and beta-chains characteristic of DR antigens from them. Cytotoxic activity of the antibody segregates with the DR5-bearing haplotype in a family. The antibody reacted with the cells of 16 of 17 DR5 individuals and was negative on all DR5-negative cells tested. SFR3-DR5 reacted weakly with PWM-activated cells of the single DR5 individual whose B lymphocytes were unreactive with the monoclonal antibody by cytotoxicity. Possible interpretations of these results are discussed.
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The biosynthesis and glycosylation of the invariant (I) chain associated with HLA-DR alpha-beta chain complexes has been examined in human B lymphoblastoid cell lines. Two-dimensional polyacrylamide gel electrophoresis of HLA-DR immune... more
The biosynthesis and glycosylation of the invariant (I) chain associated with HLA-DR alpha-beta chain complexes has been examined in human B lymphoblastoid cell lines. Two-dimensional polyacrylamide gel electrophoresis of HLA-DR immune precipitates from extracts of metabolically labeled cells demonstrated a processes form of the I chain, called Ip, which consists of a series of spots beginning near the basic I chain spot and ending near the acidic alpha spots. Pulse/chase labeling yielded information on the kinetics of processing of the invariant chain, as well as the finding that the association of Ip with alpha-beta chain complexes appears to be transient. Neuraminidase treatment of immune precipitates confirmed that Ip was a form of the I chain containing up to eight sialic acid residues. Labeling in the presence of tunicamycin suggested the presence of both N-linked and O-linked oligosaccharide units on the invariant chain. This processing pattern does not appear to be an artifa...
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SARS-CoV-2 infection induces severe disease in a subpopulation of patients, but the underlying mechanisms remain unclear. We demonstrate robust IgM autoantibodies that recognize angiotensin converting enzyme-2 (ACE2) in 18/66 (27%)... more
SARS-CoV-2 infection induces severe disease in a subpopulation of patients, but the underlying mechanisms remain unclear. We demonstrate robust IgM autoantibodies that recognize angiotensin converting enzyme-2 (ACE2) in 18/66 (27%) patients with severe COVID-19, which are rare (2/52; 3.8%) in hospitalized patients who are not ventilated. The antibodies do not undergo class-switching to IgG, suggesting a T-independent antibody response. Purified IgM from anti-ACE2 patients activates complement. Pathological analysis of lung obtained at autopsy shows endothelial cell staining for IgM in blood vessels in some patients. We propose that vascular endothelial ACE2 expression focuses the pathogenic effects of these autoantibodies on blood vessels, and contributes to the angiocentric pathology observed in some severe COVID-19 patients. These findings may have predictive and therapeutic implications.One-sentence summaryACE2 autoantibodies in severe COVID-19 have features of a T-independent im...
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In this report, we have investigated the contribution of primary sequence to the carbohydrate requirement for intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strains of vesicular... more
In this report, we have investigated the contribution of primary sequence to the carbohydrate requirement for intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strains of vesicular stomatitis virus. We used site-directed mutagenesis of the coding sequence to eliminate the two consensus sites for glycosylation in the Orsay G protein. Whereas the nonglycosylated San Juan G protein required at least one of its two asparagine-linked oligosaccharides for transport to the plasma membrane at 37 degrees C, a fraction of the Orsay G protein was transported without carbohydrate. Of the 10 amino acid differences between these two proteins, residue 172 (tyrosine in San Juan, aspartic acid in Orsay) played the major role in determining the stringency for the carbohydrate requirement. The rates at which the glycosylated and nonglycosylated Orsay G proteins were transported to the cell surface were the same, although a smaller fraction of the no...
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Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV... more
Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the t...
Research Interests: Biology, Biological Chemistry, Medicine, Biological Sciences, Dogs, and 13 moreLiver, Animals, Cell Polarity, CHEMICAL SCIENCES, Rats, Amino Acid Sequence, Base Sequence, Structure activity Relationship, Site-directed Mutagenesis, Molecular Sequence Data, Cell Membrane, Medical and Health Sciences, and In Vitro Techniques
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Research Interests:
The mixed-lineage kinases (MLK) are serine/threonine protein kinases that regulate mitogen-activated protein (MAP) kinase signaling pathways in response to extracellular signals. Recent studies indicate that MLK activity may promote... more
The mixed-lineage kinases (MLK) are serine/threonine protein kinases that regulate mitogen-activated protein (MAP) kinase signaling pathways in response to extracellular signals. Recent studies indicate that MLK activity may promote neuronal cell death through activation of the c-Jun NH2-terminal kinase (JNK) family of MAP kinases. Thus, inhibitors of MLK activity may be clinically useful for delaying the progression of neurodegenerative diseases, such as Parkinson's. In proliferating non-neuronal cells, MLK may have the opposite effect of promoting cell proliferation. In the current studies we examined the requirement for MLK proteins in regulating cell proliferation by examining MLK function during G2 and M-phase of the cell cycle. The MLK inhibitor CEP-11004 prevented HeLa cell proliferation by delaying mitotic progression. Closer examination revealed that HeLa cells treated with CEP-11004 during G2-phase entered mitosis similar to untreated G2-phase cells. However, CEP-11004...
Research Interests: Cell Cycle, Enzyme Inhibitors, Neuronal cell death, Cell Division, Cellular Signalling, and 15 moreMitosis, Cell line, Humans, Mice, Animals, Phosphorylation, Microtubules, Nuclear envelope, Histones, HeLa cells, Neurodegenerative Disease, Cell Proliferation, MAP Kinase, Biochemistry and cell biology, and Mitotic Spindle
To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a... more
To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a panel of well characterized mutant G proteins and immunoprecipitation with anti-BiP antibodies to determine if BiP interacted with newly synthesized G protein and/or mutant G proteins retained in the endoplasmic reticulum. We made three major observations: 1) BiP bound transiently to folding intermediates of wild-type G protein which were incompletely disulfide-bonded; 2) BiP did not bind stably to all mutant G proteins which remain in the endoplasmic reticulum; and 3) BiP bound stably only to mutant G proteins which do not form correct intrachain disulfide bonds.
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Nucleocapsids in quantities approaching 1 mg were purified from 109 measles virus-infected cells. They contained one polypeptide species with a molecular weight of 59,000. Antiserum was raised in rabbits against purified nucleocapsids and... more
Nucleocapsids in quantities approaching 1 mg were purified from 109 measles virus-infected cells. They contained one polypeptide species with a molecular weight of 59,000. Antiserum was raised in rabbits against purified nucleocapsids and used in a competitive radioimmunoassay. Because of their instability, purified nucleocapsids were not suitable for use in such an assay. Instead partially purified nucleocapsids from HEp-2 cells persistently infected with measles virus and labeled in vitro with [35S]methionine were used as the source of radioactive antigen. The radioimmunoassay thus developed measured less than 5 ng of nucleocapsids in infected cells.
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Research Interests:
The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid)... more
The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxO (where x is any amino acid and O is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition...
Research Interests: Kinetics, Biological Sciences, Cell line, Animals, Vesicular Stomatitis Virus, and 13 moreEndoplasmic Reticulum, P-glycoprotein, Protein Secondary Structure Prediction, Exocytosis, Amino Acid Profile, G protein, Amino Acid Sequence, Mutants, Cytoplasm, Plasma Membrane, Tyrosine, Molecular Sequence Data, and Cell Membrane
Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol... more
Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.
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Expression of the human gene A4 is enriched in the colonic epithelium and is transcriptionally activated on differentiation of colonic epithelial cells in vitro (M. M. Oliva, T. C. Wu, and V. W. Yang. Arch. Biochem. Biophys. 302: 183-192,... more
Expression of the human gene A4 is enriched in the colonic epithelium and is transcriptionally activated on differentiation of colonic epithelial cells in vitro (M. M. Oliva, T. C. Wu, and V. W. Yang. Arch. Biochem. Biophys. 302: 183-192, 1993). A4 cDNA contains an open reading frame that predicts a polypeptide of 17 kDa. To determine the function of the A4 protein, we characterized its biochemical and physiological properties. Hydropathy analysis of deduced A4 amino acid sequence revealed four putative membrane-spanning alpha-helices. The hydrophobic nature of A4 was confirmed by its being extractable with organic solvents. Immunocytochemical studies of cells expressing A4 localized it to the endoplasmic reticulum. Moreover, A4 multimerized in vivo as determined by coimmunoprecipitation experiments. The four-transmembrane topology and biophysical characteristics of A4 suggest that it belongs to a family of integral membrane proteins called proteolipids, some of which multimerize to...
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We isolated forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself... more
We isolated forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself in the membranes of the cis-Golgi network. A fraction of these virions then acquires a second envelope by enwrapping trans-Golgi network membranes to form the intracellular enveloped virus. Lipids were analyzed by high performance thin layer chromatography and digital densitometry to establish a steady-state lipid profile of viral membranes, which should reflect the compositions of the cis-Golgi network and trans-Golgi network. Phosphatidyl-inositol was slightly enriched in the cis-Golgi network of HeLa cells, whereas the trans-Golgi network showed a minor increase in phosphatidylserine and sphingomyelin. Similarly, cholesterol was only slightly more abundant in the trans-Golgi compared to the cis-Golgi. An unusual lipid, semilysobisphosphatidic ac...
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T lymphocytes are activated upon binding of their Ag receptors to a complex of Ag-derived peptides and MHC class I or class II molecules expressed on the surface of APC. It is now well established that APC degrade exogenous Ag in acidic... more
T lymphocytes are activated upon binding of their Ag receptors to a complex of Ag-derived peptides and MHC class I or class II molecules expressed on the surface of APC. It is now well established that APC degrade exogenous Ag in acidic endosomal compartments, and that Ag fragments bind to class II molecules moving through these compartments on their way to the surface of the APC. Although peptides derived from some endogenous Ag can also bind to class II molecules and subsequently be recognized by class II-restricted T cells, the intracellular trafficking pathways that enable endogenous proteins to be processed for association with class II molecules remain controversial. We have analyzed the mechanism by which the envelope (env) protein of the HIV-1 is processed in infected cells for recognition by class II-restricted T cells. A large number of env-specific class II-restricted human CTL clones were shown to lyse B-lymphoblastoid cell lines expressing the env. A novel dilutional as...
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Research Interests:
... 108, 515-520 (1981) Cells Infected with a Cell-Associated Subacute Sclerosing Panencephalitis Virus Do Not Express M Protein CAROLYN E. MACHAMER ... 3. TER MEULEN, V. HALL, WW, and KRETH, HW, In "Persistent Viruses" (JG... more
... 108, 515-520 (1981) Cells Infected with a Cell-Associated Subacute Sclerosing Panencephalitis Virus Do Not Express M Protein CAROLYN E. MACHAMER ... 3. TER MEULEN, V. HALL, WW, and KRETH, HW, In "Persistent Viruses" (JG Stevens, GJ Todaro, and CF Fox, eds.), pp. ...
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Sera from patients exposed to measles virus were investigated for the presence of antibodies against each of the viral antigens. All sera with measurable neutralizing titers contained antibodies against the two surface proteins (the... more
Sera from patients exposed to measles virus were investigated for the presence of antibodies against each of the viral antigens. All sera with measurable neutralizing titers contained antibodies against the two surface proteins (the glycoprotein and fusion protein), the nucleocapsid protein, and one of the internal proteins (P2). However, only sera from individuals with clinical symptoms of measles infection (natural measles and atypical measles) contained antibodies against the measles virus matrix protein. Levels of matrix-specific antibodies were highest in patients with atypical measles infection.
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Immunoprecipitation methods were used to analyze sera and cerebrospinal fluids from patients with multiple sclerosis for measles virus-specific antibodies. The sera contained antibodies against all virus antigens except the matrix... more
Immunoprecipitation methods were used to analyze sera and cerebrospinal fluids from patients with multiple sclerosis for measles virus-specific antibodies. The sera contained antibodies against all virus antigens except the matrix polypeptide. Of the nine cerebrospinal fluids investigated, two contained undetectable levels of measles-specific antibodies, four contained antibodies against the nucleocapsid and another internal antigen, and three contained antibodies against the surface antigens as well as the internal antigens.
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The biosynthesis and glycosylation of the invariant (I) chain associated with HLA-DR alpha-beta chain complexes has been examined in human B lymphoblastoid cell lines. Two-dimensional polyacrylamide gel electrophoresis of HLA-DR immune... more
The biosynthesis and glycosylation of the invariant (I) chain associated with HLA-DR alpha-beta chain complexes has been examined in human B lymphoblastoid cell lines. Two-dimensional polyacrylamide gel electrophoresis of HLA-DR immune precipitates from extracts of metabolically labeled cells demonstrated a processes form of the I chain, called Ip, which consists of a series of spots beginning near the basic I chain spot and ending near the acidic alpha spots. Pulse/chase labeling yielded information on the kinetics of processing of the invariant chain, as well as the finding that the association of Ip with alpha-beta chain complexes appears to be transient. Neuraminidase treatment of immune precipitates confirmed that Ip was a form of the I chain containing up to eight sialic acid residues. Labeling in the presence of tunicamycin suggested the presence of both N-linked and O-linked oligosaccharide units on the invariant chain. This processing pattern does not appear to be an artifa...
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We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two... more
We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic...
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Research Interests:
The diverse forms and functions of cellular organelles are, presumably, a consequence of their particular molecular compositions. The generation and maintenance of this diversity is achieved by the targeting of newly synthesized proteins... more
The diverse forms and functions of cellular organelles are, presumably, a consequence of their particular molecular compositions. The generation and maintenance of this diversity is achieved by the targeting of newly synthesized proteins to specific locations and their subsequent retention there. Sequences that retain proteins in the endoplasmic reticulum (ER) have been identified at the C-termini of resident ER proteins, where they are readily accessible to potential receptors. By contrast, recent results have demonstrated that retention of proteins in the Golgi complex involves sequences located within transmembrane domains. This suggests the novel possibility that the membrane composition of the Golgi complex plays a role in retention of resident Golgi proteins.
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The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered... more
The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.
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Research Interests:
Research Interests: Kinetics, Fluorescence Microscopy, Membrane Proteins, Cell Biology, Apoptosis, and 14 moreSignal Transduction, Biological Sciences, Caspases, Humans, Special functions, Caspase, Cell nucleus, Fluorescent Antibody Technique, HeLa cells, Substrate Specificity, The cell, Golgi Apparatus, Autoantigens, and Molecular Sequence Data
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The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the... more
The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.