Specificity of fatty acid acylation of cellular proteins

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.     

Fatty acylation of human platelet proteins: evidence for myristoylation of a 50 kDa peptide

Fatty acid acylation of platelet proteins was studied by measuring incorporation of [3H]palmitate and [3H]myristate after incubation at 37 degrees C for 4 h. About ten major radiolabeled proteins were detected after SDS-polyacrylamide gel electrophoresis and fluorography, for both fatty acids. Cleavage by hydroxylamine treatment indicated an ester bond of either palmitate or myristate to these proteins. Nevertheless, a single 50 kDa peptide was specifically modified by an amide-linked myristate. The functions of acylated proteins in platelets are still unknown, but their relation with DLPC-induced shape changes and vesicle shedding is excluded.     

The majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

The BC3Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins [Olson, E. N., Towler, D. A., & Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790]. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages [Olson, E. N., & Spizz, G. (1986) J. Biol. Chem. 261, 2458-2466]. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, we examined the subcellular localization of the major fatty acylated proteins in BC3Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [3H]palmitate and [3H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins.     

Acylation of cell-associated IL-1 by palmitic acid

To determine whether membrane-associated IL-1 is palmitylated, we labeled LPS-activated human monocytes with [3H]palmitic acid. The plasma membranes were isolated, and the membrane proteins extracted and analyzed simultaneously by SDS-PAGE-autoradiography and Western blot analysis from the same gel. When the monocytes were labeled with [3H]palmitate, 23- and 31-kDa bands were visualized, for membrane-associated IL-1 and its precursor, respectively. The 31- and 23-kDa bands were excised from several gels and rehydrated and analyzed again by SDS-PAGE, autoradiography, and Western blot analysis. The 23- and 31-kDa bands appeared again by both methods. To further investigate membrane-associated IL-1 acylation, human monocytes were labeled with [3H]palmitate, the plasma membranes isolated, and the membrane proteins extracted by CHAPS detergent. Immunoprecipitation of isolated membrane proteins using anti-IL-1 antibodies revealed two bands of 23 and 31 kDa after autoradiography. The studies demonstrate that both membrane-associated IL-1 and the IL-1 precursor are acylated with palmitic acid.     

Posttranslational processing of p21 ras proteins involves palmitylation of the C-terminal tetrapeptide containing cysteine-186.

The p21 proteins of ras oncogenes are synthesized as precursors in the cytosol. After processing, which involves acylation, the products are associated with the plasma membrane in eucaryotic cells. The p21 overproduced in Escherichia coli, however, is not processed by acylation. A synthetic tetrapeptide of the p21 C terminus is used to identify the acylation site in eucaryotic p21 as cysteine-186. The same peptide of bacterial p21 is not acylated. Although p21 of Harvey murine sarcoma virus-transformed NRK cells can be metabolically labeled with either [3H]palmitate or [3H]myristate, the lipid moiety of the hydrophobic peptide is identified as palmitic acid. We suggest that the enzymatic mechanism for p21 palmitylation may be different from N-terminal myristylation of many other membrane proteins.     

Selective Transacylation of 1-O-Alkylglycerophosphoethanolamine by Docosahexaenoate and Arachidonate in Rat Brain Microsomes

The mechanism involved in the enzymic acylation of 1-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) in brain microsomes was investigated in comparison with the acylation of 1-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC). Both the alkyllsophospholipids were acylated without exogenously added cofactors to similar extents. The [14C]arachidonoyl moiety of exogenously added 1-stearoyl-2-[14C]arachidonoyl-GPC was transferred to the alkyllysophospholipids and the transfer was not inhibited by exogenously added free arachidonate. These results indicated that the transferase activity was due to a transacylase that catalyzes the transfer of fatty acids between intact phospholipids. The addition of CoA increased the acylation of 1-[3H]alkyl-GPC two or three times with a high acceptor concentration, and the highest rate of acylation of 1-[3H]alkyl-GPC was observed in the presence of CoA, ATP, and Mg2+. On the other hand, the addition of such cofactors only slightly increased the acylation of 1-[3H]alkyl-GPE. HPLC analysis revealed that docosahexaenoate and arachidonate were transferred to the second position of both [3H]alkyllysophospholipids without cofactors and that other fatty acids were transferred to much lower extents. With the addition of cofactors, the acylation of 1-[3H]alkyl-GPC by both docosahexaenoate and arachidonate increased 1.5-2 times, and high amounts of palmitate, oleate, and linoleate were newly transferred. High amounts of oleate were also transferred to 1-[3H]alkyl-GPE in the presence of cofactors but the acylation by both docosahexaenoate and arachidonate scarcely increased on the addition of these cofactors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin and IGF-1 receptors contain covalently bound palmitic acid

We have studied the biosynthesis of the insulin receptor in a human hepatoma cell line, HepG2. As previously reported, these cells synthesize a disulphide-bonded alpha 2 beta 2 tetrameric insulin receptor. Labelling of HepG2 cells with [3H]palmitate or [3H]myristate followed by immunoprecipitation with a polyclonal antireceptor antibody revealed the incorporation of palmitate, but not myristate, into the beta-subunit and alpha beta-precursor of the receptor in a hydroxylamine-sensitive linkage. The extracellular alpha-subunit was not labelled, demonstrating the specificity of incorporation. Acylation of the insulin receptor was an early event as judged by fatty acid incorporation into the alpha beta-precursor and prevention by protein synthesis inhibitors. Pulse-chase studies demonstrated the expected processing of the alpha beta-precursor to mature alpha- and beta-subunits, but no evidence for preferential turnover of the fatty acid moiety was found. The site of acylation appears to be in the transmembrane or cytoplasmic domain since proteolytic treatment of intact cells produced a truncated beta-subunit still containing label. Binding studies showed that HepG2 cells contain approximately half as many insulin-like growth factor-1 receptors as insulin receptors, raising the possibility that this receptor may also be acylated. Indeed, immunoprecipitation with the antiinsulin receptor serum of MDCK cells expressing IGF-1 receptors, but not insulin receptors, revealed bands corresponding to the alpha beta-precursor, alpha- and beta-subunits, of which the alpha beta-precursor and beta-subunits incorporated [3H]palmitate but the alpha-subunit did not.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

Generation of Arachidonic Acid and Diacylglycerol Second Messengers from Polyphosphoinositides in Ischemic Fetal Brain

Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19-20-day-old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 +/- 8.2, 18.0 +/- 3.4, and 13.7 +/- 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 +/- 0.2% of the total radioactivity. Complete restriction of the maternal-fetal circulation for < or = 40 min did not affect the rate of [3H]ArA incorporation (t1/2 = 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C-mediated diacylglycerol formation at the beginning of the insult followed by a phospholipase A2-mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.     

In vivo and in vitro acylation of polypeptides in Vibrio harveyi: identification of proteins involved in aldehyde production for bioluminescence.

Incubation of soluble extracts from Vibrio harveyi with [3H]tetradecanoic acid (+ ATP) resulted in the acylation of several polypeptides, including proteins with molecular masses near 20 kilodaltons (kDa), and at least five polypeptides in the 30- to 60-kDa range. However, in growing cells pulse-labeled in vivo with [3H]tetradecanoic acid, only three of these polypeptides, with apparent molecular masses of 54, 42, and 32 kDa, were specifically labeled. When extracts were acylated with [3H] tetradecanoyl coenzyme A, on the other hand, only the 32-kDa polypeptide was labeled. When luciferase-containing dark mutants of V. harveyi were investigated, acylated 32-kDa polypeptide was not detected in a fatty acid-stimulated mutant, whereas the 42-kDa polypeptide appeared to be lacking in a mutant defective in aldehyde synthesis. Acylation of both of these polypeptides also increased specifically during induction of bioluminescence in V. harveyi. These results suggest that the role of the 32-kDa polypeptide is to supply free fatty acids, whereas the 42-kDa protein may be responsible for activation of fatty acids for their subsequent reduction to form the aldehyde substrates of the bioluminescent reaction.     

Incorporation of myristate and palmitate into the sheep reticulocyte transferrin receptor: evidence for identical sites of labeling

The ability of sheep reticulocytes and plasma membranes isolated from them to incorporate fatty acids into the transferrin receptor has been examined using both [3H]palmitate and [3H]myristate. Both fatty acids, when incorporated into the transferrin receptor, can be released by treating the protein with 1 M hydroxylamine at pH 7.0. After treatment of the 3H-acylated receptor with borohydride, an 3H-labeled alcohol is released, suggesting that the receptor-bound fatty acid is in thioester linkage. With both [3H]myristate and [3H]palmitate, Cleveland maps from immunoprecipitates of the transferrin receptor labeled in intact cells and isolated membranes show that identical peptides are labeled. No evidence was obtained for qualitatively different labeling with the two fatty acids. In intact reticulocytes, incorporation of [3H]palmitate into the transferrin receptor is approximately 3.5 times greater than the incorporation of [3H]myristate from equivalent concentrations of the labeled fatty acids. However, in isolated reticulocyte plasma membranes, there is much less difference between palmitate and myristate incorporation (with ATP) or between their acyl-CoA derivatives. The reason for the discrepancy between cells and membranes is unknown but may be due to the presence in intact cells of more than one enzyme for activating the fatty acids. Acylation of the receptor in isolated plasma membranes is fourfold greater with the CoA derivatives than with the free fatty acids. The fatty acid activating enzyme(s) as well as the acyltransferase(s) appear to be membrane bound in reticulocytes.     

Human apolipoprotein A-I. Post-translational modification by fatty acid acylation

The human apolipoproteins are secretory proteins some of which have been shown to undergo proteolytic processing and post-translational addition of carbohydrate. Apolipoprotein A-I (apo-A-I), the predominant protein associated with high density lipoproteins, undergoes co-translational proteolytic processing as well as post-translational conversion of proapo-A-I to mature apo-A-I following cellular secretion. Utilizing the human hepatoma cell line HEP-G2, we have established that, in addition to proteolytic processing, secreted nascent apo-A-I is acylated with palmitate. Uniformly labeled [14C]palmitate and [1-14C]palmitate were each incorporated into apo-A-I when analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The acylation of apo-A-I with palmitate was confirmed by immunoprecipitation and gas chromatography/mass spectrometry. Hydroxylamine treatment resulted in the deacylation of apo-A-I. Although three of the apo-A-I isoforms analyzed by two-dimensional gel electrophoresis were shown to contain radio-labeled palmitate, 80% of acylated apo-A-I was in the proapolipoprotein A-I isoform. [14C]Oleate was not incorporated in secreted apo-A-I, indicating the specificity of the acylation of apo-A-I. Incubation of [14C] palmitate-acylated apo-A-I in serum and plasma under conditions in which proapo-A-I is proteolytically cleaved to mature apo-A-I did not result in deacylation. These data establish that fatty acid acylation occurs in human secretory proteins in addition to the previously reported acylation of cellular membrane proteins. These results suggest that the covalent linkage of lipids to apolipoproteins may play a critical role in apolipoprotein and lipoprotein metabolism.     

Fatty acid acylation of vaccinia virus proteins.

Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.     

A surface antigen of Giardia lamblia with a glycosylphosphatidylinositol anchor.

Since Giardia lamblia trophozoites are exposed to high concentrations of fatty acids in their human small intestinal milieu, we determined the pattern of incorporation of [3H]palmitic acid and myristic acid into G. lamblia proteins. The pattern of fatty acylation was unusually simple since greater than 90% of the Giardia protein biosynthetically labeled with either [3H]palmitate or myristate migrated at approximately 49 kDa (GP49) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis during both growth and differentiation. GP49, which partitions into the Triton X-114 detergent phase, is localized on the cell surface since it is 125I-surface-labeled. GP49 was also biosynthetically labeled with [14C]ethanolamine and [3H]myoinositol, suggesting that it has a glycosylphosphatidylinositol (GPI) anchor. Moreover, phospholipase A2 (PLA2) or mild alkaline treatment released free fatty acids, indicating a diacylglycerol moiety with ester linkages. Finally, a 3H- and 14C-labeled species was released by nitrous acid deamination from [14C]palmitate- and [3H]myoinositol-labeled GP49. The GPI anchor of GP49 is unusual, however, because purified GP49 was cleaved by Bacillus cereus phosphatidylinositol (PI)-specific PLC, but not by Staphylococcus aureus PI-PLC, or plasma PLD, and did not react with antibody against the variant surface glycoprotein cross-reactive determinant. Moreover, the double-labeled deaminated GP49 anchor migrated faster than authentic PI in TLC and produced [3H]glycerophosphoinositol after deacylation. In contrast to the variable cysteine-rich G. lamblia surface antigens described previously, GP49 was identified in Western blots of every isolate tested, as well as in subclones of a single isolate which differ in expression of a major cysteine-rich 85/66-kDa surface antigen, which does not appear to be GPI-anchored. These observations suggest that GP49, the first common surface antigen to be described in G. lamblia, may play an important role in the interaction of this parasite with its environment.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Fatty acylation of cellular proteins. Temporal and subcellular differences between palmitate and myristate acylation

Previous studies demonstrated that palmitate and myristate are covalently linked to distinct sets of cellular proteins and that the linkages through which these fatty acids are attached to the polypeptide chains are different (Olson, E. N., Towler, D. A., and Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790). In the present study, the kinetics and subcellular sites of acylation of proteins with palmitate and myristate were examined in the BC3H1 muscle cell line. Acylation with myristate was an extremely early modification that appeared to take place cotranslationally or shortly thereafter for a variety of soluble and membrane-bound proteins. In contrast, acylation of proteins with palmitate was a post-translational event that occurred exclusively on membrane proteins. To begin to understand the intracellular pathways that acyl proteins follow during their maturation, the degree of glycosylation, and the nature of the interaction of these proteins with membranes were examined. The majority of acyl proteins were tightly associated with membranes and could not be removed by conditions that release peripheral proteins from membranes. However, only a minor fraction of acylated proteins were N-glycosylated. These data suggest that the acyltransferases that attach palmitate and myristate to proteins are present in different subcellular locations and demonstrate that these fatty acids are attached to newly synthesized acyl proteins at different times during their maturation. The lack of carbohydrate on the majority of integral membrane acyl proteins suggests that these proteins may follow intracellular pathways that are different from those followed by cell surface glycoproteins.     

Acylation of disc membrane rhodopsin may be nonenzymatic

Bovine retinal rod outer segments (ROS) support the incorporation of [3H]palmitate into rhodopsin. [14C] Palmitoyl-CoA serves as the donor with an apparent Km of 40 microM. Solubilization of ROS in the detergent, Emulphogene, results in increased incorporation of label into rhodopsin. A further increase is found when ConA-Sepharose-purified rhodopsin is used as the source of both "enzyme" and acceptor. Failure to separate enzyme from acceptor suggested the possibility of a nonenzymatic reaction. This was confirmed when boiled rhodopsin was found to support the reaction. However, the acylation of rhodopsin is not an artifact since analysis of purified native rhodopsin reveals the presence of covalently bound palmitate and we showed that whole bovine retinas incubated with [3H] palmitate incorporated the fatty acid into rhodopsin (O'Brien, P.J., and Zatz, M. (1984) J. Biol. Chem. 259, 5054-5057). Furthermore, in vivo experiments with rat retinas have revealed that opsin is acylated both in the rod inner and outer segments (St. Jules, R. S., and O'Brien, P.J. (1986) Exp. Eye Res. 43, 929-940). Incubation of labeled rhodopsin with mercaptoethanol resulted in release of the labeled palmitate indicating the presence of a thioester bond. This also illustrates the ease with which a thioester, such as palmitoyl cysteine or palmitoyl-CoA, can transfer the fatty acyl group to a free thiol, such as cysteine or mercaptoethanol.     

Autoacylation of myelin proteolipid protein with acyl coenzyme A

Rat brain myelin proteolipid protein (PLP) is known to contain long chain, covalently bound fatty acids. In the course of characterizing the mechanism of acylation, we found that the isolated PLP, in the absence of any membrane fraction, was esterified after incubation with [3H]palmitoyl coenzyme A (CoA). This observation demonstrated that the protein acts as both an acylating enzyme and an acceptor. Thus, acylation occurs by an autocatalytic process. The possibility of a separate acyltransferase that copurifies with PLP was essentially excluded by adding brain subcellular fractions to the reaction mixtures and by changing the isolation procedure. After deacylation, the protein was acylated at a 4-fold greater rate, suggesting that the original sites were reacylated. The palmitoyl-CoA concentration followed Michaelis kinetics, confirming that spontaneous acylation was not occurring. Pulse-chase experiments indicated that the reaction entails net addition of acyl groups. Although fatty acids are bound via an O-ester linkage, free SH groups are required in the reaction. Denaturation of the protein by sodium dodecyl sulfate or heat inhibits the reaction, whereas cerulenin has little or no effect. PO, the major protein in peripheral nerve myelin, is also an acylated protein, but it was not labeled upon incubation of either peripheral myelin or the isolated protein with [3H]palmitoyl-CoA, demonstrating that it is acylated by a different route. Several synthetic peptides derived from PLP sequences with sites known to be acylated in vivo as well as a series of deacylated PLP tryptic peptides were not labeled, indicating that integrity of the protein is required for acylation. Limited proteolysis and peptide mapping showed that the same sites are acylated in vitro or in vivo, suggesting that the autocatalytic acylation reaction is physiological.     

Dynamic fatty acylation of p21N-ras.

To study the acylation of p21N-ras with palmitic acid we have used cells which express the human N-ras gene to high levels under control of the steroid-inducible MMTV--LTR promoter. Addition of [3H]palmitate to these cells resulted in detectable incorporation of label into p21N-ras within 5 min, which continued linearly for 30-60 min. Inhibition of protein synthesis for up to 24 h before addition of [3H]palmitate had no effect on acylation of p21N-ras, suggesting that this can occur as a late post-translational event. Acylated p21N-ras with a high SDS--PAGE mobility is found only in the membrane fraction, whereas approximately 50% of the [35S]methionine-labelled p21N-ras is cytoplasmic and has a lower mobility. Conversion of the acylated high mobility form to a deacylated form of slightly lower mobility can be achieved with neutral hydroxylamine, which is known to cleave thioesters. This treatment also results in partial removal of p21N-ras from the membranes. A remarkably high rate of turnover of the palmitate moiety can be demonstrated by pulse--chase studies (t1/2 approximately 20 min in serum-containing medium) which cannot be attributed to protein degradation. The data suggest an active acylation--deacylation cycle for p21N-ras, which may be involved in its proposed function as a signal transducing protein.     

The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation: role in membrane targeting, cell adhesion, and spreading

The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function.