Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0038362 (stomatitis)
 8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ER to Golgi transport requires the function of two distinct vesicle coat complexes, termed COPI (coatomer) and COPII, whose assembly is regulated by the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, respectively. To address their individual roles in transport, we have developed a new assay using mammalian microsomes that reconstitute the formation of ER-derived vesicular carriers. Vesicles released from the ER were found to contain the cargo molecule vesicular stomatitis virus glycoprotein (VSV-G) and p58, an endogenous protein that continuously recycles between the ER and pre-Golgi intermediates. Cargo was efficiently sorted from resident ER proteins during vesicle formation in vitro. Export of VSV-G and p58 were found to be exclusively mediated by COPII. Subsequent movement of ER-derived carriers to the Golgi stack was blocked by a trans-dominant ARF1 mutant restricted to the GDP-bound state, which is known to prevent COPI recruitment. To establish the initial site of coatomer assembly after export from the ER, we immunoisolated the vesicular intermediates and tested their ability to recruit COPI. Vesicles bound coatomer in a physiological fashion requiring an ARF1-guanine nucleotide exchange activity. These results suggest that coat exchange is an early event preceding the targeting of ER-derived vesicles to pre-Golgi intermediates.
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PMID:COPII vesicles derived from mammalian endoplasmic reticulum microsomes recruit COPI. 892 75

Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5 degrees C instead of the normal growth temperature of 34 degrees C in order to allow for the isolation of temperature-sensitive lesions. This selection/screening procedure resulted in the isolation of M15-4 cells, which had three- to five-fold lower incorporation of [2-3H]mannose into mannose 6-phosphate, mannose 1-phosphate, GDP-mannose, oligosaccharide-lipid, and glycoprotein at 41.5 degrees C. We detected no difference in the qualitative pattern of mannose-labeled lipid-linked oligosaccharides compared to parental cells. M15-4 cells synthesized dolichol. The defect of M15-4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight- to nine-fold lower in hexokinase activity in M15-4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP-mannose in M15-4 cells was 70% of normal. The phenotype of M15-4 was a lower specific activity of labeled GDP-mannose, not a substantial reduction in the level of GDP-mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low-glucose medium.
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PMID:Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels. 1002 67

Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound-activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of beta-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated beta-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with beta-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, beta-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15 degrees C. These data suggest that the Rab2 protein plays a role in the low-temperature-sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.
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PMID:A Rab2 mutant with impaired GTPase activity stimulates vesicle formation from pre-Golgi intermediates. 1035

Arl1 is a member of the ARF-like protein (Arl) subfamily of small GTPases. Nothing is known about the function of Arl1 except for the fact that it is essential for normal development in Drosophila and that it is associated with the Golgi apparatus. In this study, we first demonstrate that Arl1 is enriched at the trans side of the Golgi, marked by AP-1. Association of Arl1 with the Golgi is saturable in intact cells and depends on N-terminal myristoylation. Over-expression of Arl1(T31N), which is expected to be restricted to the GDP-bound form and thus function as a dominant-negative mutant, causes the disappearance of the Golgi apparatus (marked by Golgi SNARE GS28), suggesting that Arl1 is necessary for maintaining normal Golgi structure. Overexpression of Arl1(Q71L), a mutant restricted primarily to the activated GTP-bound form, causes an expansion of the Golgi apparatus with massive and stable Golgi association of COPI and AP-1 coats. Interestingly, Golgi ARFs also become stably associated with the expanded Golgi. Transport of the envelope protein of vesicular stomatitis virus (VSV-G) along the secretory pathway is arrested at the expanded Golgi upon expression of Arl1(Q71L). The structure of stacked cisternae of the Golgi is disrupted in cells expressing Arl1(Q71L), resulting in the transformation of the Golgi into an extensive vesicule-tubule network. In addition, the GTP form of Arl1 interacts with arfaptin-2/POR1 but not GGA1, both of which interact with GTP-restricted ARF1, suggesting that Arl1 and ARF1 share some common effectors in regulating cellular events. On the basis of these observations, we propose that one of the mechanisms for the cell to regulate the structure and function of the Golgi apparatus is through the action of Arl1.
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PMID:Regulation of Golgi structure and function by ARF-like protein 1 (Arl1). 1179 19

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo. We have previously shown that the P protein of VSV, expressed in E. coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII). In the present study we present evidence that the P protein, in addition to being phosphorylated, binds covalently to GTP only when it is phosphorylated. Competition experiments show that ATP, ADP, GTP, and GDP can compete for the binding site(s) of GTP but not AMP, GMP, CTP, or UTP. Interestingly, once GTP is bound to P protein it cannot be displaced by unlabeled GTP. The GTP binding site has been mapped within the domain where the phosphorylation of P protein by CKII occurs. Finally, we show that phosphorylation negative P mutants P3A (P60A, P62A, P64A), P3E (P60E, P62E, P64E), and P3R (P60R, P62R, P64R) failed to bind to GTP, indicating that phosphorylation of P is indeed essential for binding to GTP. Although the precise role of binding of GTP to P is unclear, it appears that phosphorylation of P may initiate a structural change within the P protein allowing GTP to bind, thus manifesting biological function to the transcription factor.
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PMID:Novel binding of GTP to the phosphoprotein (P) of vesicular stomatitis virus. 1217 45

Nonsegmented negative-sense (nsNS) RNA viruses typically replicate within the host cell cytoplasm and do not have access to the host mRNA capping machinery. These viruses have evolved a unique mechanism for mRNA cap formation in that the guanylyltransferase transfers GDP rather than GMP onto the 5' end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype nsNS RNA virus, we now provide genetic and biochemical evidence that its mRNA cap methylase activities are also unique. Using recombinant VSV, we determined the function in mRNA cap methylation of a predicted binding site in the polymerase for the methyl donor, S-adenosyl-l-methionine. We found that amino acid substitutions to this site disrupted methylation at the guanine-N-7 (G-N-7) position or at both the G-N-7 and ribose-2'-O (2'-O) positions of the mRNA cap. These studies provide genetic evidence that the two methylase activities share an S-adenosyl-l-methionine-binding site and show that, in contrast to other cap methylation reactions, methylation of the G-N-7 position is not required for 2'-O methylation. These findings suggest that VSV evolved an unusual strategy of mRNA cap methylation that may be shared by other nsNS RNA viruses.
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PMID:A unique strategy for mRNA cap methylation used by vesicular stomatitis virus. 1670 77

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.
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PMID:Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus. 1721 73

The small GTPase Rab22B (or Rab31) has been suspected to be involved in trafficking at trans-Golgi network. However, its exact cellular localization, tissue expression profile, and functions have not been uncharacterized. Specific antibody raised against Rab22B's protein revealed that Rab22B is brain-enriched, but is also present in substantial levels in spleen and intestine. In HeLa cells, endogenous Rab22B is largely associated with the trans-Golgi network (TGN). Over-expression of a GDP-binding mutant (Rab22BSN), but not wild-type Rab22B, specifically disrupts the TGN localization of TGN46, a dynamic marker which cycles between the TGN and the plasma membrane. The TGN resident membrane protein syntaxin 16, cis-Golgi markers such as GM130 and syntaxin 5, as well as the TGN/late endosome marker mannose 6-phosphate receptor (M6PR) are not affected by Rab22BSN, neither was endosomal-TGN transport of the Shiga toxin B subunit. The disruption of TGN46 staining by Rab22BSN could be specifically attributed to a domain at the C-terminal portion of Rab22B, where its sequence deviates the most from Rab22A. Over-expression of Rab22BSN inhibits the cell surface transport of the vesicular stomatitis virus G protein. Thus, Rab22B may have a role in anterograde exit from the TGN.
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PMID:Rab22B's role in trans-Golgi network membrane dynamics. 1767 23

Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5' end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific cis-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.
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PMID:Vesicular stomatitis virus mRNA capping machinery requires specific cis-acting signals in the RNA. 1768 69

Nonsegmented negative-sense (NNS) RNA viruses cap their mRNA by an unconventional mechanism. Specifically, 5' monophosphate mRNA is transferred to GDP derived from GTP through a reaction that involves a covalent intermediate between the large polymerase protein L and mRNA. This polyribonucleotidyltransferase activity contrasts with all other capping reactions, which are catalyzed by an RNA triphosphatase and guanylyltransferase. In these reactions, a 5' diphosphate mRNA is capped by transfer of GMP via a covalent enzyme-GMP intermediate. RNA guanylyltransferases typically have a KxDG motif in which the lysine forms this covalent intermediate. Consistent with the distinct mechanism of capping employed by NNS RNA viruses, such a motif is absent from L. To determine the residues of L protein required for capping, we reconstituted the capping reaction of the prototype NNS RNA virus, vesicular stomatitis virus, from highly purified components. Using a panel of L proteins with single-amino-acid substitutions to residues universally conserved among NNS RNA virus L proteins, we define a new motif, GxxT[n]HR, present within conserved region V of L protein that is essential for this unconventional mechanism of mRNA cap formation.
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PMID:A conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense RNA viruses that is essential for mRNA capping. 1800 31


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