Synthesis of the palmitoylated and prenylated C-terminal lipopeptides of the human R- and N-Ras proteins.

For the study of biological phenomena influenced by the R- and N-Ras proteins, characteristic peptides which embody the correct lipid modifications of their parent proteins (palmitoyl thioesters, geranylgeranyl thioethers, and farnesyl thioethers), as well as analogues thereof, may serve as efficient tools. For the construction of such acid- and base labile peptide conjugates the allyl ester was developed as C-terminal protecting group. Allyl esters are cleaved selectively and in high yields from lipidated peptides by Pd(0)-mediated allyl transfer to accepting N- or C-nucleophiles like morpholine and N,N'-dimethylbarbituric acid. This protecting group technique formed the key step in the synthesis of the characteristic S-palmitoylated and S-isoprenylated C-terminus of human R-Ras and human N-Ras proteins, as well as several analogues thereof. Deprotections are so mild that no undesired side reactions of the lipid conjugates are observed.     

Proteomic identification of palmitoylated proteins

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.     

Plasma membrane localization of palmitoylated tubulin

PC12 pheochromocytoma cells incorporate [(3)H]palmitic acid into tubulin in a time- and cell-density-dependent manner. The plasma membrane-enriched fraction contains most of the radioactivity of the membrane pellet. While palmitoylated tubulin is found in both the cytoplasm and particulate fraction, the bulk of [(3)H]palmitic acid bound to tubulin is present in the crude membrane pellet and the tubulin extracted from the plasma membrane is more heavily palmitoylated than that extracted from endoplasmic reticulum. Detergent-extracted tubulin from plasma membrane is, to a large extent, polymerization competent; a substantial fraction, increasing as a function of labeling time, is not hydroxylamine-labile. The requirement for detergent extraction, the accompanying changes in tubulin properties and the present findings of preferential incorporation of labeled tubulin into plasma membranes, make it clear that direct incorporation of tubulin into the plasma membrane can occur.     

Fluorescence-based methods to image palmitoylated proteins

A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.     

Novel structural tree of β-proteins containing abcd units

A database of 528 β-proteins and β-domains containing abcd units, including 244 nonhomologous ones, was compiled from the Protein Data Bank (total 1511 PDB entries). A novel structural tree of this class of proteins was constructed to include 153 possible polypeptide chain folds. A structural classification of β-proteins containing abcd units was proposed on the basis of the tree. The database and the structural tree are available at http://strees.protres.ru/.     

Membrane palmitoylated proteins regulate trafficking and processing of nectins

Nectins are cell-cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell-cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell-cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell-cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell-cell junctions and that these associations may regulate trafficking and processing of nectins.     

RGS7 is palmitoylated and exists as biochemically distinct forms

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners Galpha and Gbeta5 are enriched in brain, but biochemical mechanisms governing RGS7/Galpha/Gbeta5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane-bound fractions (salt-extractable, detergent-extractable, and detergent-insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and Gbeta5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane-bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. Gbeta5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane-derived) forms of RGS7, when complexed with Gbeta5, are equally effective stimulators of Galpha(o) GTPase activity, suggesting that palmitoylation does not prevent RGS7/Galpha(o) interactions. The isolated core RGS domain of RGS7 selectively binds activated Galpha(i/o) in brain extracts and is an effective stimulator of both Galpha(o) and Galpha(i1) GTPase activities in vitro. In contrast, the RGS7/Gbeta5 complex selectively interacts with Galpha(o) only, suggesting that features outside the RGS domain and/or Gbeta5 association dictate RGS7-Galpha interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated.     

SNAP-23 and SNAP-25 are palmitoylated in vivo.

The neuronal presynaptic membrane t-SNARE complex consists of the transmembrane protein syntaxin with the palmitoylated protein SNAP-25. In non-neuronal tissues, SNAP-23 replaces SNAP-25 in the t-SNARE complex, although the mechanism of membrane anchoring of SNAP-23 has not been determined. We now report that like SNAP-25, SNAP-23 is palmitoylated in vivo on one or more cysteine residues present in a central "palmitoylation domain." Interestingly, SNAP-23 is palmitoylated less well than SNAP-25, and in vivo binding studies indicate a correlation between the extent of palmitoylation and the ability of SNAP-23 or SNAP-25 to bind to syntaxin in vivo.     

Membrane targeting of palmitoylated Wnt and Hedgehog revealed by chemical probes

Palmitoylation of the Wnt and Hedgehog proteins is critical for maintaining their physiological functions. To date, there are no reported studies that characterize the cellular distribution of the palmitoylated forms of these proteins. Here, we describe the subcellular localization of palmitoylated Wnt and Sonic Hedgehog by using a highly sensitive and non-radioactive labeling method that utilizes alkynyl palmitic acid. We show that palmitoylated Wnt and Sonic Hedgehog localize to cellular membrane fractions only, highlighting a role for palmitoylation in the membrane association of these proteins. The method described herein has the utility to validate inhibitors of Wnt and Hedgehog acyltransferases in drug discovery, and enables further investigations of the role of palmitoylation in the secretion and signaling of these proteins.     

Membrane association and conformational change of palmitoylated G(o)alpha

Bovine brain G(o)alpha was specifically palmitoylated in vitro. The apparent dissociation constant for depalmitoylated G(o)alpha (dG(o)alpha) was 0.273 microM, while that for palmitoylated G(o)alpha (pG(o)alpha) was 5.77 nM. The dissociation rate constant (K(21)) and dissociation half-life for dG(o)alpha were 8.4x10(-4) min and 825 min respectively, while no significant dissociation of pG(o)alpha was detected. The limiting membrane insertion pressures for pG(o)alpha and dG(o)alpha were 44.4 mN/m and 41.3 mN/m respectively. These data suggested that palmitoylation facilitated the membrane association of G(o)alpha. Conformational changes of dG(o)alpha and pG(o)alpha detected by monitoring fluorescence spectra and fluorescence quenching were significantly different after they were associated with the membrane. It was suggested that conformational changes of G(o)alpha upon membrane association might be related to regulation of G(o)alpha signaling by palmitoylation.     

Purification and immunochemical characterization of the two adrenal cortex mitochondrial cytochrome P-450-proteins.

A procedure for the separation and purification of two distinct P-450 cytochromes, termed P-450_scc and P-450_11β, solubilized from bovine adrenal cortex mitochondria is reported. Important features of the purification procedure are uses of aniline-substituted Sepharose chromatography and utilization of the markedly different characteristics in stability and solubility of each cytochrome. Polyacrylamide gel electrophoresis of the two purified preparations in sodium dodecyl sulfate reveals single protein bands. The P-450_scc, which catalyzes the formation of pregnenolone from cholesterol, has a turnover number of 16 mol of pregnenolone formed per minute per mole of P-450-heme. The P-450_11β catalyzes the hydroxylation of deoxycorticosterone at 11β- and 18-positions with turnover numbers of 110 and 18, and of 4-androstene-3,17-dione at 11β- and 19-positions with turnover numbers of 41 and 12, respectively. The recoveries of the P-450_scc and P-450_11β, in terms of the catalytic activities, are 20% and 15%, respectively, from the crude extract which contains the two activities in a ratio of roughly 2:1. Each rabbit antibody prepared against the two purified P-450-proteins interacts with respective, but not alternative cytochrome P-450, whether in the crude mitochondrial preparation or in the purified preparation. The observed patterns of immunoprecipitation and inhibition of catalytic activity indicated that the two P-450 proteins are immunochemically different from each other. Neither antibody immunoprecipitates with a highly purified bacterial cytochrome P-450, P-450_cam.     

Surfactant protein C precursor is palmitoylated and associates with subcellular membranes.

Surfactant protein C (SP-C) is a 3.7 kDa, hydrophobic protein that enhances the adsorption of phospholipids in pulmonary surfactant. SP-C is generated by proteolytic processing of a 21 kDa precursor. Murine fetal lung explant cultures and a Chinese hamster ovary cell line expressing recombinant human SP-C gene (CHO/SPC) were used to determine the subcellular location and post-translational modification(s) of proSP-C. After in vitro translation, proSP-C of Mr = 21,000 was generated. ProSP-C was associated with canine pancreatic microsomes during co-translation and was partially protected from digestion with proteinase K, supporting the concept that proSP-C enters but does not completely traverse the membrane of the endoplasmic reticulum (ER). Association of proSP-C isoforms of 21 and 26 kDa with intracellular membranes was demonstrated by subcellular fractionation of CHO/SPC cells. Pulse/chase experiments demonstrated that the 21 kDa SP-C proprotein was synthesized first and after 15 min was modified to produce a 26 kDa isoform in CHO/SPC cells or a 24 kDa isoform in murine fetal lung. Both the 21 and 26 kDa proSP-C isoforms were detected after labelling CHO/SPC cells with [3H]palmitic acid. The formation of the 26 kDa proSP-C isoform in CHO/SPC cells and the 24 kDa proSP-C isoform in murine fetal lung was blocked by cerulenin, an inhibitor of fatty acid synthesis. In conclusion, proSP-C is associated with subcellular membranes. ProSP-C is palmitoylated and undergoes additional post-translational modification that is blocked by an inhibitor of fatty acid synthesis.     

Galpha(s) is palmitoylated at the N-terminal glycine

Covalent lipid attachments are essential co- and post-translational modifications for signalling proteins. Galpha(s), the alpha-subunit of the heterotrimeric G protein that activates adenylyl cyclase, is known to be palmitoylated at the third N-terminal amino acid, a cysteine. Palmitoylation is involved in anchoring Galpha(s) to the membrane by increasing its intrinsic hydrophobicity. We identified by mass spectrometry a second, functionally even more important, covalent modification. It consists of another palmitoyl residue attached to the preceding glycine (Gly(2)). Palmitoylation at this position has profound consequences for levels of signal transduction. It sensitizes the cell up to 200-fold for adenylyl cyclase-stimulating agents. The inhibitory inputs mediated by Galpha(i) are downregulated to <10%. Thereby, Gly(2)-palmitoylation of Galpha(s) relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via Galpha(i) more difficult.     

Establishment of LC-MS methods for the analysis of palmitoylated surfactant proteins

The surfactant proteins (SPs), SP-B and SP-C, are important components of pulmonary surfactant involved in the reduction of alveolar surface tension. Quantification of SP-B and SP-C in surfactant drugs is informative for their quality control and the evaluation of their biological activity. Western blot analysis enabled the quantification of SP-B, but not SP-C, in surfactant drugs. Here, we report a new procedure involving chemical treatments and LC-MS to analyze SP-C peptides. The procedure enabled qualitative analysis of SP-C from different species with discrimination of the palmitoylation status and the artificial modifications that occur during handling and/or storage. In addition, the method can be used to estimate the total amount of SP-C in pulmonary surfactant drugs. The strategy described here might serve as a prototype to establish analytical methods for peptides that are extremely hydrophobic and behave like lipids. The new method provides an easy measurement of SP-C from various biological samples, which will help the characterization of various experimental animal models and the quality control of surfactant drugs, as well as diagnostics of human samples.     

All ras proteins are polyisoprenylated but only some are palmitoylated

The C-terminal CAAX motif of the yeast mating factors is modified by proteolysis to remove the three terminal amino acids (-AAX) leaving a C-terminal cysteine residue that is polyisoprenylated and carboxyl-methylated. Here we show that all ras proteins are polyisoprenylated on their C-terminal cysteine (Cys186). Mutational analysis shows palmitoylation does not take place on Cys186 as previously thought but on cysteine residues contained in the hypervariable domain of some ras proteins. The major expressed form of c-K-ras (exon 4B) does not have a cysteine residue immediately upstream of Cys186 and is not palmitoylated. Polyisoprenylated but nonpalmitoylated H-ras proteins are biologically active and associate weakly with cell membranes. Palmitoylation increases the avidity of this binding and enhances their transforming activity. Polyisoprenylation is essential for biological activity as inhibiting the biosynthesis of polyisoprenoids abolishes membrane association of p21ras.     

Somatostatin receptor 5 is palmitoylated by the interacting ZDHHC5 palmitoyltransferase

Many G-protein coupled receptors are palmitoylated in their C-terminal, intracellular regions. So far no enzymes responsible for this modification have been described. We identified an interaction of the membrane proximal helix 8 of somatostatin receptor 5 (SSTR5) with the N-terminal region of the putative palmitoyltransferase ZDHHC5 using the Ras recruitment interaction screening system. ZDHHC5 and SSTR5 are colocalized at the plasma membrane and can be efficiently coimmunoprecipitated from transfected cells. Coexpression of ZDHHC5 in HEK293 cells increased palmitoylation of SSTR5 whereas knock-down of endogenous ZDHHC5 by siRNAs decreased it. Our data identify the first palmitoyltransferase for a G-protein coupled receptor.     

Intracellular trafficking of a palmitoylated membrane-associated protein component of enveloped vaccinia virus

The F13L protein of vaccinia virus, an essential and abundant palmitoylated peripheral membrane component of intra- and extracellular enveloped virions, associates with Golgi, endosomal, and plasma membranes in the presence or absence of other viral proteins. In the present study, the trafficking of a fully functional F13L-green fluorescent protein (GFP) chimera in transfected and productively infected cells was analyzed using specific markers and inhibitors. We found that Sar1(H79G), a trans-dominant-negative protein inhibitor of cargo transport from the endoplasmic reticulum, had no apparent effect on the intracellular distribution of F13L-GFP, suggesting that the initial membrane localization occurs at a downstream compartment of the secretory pathway. Recycling of F13L-GFP from the plasma membrane was demonstrated by partial colocalization with FM4-64, a fluorescent membrane marker of endocytosis. Punctate F13L-GFP fluorescence overlapped with clathrin and Texas red-conjugated transferrin, suggesting that endocytosis occurred via clathrin-coated pits. The inhibitory effects of chlorpromazine and trans-dominant-negative forms of dynamin and Eps15 protein on the recycling of F13L-GFP provided further evidence for clathrin-mediated endocytosis. In addition, the F13L protein was specifically coimmunoprecipitated with alpha-adaptin, a component of the AP-2 complex that interacts with Eps15. Nocodazole and wortmannin perturbed the intracellular trafficking of F13L-GFP, consistent with its entry into late and early endosomes through the secretory and endocytic pathways, respectively. The recycling pathway described here provides a mechanism for the reutilization of the F13L protein following its deposition in the plasma membrane during the exocytosis of enveloped virions.     

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-A resolution. BET3 adopts an alpha/beta-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein-protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.