Volume 271, Number 43,
Issue of October 25, 1996
pp. 26810-26818
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Determinants of Membrane Association for Poliovirus Protein
3AB
(Received for publication, June 5, 1996, and in revised form, August 13, 1996)
Jonathan S.
Towner
,
Tri
V. Ho
and
Bert L.
Semler
From the Department of Microbiology and Molecular Genetics, College
of Medicine, University of California, Irvine, California 92697
Poliovirus protein 3AB may serve as the
lipophilic carrier of a protein primer (VPg or 3B) used for the
initiation of genomic viral RNA synthesis. In order to study the
membrane-protein interactions of 3AB required for its role in
poliovirus RNA replication, we have developed an in vitro
membrane association assay capable of distinguishing membrane-bound
from non-membrane-bound proteins that are cotranslated together
in the presence of canine microsomal membranes. This assay utilizes
equilibrium sedimentation analysis in high density sucrose gradients to
measure membrane association of both wild type and mutated forms of
3AB. Using this assay and other biochemical assays, we have identified
the following properties of the 3AB-membrane interaction:
(a) 3AB is able to post-translationally associate with
microsomal membranes, (b) 3AB is able to associate with
membranes in a manner consistent with that of an integral
membrane protein, (c) 3AB contains a critical hydrophobic
sequence within the carboxyl-terminal half of the protein that is
required for membrane association, and (d) the introduction
of charged residues into this hydrophobic sequence disrupts the 3AB
membrane-protein interaction. Taken together, these studies indicate
that poliovirus protein 3AB associates tightly with biological
membranes de novo in a manner that would allow it to serve
as a lipophilic anchor for the assembly of the poliovirus RNA
replication complex.
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