The sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza C virus CM2 protein.
The sites for fatty acylation, disulphide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine mutant can form multimers.
Li
Zhu-Nan
ZN
Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1.
Hongo
Seiji
S
Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1.
Sugawara
Kanetsu
K
Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1.
Sugahara
Kazuhiko
K
Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1.
Tsuchiya
Emi
E
Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1.
Matsuzaki
Yoko
Y
Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1.
Nakamura
Kiyoto
K
Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1.
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides; D006801:Humans; D009982:Gammainfluenzavirus / Q000235:genetics / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism*; D010766:Phosphorylation; D013395:Sucrose; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*
C090877:CM2 protein, Influenza C virus; D003432:Cross-Linking Reagents; D004220:Disulfides; D014763:Viral Matrix Proteins; D019308:Palmitic Acid; D013395:Sucrose; D003545:Cysteine