Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(alpha, beta)-methylene-diphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.     

Chemical identification of cysteine as palmitoylation site in a transmembrane protein (Semliki Forest virus E1)

The palmitoylation site of the membrane glycoprotein E1 of Semliki Forest virus (SFV) has been identified by chemical analysis of an acylpeptide. 3H-Palmitoylated E1 isolated from SFV grown in baby hamster kidney cells was digested with chymotrypsin and the resulting peptides subjected to high performance liquid chromatography on a wide-pore column. The 3H-acylated peptide fraction peaked at above 60% 2-propanol in the eluent, indicating its hydrophobic character. Polyacrylamide gel electrophoresis analysis revealed a molecular weight of about Mr = 6000 for the radiolabeled peptide. Manual sequencing of this material by the 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate procedure on solid phase revealed the amino-terminal sequence Ala-Ala-Ser-His-Ser-Asn-Val-Val-Phe-Pro. The same peptide also labels with [35S]cysteine. Comparison with the deduced amino acid sequence of E1 revealed that the palmitoylated peptide contains at least 43 amino acid residues, and thus includes the membrane spanning region down to the only cysteine residue five positions up from the carboxyl terminus of E1. Since [3H]palmitic acid was cleaved from E1 with thiol reagents, and since the peptide labels with [14C]iodoacetamide only after the release of fatty acids by hydroxylamine treatment, cysteine in position 433 represents the palmitoylation site in SFV E1.     

Alkylation of cysteine-containing peptides to mimic palmitoylation.

Numerous proteins that are involved in cell signaling and viral replication require post-translational modification by palmitoylation to function properly. The molecular details by which this palmitoyl modification affects protein function remain poorly understood. To facilitate in vitro biochemical and structural studies of the role of palmitoylation on protein function, a method was developed for alkylating peptides with saturated C16 groups at cysteine residues and demonstrated using peptides derived from the palmitoylated region of Sindbis virus E2 glycoprotein. The synthetic approach takes advantage of disulfide chemistry to specifically modify only the cysteine residues within peptides and covalently links C16 groups via disulfide bridges using a new thioalkylating reagent, hexyldexyldithiopyridine. The chemistry presented here takes place in solution under mild conditions without the need for protection of the peptide functional groups. A method for purifying these modified peptides is also described. This protocol can be of general use to investigators studying the role of palmitoylation in biological systems.     

Acyl-biotinyl Exchange Chemistry and Mass Spectrometry-Based Analysis of Palmitoylation Sites of In Vitro Palmitoylated Rat Brain Tubulin

Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on ³H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated.     

Chemical and genetic probes for analysis of protein palmitoylation

Reversible protein palmitoylation is one of the most important posttranslational modifications that has been implicated in the regulation of protein signaling, trafficking, localizing and enzymatic activities in cells and tissues. In order to achieve a precise understanding of mechanisms and functions of protein palmitoylation as well as its roles in physiological processes and disease progression, it is necessary to develop techniques that can qualitatively and quantitatively monitor the dynamic protein palmitoylation in vivo and in vitro. This review will highlight recent advances in both chemical and genetic encoded probes that have been developed for accurate analysis of protein palmitoylation, including identification and quantification of acyl moieties and palmitoylated proteins, localization of amino acid residues on which acyl moieties are attached, and imaging of cellular distributions of palmitoylated proteins. The role of major techniques of fluorescence microscopy and mass spectrometry in facilitating the analysis of protein palmitoylation will also be explored.     

The complete covalent structure of a cardiotoxin from the venom of Naja nigricollis (African black-necked spitting cobra).

The complete covalent structure of a small, basic protein with cardiotoxic activity is described. This has been isolated from the venom of Naja nigricollis by gel filtration on Sephadex G-75 and gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin, molecular weight 6806 from amino acid composition, consists of 60 amino acids, cross-linked by four disulfide bridges, connecting 3-21, 14-38, 42-53, and 54-59. The protein contains one residue of tryptophan, phenylalanine, and glutamic acid, two residues of arginine and tyrosine, four residues of methionine, and nine residues of lysine. Histidine is absent. The chymotryptic peptides of the oxidized and S-carboxymethylated protein were isolated by gel filtration on Sephadex G-25 and zone electrophoresis on a cellulose column. The sequence was determined by Edman degradation, using the (manual) direct phenylthiohydantoin method and with the use of carboxypeptidase A. Disulfide pairing was determined on thermolysin cleaved peptides from the native protein. The sequence is shown to be homologous to other cardiotoxins and a lytic factor from snake venoms and also shows homology, both in sequence and disulfide pairing to neurotoxins. A partial reduction experiment in the absence of denaturing agent using 14-C-labeled iodoacetic acid as S-carboxymethylating agent shows that disulfide bonds 14-38 and 42-53 were reduced fastest followed marginally by 54-59, and then bond 3-21.     

Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence

Purification of C4a from heat-activated bovine plasma by elution from CM-Sephadex C-50 at pH 7.4 and gel filtration on Sephadex G-50 gives a 20% yield of pure C4a. The complete amino acid sequence of bovine C4a has been determined by automatic sequencer degradation of CNBr and enzymic fragments, and by carboxypeptidase digestion. The 77-residue bovine sequence shows 12 differences from the human sequence with five of these differences occurring in the C-terminal 11 residues. The sequence of C4a confirms earlier suggestions of homology with C3a and C5a: the three sequences show an almost equal number of identities with each other. The six cysteine residues of the 'disulphide knot' are conserved as well as seven other residues including the C-terminal arginine.     

Amino acid sequence of a cyanogen bromide fragment containing the two tryptophanyl residues of lobster arginine kinase (Homarus vulgaris).

Lobster arginine kinase [EC 2.7.3.3] contains 2 tryptophanyl residues and 9 methionyl residues. The whole carboxymethylated protein was first subjected to CNBr cleavage and the resulting fragments were isolated by gel filtration and other experimental approaches. One fragment, CB5, which contains 60 residues including the two tryptophanyl residues and two of the five cysteinyl residues of the protein, was characterized and the results are reported inthis paper. The overall strategy for the establishment of the complete sequence of this fragment was based on the use of three types of peptides: (a) whole cyanogen bromide peptide CB5 which was partially characterized by automatic Edman degradation using a sequencer: 42 steps were performed out of 60 residues, (b) tryptic peptides of CB5, (c) peptides formed by cleavage of S-carboxymethylated arginine kinase (whole protein) at the two tryptophanyl residues with BNPS-skatole. The complete amino acid sequence of the CNBr polypeptide (CB5) which contains the two tryptophanyl residues of the whole protein was established.     

Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide

Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly palmitoylated GAP-peptide product was confirmed by mass spectrometry. The GAP-peptide substrate was separated from the palmitoylated peptide product in less than 7 min by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.     

Single site alpha-tubulin mutation affects astral microtubules and nuclear positioning during anaphase in Saccharomyces cerevisiae: possible role for palmitoylation of alpha-tubulin

We generated a strain of Saccharomyces cerevisiae in which the sole source of alpha-tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S alpha-tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15 degrees C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in alpha-tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated alpha-tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated alpha-tubulin in C377S tub1 cells. Our results suggest that cys-377 of alpha-tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle.     

An electrostatic switch controls palmitoylation of the large conductance voltage- and calcium-activated potassium (BK) channel

Protein palmitoylation is a major dynamic posttranslational regulator of protein function. However, mechanisms that control palmitoylation are poorly understood. In many proteins, palmitoylation occurs at cysteine residues juxtaposed to membrane-anchoring domains such as transmembrane helices, sites of irreversible lipid modification, or hydrophobic and/or polybasic domains. In particular, polybasic domains represent an attractive mechanism to dynamically control protein palmitoylation, as the function of these domains can be dramatically influenced by protein phosphorylation. Here we demonstrate that a polybasic domain immediately upstream of palmitoylated cysteine residues within an alternatively spliced insert in the C terminus of the large conductance calcium- and voltage-activated potassium channel is an important determinant of channel palmitoylation and function. Mutation of basic amino acids to acidic residues within the polybasic domain results in inhibition of channel palmitoylation and a significant right-shift in channel half maximal voltage for activation. Importantly, protein kinase A-dependent phosphorylation of a single serine residue within the core of the polybasic domain, which results in channel inhibition, also reduces channel palmitoylation. These data demonstrate the key role of the polybasic domain in controlling stress-regulated exon palmitoylation and suggests that phosphorylation controls the domain by acting as an electrostatic switch.     

The complete amino acid sequence of a cardiotoxin from the venom of Naja naja (Cambodian Cobra).

The complete amino acid sequence of a small, basic protein with cardiotoxic activity is described. This toxin, designated Naja naja F8, was isolated from the venom of Naja naja, of Cambodian origin, by gel filtration on Sephadex G-75 followed by gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin F8, molecular weight 6727 from amino acid composition, consists of 60 amino acids in a single peptide chain cross-linked by four disulfide bridges and is devoid of histidine, tryptophan, and glutamic acid. The chymotryptic and tryptic peptides from the performic acid oxidized toxin were separated by gel filtration on Sephadex G-25 and zone electrophoresis in columns of cellulose powder. The sequence was established by Edman degradation, using the direct phenylthiohydantoin method, and with the aid of carboxypetidase A, and is similar to the consequences reported for other cardiotoxins, cytotoxins, and/or lytic factors from cobra venoms, all of which show considerable homology with the functionally distinct neurotoxins.     

Global profiling of dynamic protein palmitoylation

The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.     

Isolation of immunizing cyanogen bromide-peptides of foot-and-mouth disease virus.

The isolated structural protein with the N-terminal amino acid threonine of foot-and-mouth disease virus, type O₁ strain Kaufbeuren was treated with CNBr and the cleavage peptides were separated by gel filtration on Sephadex G-100 followed by ion exchange chromatography on phosphocellulose. Two peptides with molecular weights of about 5.200 daltons still capable of inducing antibodies in guinea pigs were purified. The antibodies were found to neutralize the homologous foot-and-mouth disease virus as detected by the neutralization test in suckling mice. The findings strongly suggest that the primary structure of small CNBr cleavage peptides carries antigenic determinants similar to those on the native virus protein with the N-terminale amino acid threonine.     

Snake venom toxins. The amino-acid sequences of three toxins (CM-8, CM-11 and CM-13a) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-8, CM-11, and CM-13a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 61 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-11 resemble the corresponding properties of the cytotoxin group. The sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group.     

Differential effect of brefeldin A on the palmitoylation of surfactant protein C proprotein mutants.

The surfactant protein C precursor (proSP-C) is palmitoylated on two cysteines adjacent to its transmembrane domain. We showed previously that palmitoylation of proSP-C occurs in a postendoplasmic reticulum compartment and is not affected by the Golgi-disturbing agent brefeldin A (BFA). In contrast, the investigations presented here showed that BFA almost completely abolished palmitoylation of proSP-C mutants that contained alterations in the region between the palmitoylated cysteines and the transmembrane domain, including a Pro 30 to Leu mutant associated with interstitial lung disease. This differential effect of BFA was not caused by differences in the palmitoylation kinetics between wild-type proSP-C and the mutants and was not mimicked by nocodazole and monensin. However, differences between the mutants and wild-type proSP-C in the relative degree of processing suggest that BFA may unmask a difference in routing. This would imply that the amino acids just N-terminal of the transmembrane domain may be important for a proper sorting of proSP-C.     

Regulation of NCX1 by palmitoylation

Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle.     

Autopalmitoylation of tubulin

Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2-3 h at 36-37 degrees C under native conditions. Both alpha and beta tubulin are labeled, and 63-73% of the label was hydroxylamine-labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of approximately 13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of alpha tubulin toward the beta band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.     

The position of cysteine relative to the transmembrane domain is critical for palmitoylation of H1, the major subunit of the human asialoglycoprotein receptor

The mammalian hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes where they are degraded. The human ASGP-R is a hetero-oligomeric complex composed of two subunits designated H1 and H2. Both subunits are palmitoylated at the cytoplasmic Cys residues near their transmembrane domains (TMD). The cytoplasmic Cys(36) in H1 is located at a position that is five amino acids from the transmembrane junction. Because the sequences of subunits in all mammalian ASGP-R species are highly conserved especially at the region near the palmitoylated Cys, we sought to identify a recognition signal for the palmitoylation of H1. Various types of H1 mutants were created by site-directed or deletion mutagenesis including alteration of the amino acids surrounding Cys(36), replacing portions of the TMD with that of a different protein and partial deletion of the cytoplasmic domain as well as transposing the palmitoylated Cys to positions further away from the TMD. Mutant H1 cDNAs were transiently expressed in COS-7 cells, and the H1 proteins were analyzed after metabolic labeling with [(3)H]palmitate. The results indicate that neither the native amino acid sequence surrounding Cys(36) nor the majority of the cytoplasmic domain sequence is critical for palmitoylation. Palmitoylation was also not dependent on the native TMD of H1. In contrast, the attachment of palmitate was abolished if the Cys residue was transposed to a position that was 30 amino acids away from the transmembrane border. We conclude that the spacing of a Cys residue relative to the TMD in the primary protein sequence of H1 is the major determinant for successful palmitoylation.     

Global analysis of protein palmitoylation in African trypanosomes

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.