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Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its temperature-conditional cell-surface expression 总被引:6,自引:2,他引:4 下载免费PDF全文
In the preceding paper (Hearing, J., E. Hunter, L. Rodgers, M.-J. Gething, and J. Sambrook. 1989. J. Cell Biol. 108:339-353) we described the isolation and initial characterization of seven Chinese hamster ovary cell lines that are temperature conditional for the cell-surface expression of influenza virus hemagglutinin (HA) and other integral membrane glycoproteins. Two of these cell lines appeared to be defective for the synthesis and/or addition of mannose-rich oligosaccharide chains to nascent glycoproteins. In this paper we show that at both 32 and 39 degrees C in two mutant cell lines accumulate a truncated version, Man5GlcNAc2, of the normal lipid-linked precursor oligosaccharide, Glc3Man9GlcNAc2. This is possibly due to a defect in the synthesis of dolichol phosphate because in vitro assays indicate that the mutant cells are not deficient in mannosylphosphoryldolichol synthase at either temperature. A mixture of truncated and complete oligosaccharide chains was transferred to newly synthesized glycoproteins at both the permissive and restrictive temperatures. Both mutant cell lines exhibited altered sensitivity to cytotoxic plant lectins when grown at 32 degrees C, indicating that cellular glycoproteins bearing abnormal oligosaccharide chains were transported to the cell surface at the permissive temperature. Although glycosylation was defective at both 32 and 39 degrees C, the cell lines were temperature conditional for growth, suggesting that cellular glycoproteins were adversely affected by the glycosylation defect at the elevated temperature. The temperature-conditional expression of HA on the cell surface was shown to be due to impairment at 39 degrees C of the folding, trimerization, and stability of HA molecules containing truncated oligosaccharide chains. 相似文献
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Simon I. Hay Marianne E. Sinka Robi M. Okara Caroline W. Kabaria Philip M. Mbithi Carolynn C. Tago David Benz Peter W. Gething Rosalind E. Howes Anand P. Patil William H. Temperley Michael J. Bangs Theeraphap Chareonviriyaphap Iqbal R. F. Elyazar Ralph E. Harbach Janet Hemingway Sylvie Manguin Charles M. Mbogo Yasmin Rubio-Palis H. Charles J Godfray 《PLoS medicine》2010,7(2)
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The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins 下载免费PDF全文
Goffin L Vodala S Fraser C Ryan J Timms M Meusburger S Catimel B Nice EC Silver PA Xiao CY Jans DA Gething MJ 《Molecular biology of the cell》2006,17(12):5309-5323
The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin alpha (Kap60p) and a novel betaNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin beta homologues. Kinetic binding data suggest that binding to importin beta proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling. 相似文献
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Relationship between the messenger RNAs transcribed from two overlapping genes of influenza virus. 总被引:2,自引:2,他引:2 下载免费PDF全文
The relationship of the mRNAs encoding the NS1 and NS2 polypeptides of influenza virus has been investigated through synthesis and characterisation of complementary DNA copies of the mRNAs. Previous work had shown that both mRNAs are encoded by virion RNA segment 8, and that the sequences comprising the smaller of the two mRNAs (the NS2 mRNA) were also present on the NS1 mRNA. Our results indicate that the mRNA encoding the NS2 polypeptide of the avian influenza, fowl plague virus, is approximately 400 ntds long, and that its sequences correspond largely with the 3'-terminal region of the NS1 mRNA. 相似文献
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The contribution of each of the seven asparagine-linked oligosaccharide side chains on the hemagglutinin of the A/Aichi/68 (X31) strain of influenza virus was assessed with respect to its effect on the folding, intracellular transport, and biological activities of the molecule. Twenty mutant influenza virus hemagglutinins were constructed and expressed, each of which had one or more of the seven glycosylation sites removed. Investigations of these mutant hemagglutinins indicated that (i) no individual oligosaccharide side chain is necessary or sufficient for the folding, intracellular transport, or function of the molecule, (ii) at least five oligosaccharide side chains are required for the X31 hemagglutinin molecule to move along the exocytic pathway to the plasma membrane, and (iii) mutant hemagglutinins having less than five oligosaccharide side chains form intracellular aggregates and are retained in the endoplasmic reticulum. 相似文献
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Molecular chaperones: Clasping the prize 总被引:1,自引:0,他引:1
Mary-Jane Gething 《Current biology : CB》1996,6(12):1573-1576
The three-dimensional structure of the substrate-binding domain of DnaK, a bacterial Hsp70, shows how such molecular chaperones can be so promiscuous in recognizing different proteins, yet so accurate in discriminating between unfolded and folded forms of their polypeptide substrates. 相似文献