The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo.
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Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules. (+info)
S-myristoylation of a glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei.
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Covalent modification with lipid can target cytosolic proteins to biological membranes. With intrinsic membrane proteins, the role of acylation can be elusive. Herein, we describe covalent lipid modification of an integral membrane glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from the kinetoplastid Trypanosoma brucei. Myristic acid was detected on cysteine residue(s) (i.e. thiomyristoylation). Thiomyristoylation occurred both co- and post-translationally. Acylated GPI-PLC was active against variant surface glycoprotein (VSG). The half-life of fatty acid on GPI-PLC was 45 min, signifying the dynamic nature of the modification. Deacylation in vitro decreased activity of GPI-PLC 18-30-fold. Thioacylation, from kinetic analysis, activated GPI-PLC by accelerating the conversion of a GPI-PLC.VSG complex to product. Reversible thioacylation is a novel mechanism for regulating the activity of a phospholipase C. (+info)
SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells.
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Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effective treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and [3H]palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833) activates ceramide synthesis. In a short time course experiment, ceramide was generated as early as 15 min (40% increase) after the addition of PSC 833 (5.0 microM), and by 3 h, [3H]ceramide was >3-fold that of control cells. A 24-h dose-response experiment showed that at 1.0 and 10 microM PSC 833, ceramide levels were 2.5- and 13.6-fold higher, respectively, than in untreated cells. Concomitant with the increase in cellular ceramide was a progressive decrease in cell survival, suggesting that ceramide elicited a cytotoxic response. Analysis of DNA in cells treated with PSC 833 showed oligonucleosomal DNA fragmentation, characteristic of apoptosis. The inclusion of fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Assessment of ceramide mass by TLC lipid charring confirmed that PSC 833 markedly enhanced ceramide synthesis, not only in KB-V-1 cells but also in wild-type KB-3-1 cells. The capacity of PSC 833 to reverse drug resistance was demonstrated with vinblastine. Whereas each agent at a concentration of 1.0 microM reduced cell survival by approximately 20%, when PSC 833 and vinblastine were coadministered, cell viability fell to zero. In parallel experiments measuring ceramide metabolism, it was shown that the PSC 833/vinblastine combination synergistically increased cellular ceramide levels. Vinblastine toxicity, also intensified by PSC 833 in wild-type KB-3-1 cells, was as well accompanied by enhanced ceramide formation. These data demonstrate that PSC 833 has mechanisms of action in addition to P-glycoprotein chemotherapy efflux pumping. (+info)
A lipid modified ubiquitin is packaged into particles of several enveloped viruses.
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An anti-ubiquitin cross-reactive protein which migrates more slowly (6.5 kDa) by SDS-PAGE than ubiquitin was identified in African swine fever virus particles. This protein was extracted into the detergent phase in Triton X-114 phase separations, showing that it is hydrophobic, and was radiolabelled with both [3H]palmitic acid and [32P]orthophosphate. This indicates that the protein has a similar structure to the membrane associated phosphatidyl ubiquitin described in baculovirus particles. A similar molecule was found in vaccinia virus and herpes simplex virus particles, suggesting that it may be a component of uninfected cell membranes, which is incorporated into membrane layers in virions during morphogenesis. (+info)
Epidermal growth factor regulates fatty acid uptake and metabolism in Caco-2 cells.
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Epidermal growth factor (EGF) has been reported to stimulate carbohydrate, amino acid, and electrolyte transport in the small intestine, but its effects on lipid transport are poorly documented. This study aimed to investigate EGF effects on fatty acid uptake and esterification in a human enterocyte cell line (Caco-2). EGF inhibited cell uptake of [14C]palmitate and markedly reduced its incorporation into triglycerides. In contrast, the incorporation in phospholipids was enhanced. To elucidate the mechanisms involved, key steps of lipid synthesis were investigated. The amount of intestinal fatty acid-binding protein (I-FABP), which is thought to be important for fatty acid absorption, and the activity of diacylglycerol acyltransferase (DGAT), an enzyme at the branch point of diacylglycerol utilization, were reduced. EGF effects on DGAT and on palmitate esterification occurred at 2-10 ng/ml, whereas effects on I-FABP and palmitate uptake occurred only at 10 ng/ml. This suggests that EGF inhibited palmitate uptake by reducing the I-FABP level and shifted its utilization from triglycerides to phospholipids by inhibiting DGAT. This increase in phospholipid synthesis might play a role in the restoration of enterocyte absorption function after intestinal mucosa injury. (+info)
Comparisons of flux control exerted by mitochondrial outer-membrane carnitine palmitoyltransferase over ketogenesis in hepatocytes and mitochondria isolated from suckling or adult rats.
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The primary aim of this paper was to calculate and report flux control coefficients for mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I) over hepatic ketogenesis because its role in controlling this pathway during the neonatal period is of academic importance and immediate clinical relevance. Using hepatocytes isolated from suckling rats as our model system, we measured CPT I activity and carbon flux from palmitate to ketone bodies and to CO2 in the absence and presence of a range of concentrations of etomoxir. (This is converted in situ to etomoxir-CoA which is a specific inhibitor of the enzyme.) From these data we calculated the individual flux control coefficients for CPT I over ketogenesis, CO2 production and total carbon flux (0.51 +/- 0.03; -1.30 +/- 0.26; 0.55 +/- 0.07, respectively) and compared them with equivalent coefficients calculated by similar analyses [Drynan, L., Quant, P.A. & Zammit, V.A. (1996) Biochem. J. 317, 791-795] in hepatocytes isolated from adult rats (0.85 +/- 0.20; 0.23 +/- 0.06; 1.06 +/- 0.29). CPT I exerts significantly less control over ketogenesis in hepatocytes isolated from suckling rats than those from adult rats. In the suckling systems the flux control coefficients for CPT I over ketogenesis specifically and over total carbon flux (< 0.6) are not consistent with the enzyme being rate-limiting. Broadly similar results were obtained and conclusions drawn by reanalysis of previous data {from experiments in mitochondria isolated from suckling or adult rats [Krauss, S., Lascelles, C.V., Zammit, V.A. & Quant, P.A. (1996) Biochem. J. 319, 427-433]} using a different approach of control analysis, although it is not strictly valid to compare flux control coefficients from different systems. Our overall conclusion is that flux control coefficients for CPT I over oxidative fluxes from palmitate (or palmitoyl-CoA) differ markedly according to (a) the metabolic state, (b) the stage of development, (c) the specific pathway studied and (d) the model system. (+info)
Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs.
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During synaptic development, proteins aggregate at specialized pre- and postsynaptic structures. Mechanisms that mediate protein clustering at these sites remain unknown. To investigate this process, we analyzed synaptic targeting of a postsynaptic density protein, PSD-95, by expressing green fluorescent protein- (GFP-) tagged PSD-95 in cultured hippocampal neurons. We find that postsynaptic clustering relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. In contrast, disruptions of PDZ3, SH3, or guanylate kinase (GK) domains do not affect synaptic targeting. Palmitoylation is sufficient to target the diffusely expressed SAP-97 to synapses, and palmitoylation cannot be replaced with alternative membrane association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic clustering. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting. (+info)
Muscle contractile activity increases fatty acid metabolism and transport and FAT/CD36.
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We have examined whether 1) fatty acid (FA) uptake, 2) FA transporter expression, and 3) FA metabolism are increased when the oxidative capacity of skeletal muscle is increased. The oxidative capacities of red and white tibialis anterior and extensor digitorum longus muscles were increased via chronic stimulation (10 Hz, 24 h/day for 7 days). The contralateral muscles served as controls. After 7 days of increased muscle activity 1) palmitate uptake by giant sarcolemmal vesicles was increased twofold (P < 0.05), 2) the expression of FA translocase (FAT)/CD36 was increased at both the mRNA (3.2- to 10-fold) and protein (3.4-fold) levels, and 3) palmitate oxidation and esterification into triacylglycerols and phospholipids were increased 1.5-, 2.7-, and 1.7-fold, respectively (P < 0.05). These data show that when the oxidative capacity of muscle is increased, there is a parallel increase in the rate of FA transport and FA transporters at the sarcolemmal membrane, which is associated with the enhanced expression of the membrane transporter FAT/CD36. (+info)