The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme.
The nonstructural protein Nsp1 of Semliki Forest virus has guanine-7-methyltransferase and guanylyltransferase-like activities, required in the capping of viral mRNAs. It is palmitoylated and tightly associated with the cytoplasmic surface of the plasma membrane, endosomes, and lysosomes. To localize the acylation site(s) and the putative membrane-targeting domain, a number of deletions were made in the nsp1 gene. Most deletions resulted in the expression of nonpalmitoylated, enzymatically inactive, cytoplasmic protein. Palmitate could be released from Nsp1 with neutral hydroxylamine, indicating a thioester linkage to a cysteine residue. Therefore we mutated the conserved cysteine residues of Nsp1 to alanine. Triple mutation of Cys418, Cys419, and Cys420 resulted in nonpalmitoylated Nsp1, which was enzymatically active and still associated with membranes. However, it could be released from the membranes with 1 M NaCl, whereas 50 mM sodium carbonate (pH 12) was required to release wild type Nsp1, suggesting a conversion from an integral to a peripheral membrane protein. Indirect confocal immunofluorescence microscopy showed that the nonpalmitoylated Nsp1 colocalized with the plasma membrane marker, concanavalin A. However, it was not detected in filopodia, which were heavily stained in cells expressing wild type Nsp1. These results indicate that the acylation of Nsp1 was not needed for its targeting to the plasma membrane, but it was necessary for the migration to the filopodial extensions of the plasma membrane.
Laakkonen
P
P
Institute of Biotechnology, University of Helsinki, P. O. Box 56, Viikinkaari 9, FIN-00014 Helsinki, Finland. pirjo.laakkonen@helsinki.fi
Ahola
T
T
Kääriäinen
L
L
D000215:Acylation; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D017830:Octoxynol; D019308:Palmitic Acid / Q000494:pharmacology*; D011092:Polyethylene Glycols; D012367:RNA, Viral / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012672:Semliki forest virus / Q000235:genetics*; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000737:chemistry; D014162:Transfection
D002135:Calcium-Binding Proteins; D005656:Fungal Proteins; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D012367:RNA, Viral; D029701:Saccharomyces cerevisiae Proteins; D019308:Palmitic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40