Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein.
Human immunodeficiency virus type 1 Nef protein is N-terminally myristoylated, a modification reported to be required for the association of Nef with cytoplasmic membranes. As myristate alone is not sufficient to anchor a protein stably into a membrane, it has been suggested that N-terminal basic residues contribute to Nef membrane association via electrostatic interactions with acidic phospholipids. Here, data are presented pertaining to the role of the myristate and basic residues in Nef membrane association, subcellular localization and function. Firstly, by using a biochemical assay for membrane association it was shown that, whereas myristoylation of Nef was not essential, mutation of a cluster of four arginines between residues 17 and 22 reduced membrane association dramatically. Mutation of two lysines at residues 4 and 7 had negligible effect alone, but when combined with the arginine substitutions, abrogated membrane association completely. By using indirect immunofluorescence, it was demonstrated that mutation of either of the two basic clusters altered the subcellular distribution of Nef dramatically. Thirdly, the requirement of the arginine and lysine clusters for Nef-mediated CD4 down modulation was shown to correlate precisely with membrane association. These data suggest that membrane localization and subcellular targeting of Nef are controlled by a complex interplay of signals at the N terminus of the protein.
Bentham
Matthew
M
Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Mazaleyrat
Sabine
S
Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Harris
Mark
M
Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D015704:CD4 Antigens / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D015536:Down-Regulation; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015497:HIV-1; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus
D015704:CD4 Antigens; D015693:Gene Products, nef; D054311:nef Gene Products, Human Immunodeficiency Virus; D019814:Myristic Acid; D001120:Arginine; D008239:Lysine