(1/750) S-myristoylation of a glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei.

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)

(2/750) Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus.

Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55(Gag), to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55(Gag), a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.  (+info)

(3/750) Flagellar protein localization mediated by a calcium-myristoyl/palmitoyl switch mechanism.

The mechanisms by which proteins are targeted to flagella and cilia are poorly understood. We set out to determine the basis for the specific localization of a 24 kDa flagellar calcium-binding protein (FCaBP) expressed in all life cycle stages of Trypanosoma cruzi. Through the study of trypanosome transfectants expressing various FCaBP deletion mutants, we found that the N-terminal 24 amino acids of the protein are necessary and sufficient for flagellar localization. Subsequent experiments revealed that FCaBP is myristoylated and palmitoylated and, in fact, is one of very few proteins in the cell possessing these acyl modifications. Both fatty acids are required for flagellar localization, suggesting that FCaBP localization may be mediated through association with the flagellar plasma membrane. Indeed, FCaBP associates with the flagellar membrane in a calcium-dependent manner, reminiscent of the recoverin family of calcium-myristoyl switch proteins. Thus, FCaBP is a novel member of the calcium-acyl switch protein family and is the only member described to date that requires two fatty acid modifications for specific membrane association. Its unique localization mechanism is the first described for any flagellar protein. The existence of such a protein in this protozoan suggests that acylation and calcium switch mechanisms for regulated membrane association are conserved among eukaryotes.  (+info)

(4/750) Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction.

Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser5) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.  (+info)

(5/750) Dual fatty acylation of p59(Fyn) is required for association with the T cell receptor zeta chain through phosphotyrosine-Src homology domain-2 interactions.

The first 10 residues within the Src homology domain (SH)-4 domain of the Src family kinase Fyn are required for binding to the immune receptor tyrosine-based activation motif (ITAM) of T cell receptor (TCR) subunits. Recently, mutation of glycine 2, cysteine 3, and lysines 7 and 9 was shown to block binding of Fyn to TCR zeta chain ITAMs, prompting the designation of these residues as an ITAM recognition motif (Gauen, L.K.T., M.E. Linder, and A.S. Shaw. 1996. J. Cell Biol. 133:1007-1015). Here we show that these residues do not mediate direct interactions with TCR ITAMs, but rather are required for efficient myristoylation and palmitoylation of Fyn. Specifically, coexpression of a K7,9A-Fyn mutant with N-myristoyltransferase restored myristoylation, membrane binding, and association with the cytoplasmic tail of TCR zeta fused to CD8. Conversely, treatment of cells with 2-hydroxymyristate, a myristoylation inhibitor, blocked association of wild-type Fyn with zeta. The Fyn NH2 terminus was necessary but not sufficient for interaction with zeta and both Fyn kinase and SH2 domains were required, directing phosphorylation of zeta ITAM tyrosines and binding to zeta ITAM phosphotyrosines. Fyn/zeta interaction was sensitive to octylglucoside and filipin, agents that disrupt membrane rafts. Moreover, a plasma membrane bound, farnesylated Fyn construct, G2A,C3S-FynKRas, was not enriched in the detergent insoluble fraction and did not associate with zeta. We conclude that the Fyn SH4 domain provides the signals for fatty acylation and specific plasma membrane localization, stabilizing the interactions between the Fyn SH2 domain and phosphotyrosines in TCR zeta chain ITAMs.  (+info)

(6/750) Gbetagamma and palmitate target newly synthesized Galphaz to the plasma membrane.

The subcellular location of a signaling protein determines its ability to transmit messages accurately and efficiently. Three different lipid modifications tether heterotrimeric G proteins to membranes: alpha subunits are myristoylated and/or palmitoylated, and gamma subunits are prenylated. In a previous study, we examined the role of lipid modifications in maintaining the membrane attachment of a G protein alpha subunit, alphaz, which is myristoylated and palmitoylated (Morales, J., Fishburn, C. S., Wilson, P. T., and Bourne, H. R. (1998) Mol. Biol. Cell 9, 1-14). Now we extend this analysis by characterizing the mechanisms that target newly synthesized alphaz to the plasma membrane (PM) and analyze the role of lipid modifications in this process. In comparison with newly synthesized alphas, which is palmitoylated but not myristoylated, alphaz moves more rapidly to the membrane fraction following synthesis in the cytosol. Newly synthesized alphaz associates randomly with cellular membranes, but with time accumulates at the PM. Palmitoylated alphaz is present only in PM-enriched fractions, whereas a nonpalmitoylated mutant of alphaz (alphazC3A) associates less stably with the PM than does wild-type alphaz. Expression of a C-terminal fragment of the beta-adrenoreceptor kinase, which sequesters free betagamma, impairs association of both alphaz and alphazC3A with the PM, suggesting that the alpha subunit must bind betagamma in order to localize at the PM. Based on these findings, we propose a model in which, following synthesis on soluble ribosomes, myristoylated alphaz associates randomly and reversibly with membranes; upon association with the PM, alphaz binds betagamma, which promotes its palmitoylation, thus securing it in the proper place for transmitting the hormonal signal.  (+info)

(7/750) ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I.

The small GTP-binding protein, ADP-ribosylation factor 1 (ARF1) is essential for the formation of coatomer-coated vesicles from the Golgi and is also an activator of phospholipase D (PLD). Moreover, ARF1-regulated PLD is part of the signal-transduction pathway that can lead to secretion. In this study, substitution and deletion mutants of ARF1 were tested for their ability to activate PLD. These map the PLD effector region of ARF1 to the alpha2 helix, part of the beta2-strand and the N-terminal helix and its ensuing loop. ARF mutants with an increased or decreased ability to activate PLD showed similar characteristics when tested for their ability to stimulate secretion from HL60 cells. ARF1, deleted of the N-terminal 17 amino acid residues (Ndel17), did not support PLD activity or secretion, and neither did it inhibit the activity of wild-type myristoylated ARF1 (myrARF1). In contrast, Ndel17 effectively competed with wild-type myrARF1 to prevent coatomer binding to membranes. This appears to define a structural role for Ndel17, as it can bind a high-molecular mass complex in cytosol. In addition, ethanol has no effect on recruitment of coatomer to membrane. We conclude that the function of ARF-regulated PLD is in the signal-transduction pathway leading to secretion of lysosomal granules, and not as an essential component of ARF1-mediated coatomer binding.  (+info)

(8/750) Augmentation of Ca2+-stimulated insulin release by glucose and long-chain fatty acids in rat pancreatic islets: free fatty acids mimic ATP-sensitive K+ channel-independent insulinotropic action of glucose.

Glucose augments Ca2+-stimulated insulin release from the pancreatic beta-cell in an ATP-sensitive K+ channel (K(ATP) channel)-independent manner. In studying the mechanisms underlying this action, we used rat pancreatic islets and examined the effects of exogenous free fatty acids (FFAs), which are precursors of long-chain acyl-CoA (LC-CoA), on KCl-induced Ca2+-stimulated insulin release. Myristate, palmitate, and stearate augmented insulin release induced by 50 mmol/l KCl in the presence of 2.8 mmol/l glucose. Added acutely, their potency was weak compared with that of glucose-induced augmentation. The FFA-induced augmentation became much greater, however, when islets were preincubated with FFAs under stringent Ca2+-free conditions (with 1 mmol/l EGTA) before the KCl stimulation. Under these conditions, 16.7 mmol/l glucose augmented 13-fold insulin release induced by 50 mmol/l KCl, whereas palmitate or myristate (both at a free concentration of 10 micromol/l) produced 5.8- and 5.2-fold augmentations. Effects of FFAs and glucose were concentration-dependent. The temporal profiles of augmentation induced by 11.1 mmol/l glucose and 10 micromol/l palmitate were similar. Glucose and palmitate caused almost identical augmentation patterns for the initial 10 min of stimulation; subsequently, glucose augmentation was better sustained than palmitate augmentation. This suggests the existence of a longer-term glucose-specific signaling moiety that cannot be mimicked by FFAs. Our results provide direct evidence that FFAs can mimic the K(ATP) channel-independent action of glucose. Taking these results together with previous results, we conclude that glucose augments Ca2+-stimulated insulin release, at least in part, by increasing malonyl-CoA and cytosolic LC-CoA. However, one or more other glucose-specific signaling molecules are required for the full expression of augmentation.  (+info)