Partitioning of HIV-1 Gag and Gag-related proteins to membranes.
The binding of HIV-1 Gag and Gag-related proteins to model membranes was examined using three experimental systems: (i) large unilamellar phospholipid vesicles (LUVs) and recombinant Gag purified from Escherichia coli; (ii) LUVs added to a mammalian cell extract in which Gag proteins were expressed by a coupled transcription/translation system; and (iii) inside-out plasma membrane vesicles purified from human red blood cells (RBC) and recombinant, purified Gag from E. coli. Several novel aspects of HIV-1 Gag membrane interactions were observed: (i) Gag proteins bound with high affinity to both model membranes with a negatively charged surface and to RBC membranes. (ii) Binding of the Gag precursor and mature Gag proteins exhibited different sensitivities to ionic strength indicating that the precursor directed membrane binding through interactions that were qualitatively and quantitatively distinct from those of any of its individual domains. Studies using energy transfer between tryptophan residues in the proteins and anthroyloxy-containing probes inserted in the LUVs indicated that the orientation of the precursor and of the mature proteins on the membrane surface were distinct; (iii) Gag oligomers appear to have facilitated high-affinity binding under high salt conditions, suggesting that protein-protein interactions led to formation of stronger electrostatic or new hydrophobic membrane binding determinants. Since binding studies with model membranes permit quantitative analysis, these experimental approaches may permit identification of interactions that drive Gag assembly on the membrane.
Ehrlich
L S
LS
Department of Molecular Genetics & Microbiology, State University of New York at Stony Brook, New York, 11794, USA.
Fong
S
S
Scarlata
S
S
Zybarth
G
G
Carter
C
C
D003001:Cloning, Molecular; D004735:Energy Transfer; D004926:Escherichia coli; D015683:Gene Products, gag / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015750:Genes, gag; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D008954:Models, Biological; D010715:Phosphatidylglycerols; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence
D015683:Gene Products, gag; D008081:Liposomes; D010715:Phosphatidylglycerols; D011498:Protein Precursors; D011994:Recombinant Proteins; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol