Oligomerization of VIP21-caveolin in vitro is stabilized by long chain fatty acylation or cholesterol.

VIP21-caveolin is one of the components which form the cytoplasmic surface of caveolae. In vivo, this integral membrane protein is found in homo-oligomers with molecular masses of approximately 200, 400 and 600 kDa. These oligomers are also formed by the addition of cytosol to the in vitro synthesized and membrane inserted VIP21-caveolin. Here we show that long chain fatty acyl coenzyme A esters can completely substitute for cytosol in inducing 200 kDa and 400 kDa complexes, whereas 25-hydroxy-cholesterol can produce the 200 kDa oligomer. In order to understand whether acylation of VIP21-caveolin itself is a prerequisite for oligomerization, we studied a mutant protein lacking all three cysteines. When analyzed by velocity sucrose gradient centrifugation in the presence of the non-ionic detergent octylglucoside, both palmitoylated and non-palmitoylated VIP21-caveolin formed oligomers that were indistinguishable. However, only the oligomers of the non-palmitoylated protein are disrupted when analyzed by SDS-PAGE without boiling. These data suggest that the protein domains of VIP21-caveolin are the primary determinants of oligomerization, but that palmitoylation of cysteine residues can increase the stability of the oligomers.

Caveolin is palmitoylated on multiple cysteine residues. Palmitoylation is not necessary for localization of caveolin to caveolae.

Caveolae are subdomains of the plasma membrane which concentrate cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-linked proteins. It has recently been demonstrated that specific members of the Src family of protein tyrosine kinases require palmitoylation of NH2-terminal cysteine residues to localize in caveolae. Here we report that caveolin, an integral membrane protein which forms part of the coat of caveolae, also incorporates palmitate through linkage to cysteine residues. Caveolin contains only three cysteine residues which are all located on the COOH-terminal side of the hydrophobic transmembrane region. Immunofluorescent staining of cells transfected with caveolin indicated that, like the NH2 terminus, this COOH-terminal region is located on the cytoplasmic side of the plasma membrane. Studies of cysteine substitution mutants showed that all three cysteines are capable of incorporating palmitate and that the juxtamembrane Cys133 residue is the predominant site of palmitoylation. Simultaneous mutation of all three cysteine residues in caveolin resulted in the loss of ability to incorporate palmitate; however, this did not affect localization of the protein. Thus, palmitoylation of cysteine residues in nonmembrane spanning Src family protein tyrosine kinases has different consequences than in the transmembrane protein caveolin.

Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix.

The negative factor, Nef, of HIV-1 was found to associate to an extent of 16-42% with the detergent insoluble cytoskeletal fraction of T lymphocytes. Furthermore, Escherichia coli expressed Nef protein was found to bind during in vitro reactions with the cytoskeletal matrix to an extent of 30-50%. Cytoskeletal association of Nef was significantly enhanced by myristoylation. The specificity of the myristoylation-enhanced binding was demonstrated by the lack of an effect of myristoylation on binding of the HIV-1 Gag protein to the cytoskeleton. Cytoskeletal binding was saturable, and inhibited by high concentrations of sodium chloride, or with SDS or urea. Binding of Nef to the cytoskeletal matrix may be important in mediating its effects on HIV-1 replication.

HIV-1 Nef protein inhibits the recruitment of AP-1 DNA-binding activity in human T-cells.

The human immunodeficiency virus type 1 long terminal repeat, HIV-1-LTR, contains binding sites for several cellular transcription factors which contribute to HIV-1 gene expression. Our previous studies on the function of the HIV-1-encoded Nef protein suggested that Nef may be an inhibitor HIV-1 transcription. To determine whether Nef affects the binding of cellular factors implicated in HIV-1 regulation, 32P-labeled oligonucleotides corresponding to the binding sites were incubated with nuclear extracts prepared from Nef-expressing T-cell lines that were not stimulated or were stimulated with T-cell mitogens. We found that Nef inhibited the recruitment of AP-1 DNA-binding activity in mitogen-stimulated human T-cells. Additionally, Nef expressing cells were transiently transfected with a plasmid in which HIV-1 AP-1 DNA recognition sequences were cloned downstream of the chloramphenicol acetyltransferase (CAT) gene. Mitogen-mediated transcriptional activation of the CAT gene in this construct was inhibited in Nef-expressing cells but not in control cells. These studies suggest that, by inhibiting AP-1 activation, Nef may play a role in regulating HIV-1 gene expression in infected T-cells.

Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells.

Human immunodeficiency virus type 1 (HIV-1) can establish a persistent and latent infection in CD4+ T lymphocytes (W. C. Greene, N. Engl. J. Med. 324:308-317, 1991; S. M. Schnittman, M. C. Psallidopoulos, H. C. Lane, L. Thompson, M. Baseler, F. Massari, C. H. Fox, N. P. Salzman, and A. S. Fauci, Science 245:305-308, 1989). Production of HIV-1 from latently infected cells requires host cell activation by T-cell mitogens (T. Folks, D. M. Powell, M. M. Lightfoote, S. Benn, M. A. Martin, and A. S. Fauci, Science 231:600-602, 1986; D. Zagury, J. Bernard, R. Leonard, R. Cheynier, M. Feldman, P. S. Sarin, and R. C. Gallo, Science 231:850-853, 1986). This activation is mediated by the host transcription factor NF-kappa B [G. Nabel and D. Baltimore, Nature (London) 326:711-717, 1987]. We report here that the HIV-1-encoded Nef protein inhibits the induction of NF-kappa B DNA-binding activity by T-cell mitogens. However, Nef does not affect the DNA-binding activity of other transcription factors implicated in HIV-1 regulation, including SP-1, USF, URS, and NF-AT. Additionally, Nef inhibits the induction of HIV-1- and interleukin 2-directed gene expression, and the effect on HIV-1 transcription depends on an intact NF-kappa B-binding site. These results indicate that defective recruitment of NF-kappa B may underlie Nef's negative transcriptional effects on the HIV-1 and interleukin 2 promoters. Further evidence suggests that Nef inhibits NF-kappa B induction by interfering with a signal derived from the T-cell receptor complex.