The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.

Budding of lentiviruses occurs at the plasma membrane, but the preceding steps involved in particle assembly are poorly understood. Since the Gag polyprotein mediates virion assembly and budding, studies on the localization of Gag within the cell should provide insight into the mechanism of particle assembly. Here, we utilize biochemical fractionation techniques as well as high-resolution confocal imaging of live cells to demonstrate that Gag is localized at the plasma membrane in a striking punctate pattern. Mutation of the N-terminal myristoylation site results in the formation of large cytosolic complexes, whereas mutation of the N-terminal basic residue cluster in the matrix domain redirects the Gag protein to a region partially overlapping the Golgi apparatus. In addition, we show that Gag and Env colocalize at the plasma membrane and that mistargeting of a mutant Gag to the Golgi apparatus alters the pattern of surface expression of Env.

The binding of Src to phospholipid membranes requires both hydrophobic insertion of its myristate into the hydrocarbon interior of the membrane and nonspecific electrostatic interaction of its N-terminal cluster of basic residues with acidic phospholipids. We provide a theoretical description of the electrostatic partitioning of Src onto phospholipid membranes. Specifically, we use molecular models to represent a nonmyristoylated peptide corresponding to residues 2-19 of Src [nonmyr-Src(2-19); GSSKSKPKDPSQRRRSLE-NH2] and a phospholipid bilayer, calculate the electrostatic interaction by solving the nonlinear Poisson-Boltzmann equation, and predict the molar partition coefficient using statistical thermodynamics. The theoretical predictions agree with experimental data obtained by measuring the partitioning of nonmyr-Src(2-19) onto phospholipid vesicles: membrane binding increases as the mole percent of acidic lipid in the vesicles is increased, the ionic strength of the solution is decreased, or the net positive charge of the peptide is increased. The theoretical model also correctly predicts the measured partitioning of the myristoylated peptide, myr-Src(2-19); for example, adding 33% acidic lipid to electrically neutral vesicles increases the partitioning of myr-Src(2-19) 100-fold. Phosphorylating either serine 12 (by protein kinase C) or serine 17 (by cAMP-dependent protein kinase) decreases the partitioning of myr-Src(2-19) onto vesicles containing acidic lipid 10-fold. We investigated the effect of phosphorylation on the localization of Src to biological membranes by expressing fusion constructs of Src's N terminus with a soluble carrier protein in COS-1 cells; phosphorylation produces a small shift in the distribution of the Src chimeras from the plasma membrane to the cytosol.