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Mutations that mimic phosphorylation of the HIV‐1 matrix protein do not perturb the myristyl switch
Author(s) -
Saad Jamil S.,
Kim Andrew,
Ghanam Ruba H.,
Dalton Amanda K.,
Vogt Volker M.,
Wu Zhibin,
Lu Wuyuan,
Summers Michael F.
Publication year - 2007
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.072987607
Subject(s) - phosphorylation , mutant , nls , microbiology and biotechnology , chemistry , nuclear transport , mutant protein , membrane protein , biology , biochemistry , nuclear localization sequence , membrane , cell nucleus , gene , cytoplasm
Abstract Recent studies indicate that the matrix domain (MA) of the HIV‐1 Gag polyprotein directs Gag to the plasma membrane for virus assembly via a phosphatidylinositol‐4,5‐bisphosphate (PIP 2 )–dependent myristyl switch mechanism. MA also has been reported to direct nuclear trafficking via nuclear import and export functions, and some studies suggest that nuclear targeting may be regulated by MA phosphorylation (although this proposal remains controversial). We have prepared and studied a series of HIV‐1 MA mutants containing Ser‐to‐Asp substitutions designed to mimic phosphorylation, including substitutions in regions of the protein involved in protein–protein interactions and known to influence the myristyl switch (S6D, S9D, S67D, S72D, S6D/S9D, and S67D/S72D). We were particularly interested in substitutions at residue 6, since conservative mutations adjacent to this site strongly perturb the myristyl switch equilibrium, and this site had not been genetically tested due to its involvement in post‐translational myristylation. Our studies reveal that none of these mutations, including S6D, influences the PIP 2 ‐ or concentration‐dependent myristyl switch equilibrium. In addition, all of the mutants bind liposomes with affinities that are only slightly reduced in comparison with the native protein. In contrast, the myristylated mutants bind liposomes with substantially greater affinity than that of the native, unmyristylated protein. These findings support the hypothesis that phosphorylation is unlikely to significantly influence membrane‐mediated intracellular trafficking.