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(Received for publication, May 30, 1996, and in revised form, August 22, 1996)
From the Institute of Biotechnology, University of Helsinki, P. O.
Box 56, Viikinkaari 9, FIN-00014 Helsinki, Finland
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.
Volume 271, Number 45,
Issue of November 8, 1996
pp. 28567-28571
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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