Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of the polyene antibiotic filipin with membrane-bound cholesterol in vesicular stomatitis (VS), influenza, and Rauscher leukemia virions was studied. Exposure of virions to filipin resulted in a series of depressions and ridges in the envelope of VS virions, with a periodicity of 15 to 20 nm perpendicular to the long axis of the particle; similar morphological alterations were observed in negatively stained preparations, in thin-sectioned virions, and in protease-treated virions that lack surface glycoproteins. This morphological effect was specific for filipin, since the envelopes of VS virions that had been treated with another polyene antibiotic, amphotericin B, exhibited markedly different morphology. Morphological alterations induced by filipin in influenza and Rauscher leukemia virions differed from those seen in VS virions. The infectivity of filipin-treated VS virions was reduced up to 500-fold, whereas influenza virions were resistant to filipin treatment. Incorporation of filipin into the virions was demonstrated, and no release of either lipids or proteins from virions was detected after filipin treatment. A stoichiometry of approximately 1 mol of bound filipin per mol of cholesterol was found in both intact and protease-treated VS virions. The equilibrium dissociation constant for filipin-cholesterol interaction was approximately 74-fold larger in intact than in protease-treated VS virions. The initial rate of association of filipin with cholesterol in intact virions was slower than that in protease-treated particles. The fluidity of lipids in VS viral membranes, as probed by a stearic acid derivative spin label, was markedly reduced when either intact or protease-treated virions were treated with filipin.
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PMID:Effects of filipin on the structure and biological activity of enveloped viruses. 20 81

The kinetics of the appearance of influenza mRNA, the distribution of mRNA between free and membrane-associated polyribosomes, its poly(A) content, and the extent to which the genome was transcribed into mRNA early in infection were determined. Polyribosomes were prepared from influenza virus-infected cells labeled for 30-min periods at various times after infection with [3H]uridine. Most of the 3H-labeled RNA extracted from these polyribosomes sedimented as a heterogeneous 8S to 20S peak in sucrose gradients, and it was largely complementary to virion RNA. By the following criteria, the complementary RNA had properties normally ascribed to mRNA: (i) it labeled rapidly with [3H]uridine; (ii) after glutaraldelyde treatment, it banded with polyribosomes in CsCl density gradients; and (iii) it contained poly(A). In chick cells at 37 C, virus mRNA was first detectable at 45 min postinfection and reached its maximal rate of appearance at 2 to 2.5 h postinfection. The free and membrane-bound polyribosomes of infected cells were separated and were found to contain the same classes of mRNA. There was no absolute segregation of mRNA sequences into either polyribosome class although each probably contained distinct ratios of the different mRNA's. From 45 min postinfection onwards, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences complementary to at least 80% of the genome RNA, whereas poly(A)-minus RNA contained sequences complementary to 90 to 100% of the genome. There was no evidence for the temporal control of transcription of influenza mRNA. At 31 C, when virus development was slowed relative to 37 C,complementary RNA first appeared at 1 h postinfection. At this time, total polysomal RNA contained sequences complementary to the whole genome.
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PMID:Characterization of the mRNA of influenza virus. 120 43

The beta-galactoside alpha 2,6-sialyltransferase has been localized to the trans cisternae of the Golgi apparatus and the trans Golgi network where it transfers sialic acid residues to terminal positions on N-linked oligosaccharides. It is a type II transmembrane protein possessing a 9-amino acid amino-terminal cytoplasmic tail, a 17-amino acid signal anchor domain, and a 35-amino acid stem region which tethers the large luminal catalytic domain to the membrane anchor. Previous work has demonstrated that the soluble sialytransferase catalytic domain is rapidly secreted from Chinese hamster ovary cells. These results suggest that the signals for Golgi apparatus localization do not reside in the catalytic domain of the enzyme but must reside in the cytoplasmic tail, signal anchor domain, and/or stem region. To determine which amino-terminal regions are required for Golgi apparatus localization, mutant sialyltransferase proteins were constructed by in vitro oligonucleotide-directed mutagenesis, expressed in Cos-1 cells, and localized by indirect immunofluorescence microscopy. Signal cleavage-sialyltransferase mutants which consist of only the stem and catalytic domain of the enzyme are not rapidly secreted but are retained intracellularly and predominantly localized to the Golgi apparatus. However, deletion of either the stem region or the cytoplasmic tail of the membrane-bound sialyltransferase does not alter its Golgi apparatus localization. In addition, sequential replacement of the amino acids of the sialyltransferase signal anchor domain with amino acids from the signal anchor domain of a plasma membrane protein, the influenza virus neuraminidase does not alter the Golgi apparatus localization of the sialyltransferase. These observations suggest that sequences in the signal anchor region and stem region allow the Golgi apparatus localization of the membrane-bound and soluble forms of the sialytransferase, respectively, and that both regions may contain Golgi apparatus localization signals.
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PMID:The signal anchor and stem regions of the beta-galactoside alpha 2,6-sialyltransferase may each act to localize the enzyme to the Golgi apparatus. 156 12

I am investigating the role of protein folding in the transport of influenza virus hemagglutinin (HA), a membrane-bound protein, along the exocytotic pathway. From a previous work (Gething, M.-J., McCammon, K., and Sambrook, J. (1986) Cell 46, 939-950), it has been shown that a subset of alterations of the COOH-terminal sequences of the HA molecule inhibit folding and impede its transport to the cell surface. Current studies establish that the integrity of the NH2-terminal sequences of the HA is essential for assembly and transport of the molecule. Mutants lacking just 1 or 2 amino acids immediately COOH-terminal to the signal cleavage site are translocated and core glycosylated, but also incorrectly folded. The mutant molecules are not terminally glycosylated and are thus confined inside the cells. A hypothesis will be presented to explain why sequences at opposite ends of the HA molecule are essential for the assembly of native structures and why correct folding is necessary for transport along the exocytotic pathway of mammalian cells.
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PMID:A single amino acid deletion at the amino terminus of influenza virus hemagglutinin causes malfolding and blocks exocytosis of the molecule in mammalian cells. 173 23

Fusion of individual human erythrocytes to fibroblasts expressing the influenza virus hemagglutinin Cells were attached to coverslips fitted in a specially designed flow chamber mounted on a microscope stage, and fusion was triggered by rapid acidification to pH less than 5.2. Fusion between single cell pairs was monitored by a fluorescence increase due to redistribution of fluorescent dyes between either membrane or cytoplasmic compartments of fusing cells. The single cell fusion events were broadly heterogenous in lag times, rise times, and overall shape of the curves. Lag times obtained with a water-soluble dye were within the range obtained with a water-soluble dye were within the range obtained with the membrane-bound fluorophores, (10-160 s). Fusion was both all-or-nothing and irreversible, in that once dye redistribution in any cell commenced, it completed, regardless of pH. Short pulses of pH 4.9 for 6-10 s led to about half of the cell pairs fusing, but pulses greater than 14 s were as effective as constant low pH. Pulses that were too short to trigger fusion did not partially activate nor deactivate the fusion process, as shown by the ability of a second acidification to cause fusion of the same cells, with similar lag times. These results indicate that the overall hemagglutinin-mediated fusion process is composed of at least two stages, one required for commitment of the hemagglutinin to a fusogenic state that is pH-dependent and a maturation stage that is pH-independent.
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PMID:Single cell fusion events induced by influenza hemagglutinin: studies with rapid-flow, quantitative fluorescence microscopy. 205 63

The influenza hemagglutinin precursor (HA0) and many other glycoproteins fold and oligomerize in the endoplasmic reticulum (ER). Only correctly folded oligomers are transported to the cell surface. To analyse the rules which determine this type of ER sorting, we have extended our analysis of hemagglutinin transport to two soluble, anchor-free recombinant HA0s derived from X31/A/Aichi/68 and A/Japan/305/57 influenza A. The results showed that individual monomers rapidly acquired a folded structure similar to that of monomeric membrane-anchored HA0. They were efficiently transported and secreted, but oligomerization was not required for secretion. Trimers or higher order complexes were either not formed (X31 HA0), or appeared during passage through the late compartments of the secretory pathway, with no effect on the rate of transport (Japan HA0). However, when initial folding was disturbed by inhibition of N-linked glycosylation, anchor-free X31 HA0 was misfolded and retained in the ER as disulfide-linked complexes associated with binding protein, BiP (GRP78). The complexes were similar to those seen for the nonglycosylated membrane-bound HA0, but instead of forming immediately after synthesis they appeared with a half-time of 6 min. Taken together, the data demonstrate that the structural criteria that makes the anchor-free HA0 transport competent are less stringent than those for the membrane form; they must fold correctly but do not need to oligomerize.
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PMID:Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly and secretion of anchor-free influenza hemagglutinin. 217 22

A recombinant vaccinia has been designed to express amino acids 366-379 of influenza nucleoprotein, previously shown to be the minimal epitope recognized by a class I-restricted cytotoxic T cell clone. Target cells infected with the recombinant vaccinia virus expressing this peptide are recognized by CTL as efficiently as target cells expressing the complete nucleoprotein. The results imply the existence of a peptide transport system that constitutively passes the products of degraded proteins from the cytoplasm into a membrane-bound compartment of the cell.
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PMID:A 15 amino acid fragment of influenza nucleoprotein synthesized in the cytoplasm is presented to class I-restricted cytotoxic T lymphocytes. 247 69

We have monitored the mixing of both aqueous intracellular and membrane-bound fluorescent dyes during the fusion of human red blood cells to influenza hemagglutinin-expressing fibroblasts using fluorescence spectroscopy and low light, image-enhanced video microscopy. The water-soluble fluorescent dye, N-(7-nitrobenzofurazan-4-yl)taurine, was incorporated into intact human red blood cells. The fluorescence of the dye in the intact red blood cell was partially quenched by hemoglobin. The lipid fluorophore, octadecylrhodamine, was incorporated into the membrane of the same red blood cell at self-quenching concentrations (Morris, S. J., D. P. Sarkar, J. M. White, and R. Blumenthal. 1989. J. Biol. Chem. 264: 3972-3978). Fusion, which allowed movement of the water-soluble dye from the cytoplasm of the red blood cell into the hemagglutinin-expressing fibroblasts, and movement of octadecylrhodamine from membranes of red blood cell to the plasma membrane of the fibroblasts, was observed by fluorescence microscopy as a spatial relocation of dyes, and monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, fluorescence increased after a delay of about 30 s at 37 degrees C, reaching a maximum within 3 min. The kinetics, pH profile, and temperature dependence were similar for both fluorescent events measured simultaneously, indicating that influenza hemagglutinin-induced fusion rapidly establishes bilayer continuity and exchange of cytoplasmic contents.
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PMID:Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing. 274 45

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.
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PMID:Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins. 282 Oct 12

The binding sites for t-[35S]butylbicyclophosphorothionate ([35S] TBPS) and [3H]flunitrazepam ([3H]Flu) were solubilized from freshly prepared and washed membranes from rat forebrain with 20 mM 3-[(cholamidopropyl)-dimethylamonio]-1-propanesulfonate in the presence of protease inhibitors. Approximately 64% of the protein, 56% of the [35S]TBPS sites and 45% of the [3H] Flu sites were solubilized. The soluble and membrane-bound binding sites for [3H]Flu were relatively stable on storage at 0 degree C or at -65 degrees C for 11 days. Binding of [35S]TBPS to membranes was increased after freezing the membranes at -65 degrees C for 1 to 11 days and was relatively unchanged if the membranes were stored at 0 degree C for 1 to 11 days. Solubilized [35S]TBPS binding sites were stable when stored at -65 degrees C but declined to 20% of control when stored at 0 degree C for 11 days. Tested shortly after preparation, [35S]TBPS binding to both soluble and membrane preparations demonstrated similar affinity, temperature dependence and noncompetitive inhibition by pentobarbital (pb). Stimulation by pb of [3H]Flu binding to the soluble fraction decayed on storage of the solubilized material at 0 degree C with a time course similar to the loss of [35S]TBPS binding. This finding might suggest that the stimulation of [3H]Flu binding by pb is mediated via its action on TBPS sites. However in membrane preparations stored at 0 degree C, [35S]TBPS binding was stable whereas the pb effects declined with time.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of pentobarbital on t-[35S]butylbicyclophosphorothionate and [3H]flunitrazepam binding to membrane-bound and solubilized preparations from rat forebrain. 298 10


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