Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells

Increased [3H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary (Wellner, R. B., Ray, B., Ghosh, P. C., and Wu, H. C. (1984) J. Biol. Chem. 259, 12788-12793) and yeast (Wen, D., and Schlesinger, M. J. (1984) Mol. Cell. Biol. 4, 688-694) mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [3H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [3H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [3H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [3H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [3H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [3H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [3H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [3H]palmitate incorporation into the 20,000 molecular weight species. Endoglycosidase H treatment of [3H]palmitate-labeled extracts from the mutant cell line resulted in the disappearance of the heavily labeled 30,000 molecular weight species and the appearance of intensely labeled 20,000 molecular weight species. Pretreatment of the mutant cell line with either castanospermine or deoxynojirimycin reduced the [3H]palmitate incorporation in to the 30,000 molecular weight species increased in untreated cells, but did not cause increased [3H]palmitate incorporation into the 20,000 molecular weight species. Our results indicate that perturbation of N-linked oligosaccharide structure results in altered incorporation of [3H]palmitate into specific proteins in CHO cells.     

Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.

Yeast secretory (sec) mutants that are blocked in the transport of secretory proteins and accumulate membrane organelles were used to study the biosynthesis of fatty acid-acylated proteins. Four proteins were labeled with [3H]palmitate in sec mutants accumulating endoplasmic reticulum membranes. Three of these (molecular weights approximately equal to 20,000, 50,000, and 120,000) were N-linked glycoproteins, based on their ability to be labeled with [3H]mannose and their sensitivity to endoglycosidase H. The fourth protein (molecular weight approximately equal to 30,000) also was labeled with [3H]mannose but was insensitive to endoglycosidase H; it appeared to contain O-linked sugars. In sec mutants accumulating Golgi membranes or post-Golgi vesicles, a 35-kilodalton protein was labeled with [3H]palmitate. Analysis of Staphylococcus aureus protease V8 digests and pulse-chase experiments indicated that the 30-kilodalton protein was a precursor of 35 kilodaltons. None of these proteins was labeled with [3H]palmitate in a sec mutant that blocked the penetration of nascent polypeptides into endoplasmic reticulum; thus, acylation occurred in endoplasmic reticulum. All four proteins could be recovered from fractions enriched for yeast membranes. Fatty acids were not released from proteins by boiling in sodium dodecyl sulfate or extraction with organic solvents but were recovered as methyl esters after proteins were treated with KOH-methanol, a reaction characteristic of an acyl ester linkage.     

Fatty acid acylation of salivary mucin in rat submandibular glands

The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with [3H]palmitic acid and [3H]proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. Both preparations gave single bands at the CsCl density of 1.48, in which carbohydrate peaks coincided with that of the labels. The [3H]palmitic acid in these glycoproteins was susceptible to cleavage by alkali and hydroxylamine, thus indicating the ester nature of the bond. With both intracellular and extracellular glycoproteins deacylation caused the glycoproteins to band in the CsCl gradient at a density of 1.55. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of [3H]palmitate and [3H]proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.     

Fatty acid modification of Newcastle disease virus glycoproteins.

The fatty acid acylation of Newcastle disease virus hemagglutininin-neuraminidase and fusion glycoproteins was assayed. [3H]palmitate label was associated with cytoplasmic fusion proteins (F0 and F1) and virion-associated F1. In contrast, there was no detectable [3H]palmitate label associated with the hemagglutin-neuraminidase protein in Newcastle disease virus-infected Chinese hamster ovary cells or chicken embryo cells or in virions released from these cells. Thus, fatty acid modification may not be important for the maturation of some glycoproteins.     

Inhibition of Vibrio harveyi bioluminescence by cerulenin: in vivo evidence for covalent modification of the reductase enzyme involved in aldehyde synthesis.

Bacterial bioluminescence is very sensitive to cerulenin, a fungal antibiotic which is known to inhibit fatty acid synthesis. When Vibrio harveyi cells pretreated with cerulenin were incubated with [3H]myristic acid in vivo, acylation of the 57-kilodalton reductase subunit of the luminescence-specific fatty acid reductase complex was specifically inhibited. In contrast, in vitro acylation of both the synthetase and transferase subunits, as well as the activities of luciferase, transferase, and aldehyde dehydrogenase, were not adversely affected by cerulenin. Light emission of wild-type V. harveyi was 20-fold less sensitive to cerulenin at low concentrations (10 micrograms/ml) than that of the dark mutant strain M17, which requires exogenous myristic acid for luminescence because of a defective transferase subunit. The sensitivity of myristic acid-stimulated luminescence in the mutant strain M17 exceeded that of phospholipid synthesis from [14C]acetate, whereas uptake and incorporation of exogenous [14C]myristic acid into phospholipids was increased by cerulenin. The reductase subunit could be labeled by incubating M17 cells with [3H]tetrahydrocerulenin; this labeling was prevented by preincubation with either unlabeled cerulenin or myristic acid. Labeling of the reductase subunit with [3H]tetrahydrocerulenin was also noted in an aldehyde-stimulated mutant (A16) but not in wild-type cells or in another aldehyde-stimulated mutant (M42) in which [3H]myristoyl turnover at the reductase subunit was found to be defective. These results indicate that (i) cerulenin specifically and covalently inhibits the reductase component of aldehyde synthesis, (ii) this enzyme is partially protected from cerulenin inhibition in the wild-type strain in vivo, and (iii) two dark mutants which exhibit similar luminescence phenotypes (mutants A16 and M42) are blocked at different stages of fatty acid reduction.     

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).     

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.     

Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production.

The antibiotic tunicamycin, which blocks the synthesis of glycoproteins, inhibited the production of infectious herpes simplex virus. In the presence of this drug, [14C]glucosamine and [3H]mannose incorporation was reduced in infected cells, whereas total protein synthesis was not affected. Gel electrophoresis of [2-3H]mannose-labeled polypeptides failed to detect glycoprotein D or any of the other herpes simplex virus glycoproteins. By use of specific antisera we demonstrated that in the presence of tunicamycin the normal precursors to viral glycoproteins failed to appear. Instead, lower-molecular-weight polypeptides were found which were antigenically and structurally related to the glycosylated proteins. Evidence is presented to show that blocking the addition of carbohydrate to glycoprotein precursors with tunicamycin results in the disappearance of molecules, possibly due to degradation of the unglycosylated polypeptides. We infer that the added carbohydrate either stabilizes the envelope proteins or provides the proper structure for correct processing of the molecules needed for infectivity.     

Proteolipid formation in Mycoplasma capricolum. Influence of cholesterol on unsaturated fatty acid acylation of membrane proteins

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph was grown on media supplemented with [3H]palmitate or [3H]oleate. The isolated bacterial membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the more than 50 membrane polypeptides revealed by Coomassie blue staining, approximately 25 were labeled with [3H]palmitate and only about 6 were labeled with [3H]oleate. Exhaustive delipidation of the membranes with chloroform:methanol did not alter the labeling pattern. Treatment of delipidated membranes by mild alkaline hydrolysis released up to 71% of the [3H]palmitate and 93% of the [3H]oleate. The data suggest that numerous membrane proteins of M. capricolum are covalently modified by acylation with saturated and unsaturated fatty acids. Cerulenin, a specific inhibitor of fatty acid synthesis had no effect on the labeling of mycoplasma membrane proteins by either [3H]palmitate or [3H]oleate. A small amount of membrane-associated cholesterol previously shown to stimulate sequentially the synthesis of unsaturated phospholipid, RNA, and protein (Dahl, J. S., and Dahl, C. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 692-696) specifically enhances the acylation of certain proteolipids by oleate but not by palmitate.     

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.     

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.     

The phenotype of five classes of T lymphoma mutants. Defective glycophospholipid anchoring, rapid degradation, and secretion of Thy-1 glycoprotein

Thy-1 glycoprotein is a member of a class of proteins which are anchored to the plasma membrane via a covalently bound glycophospholipid. The biosynthesis and anchoring of Thy-1 were investigated in a family of wild-type and mutant (complementation groups A, B, C, E, and F) T lymphomas. The mutants all synthesize Thy-1 but fail to express it on the cell surface. Analysis of the size of D-[2-3H]mannose-labeled dolichol-linked oligosaccharides showed that the class E mutant is the only cell line which does not synthesize dolichol-P-P-Glc3Man9GlcNAc2. Turnover and possible secretion of Thy-1 by mutant T lymphoma cells were documented in D-[2-3H]mannose pulse-chase experiments. The turnover of [3H]Thy-1 for all wild-type cells is considerably slower than for the mutant cells. Class B and E cells release appreciably more [3H]Thy-1 than wild-type cells. Additional experiments were performed to determine the electrophoretic mobility and hydrophobicity of cell-associated and released forms of Thy-1 labeled overnight with [3H]mannose. All wild-type and class A, C, E, and F mutant cells contain a major Triton X-114 binding species of cell-associated [3H]Thy-1. All extracellular [3H]Thy-1 was almost exclusively hydrophilic. The presence of two Thy-1 anchor components, ethanolamine and palmitate, was investigated. Biosynthetic labeling with [3H]palmitic acid showed that all of the wild-type cells but none of the mutants incorporated this anchor precursor into Thy-1. In [3H]ethanolamine-labeling experiments, incorporation was detected in the Thy-1 of all wild-type cells and in two mutants, S1A-b and T1M1-c. Based on the above studies, the phenotype of Thy-1 negative T lymphoma mutants can be re-evaluated. In classes A and F, dolichol-linked oligosaccharides appear normal and no anchor is detected. In class B, dolichol-linked oligosaccharides appear normal, a partial anchor may be present, and a substantial amount of Thy-1 is released. In class C, dolichol-linked oligosaccharides appear normal and a partial anchor may be present. In class E, truncated dolichol-linked oligosaccharides are formed, no anchor is detected, but a substantial amount of newly synthesized Thy-1 is released. These observations are discussed with reference to the possibility that the lesions which characterize the mutants pertain to the biosynthesis of the glycophospholipid moiety of Thy-1.     

Localization of the lipid intermediate pathway of protein glycosylation in oviduct cell types

Oviduct tissue slices were incubated with [3H]-leucine or [3H]-mannose in the presence and absence of tunicamycin, a specific inhibitor of lipid-mediated protein glycosylation. Conditions were established where tunicamycin had maximal effect on [3H]-mannose incorporation (greater than 90% inhibition) but a minimal effect on [3H]-leucine incorporation (less than 10% inhibition) into total TCA-insoluble products. Analysis of incubated tissues by SDS-polyacrylamide gel electrophoresis revealed that in the absence of tunicamycin, [3H]-mannose was incorporated into only a few proteins, of which ovalbumin represented the major radiolabeled component. Tunicamycin markedly reduced the incorporation of [3H]-mannose into ovalbumin and other oviduct glycoproteins. In contrast, analysis by SDS-polyacrylamide gel electrophoresis showed that [3H]-leucine was incorporated into a variety of proteins in the absence of tunicamycin. The radioactivity profile of some of these proteins was shifted toward lower Mr when oviduct slices were incubated in the presence of tunicamycin, with only a minimal decrease in protein labeling. Light microscopic autoradiograms of tissue incubated with [3H]-leucine in either the presence or absence of tunicamycin exhibited extensive labeling of tubular gland and epithelial cells. In the absence of tunicamycin, these cell types also become markedly labeled with [3H]-mannose; however, incorporation of label in both cell types was substantially reduced in the presence of tunicamycin. Qualitatively, labeling of tubular gland cells appeared greater than that of epithelial cells, largely due to the concentration of silver grains over the dense population of secretory vesicles in the tubular gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of acyl moieties of phospholipids into murein lipoprotein in intact cells of Escherichia coli by phospholipid vesicle fusion.

The biosynthesis of the acyl moieties in murein lipoprotein was studied by fusion of [3H]palmitate-labeled phospholipid vesicles with intact cells of an fadD mutant of Escherichia coli. A linear increase in the incorporation of [3H]palmitate radioactivity into both the ester- and amide-linked fatty acids in lipoprotein was observed during a 3-h chase after the fusion. Addition of chloramphenicol completely prevented the incorporation of [3H]palmitate from phospholipids to lipoprotein. These results strongly support our hypothesis that the acyl moieties in phospholipids are the precursors for the fatty acids in murein lipoprotein of E. coli. Among the major glycerophosphatides in E. coli, no specificity was observed regarding the efficacy of the donor.     

Acylated proteins in Acholeplasma laidlawii.

The covalent modification of membrane proteins by long-chain fatty acids was determined in two strains of Acholeplasma laidlawii by one-dimensional gel electrophoresis of radiolabeled membranes. Of the more than 50 membrane polypeptides detected, approximately 30 were labeled with [3H]palmitate, whereas covalent binding of [3H]oleate to membrane proteins could not be demonstrated. We suggest that in these wall-less bacteria, membrane protein acylation with saturated fatty acids may serve to ensure the structural integrity of the membrane.     

Sulfated glycoproteins synthesized by the corneal epithelium of chick embryo having the same molecular weights as cytokeratin polypeptides

The previous study from this laboratory demonstrated that the corneal epithelium of 19-d-old chick embryo synthesizes two classes of sulfated glycoconjugates consisting of sulfated glycoproteins and proteoglycans (Yonekura, H., Oguri, K., Nakazawa, K., Shimizu, S., Nakanishi, Y., & Okayama, M. (1982) J. Biol. Chem. 257, 11166-11175). The present study demonstrated that when the sulfated glycoproteins labeled metabolically with [35S]sulfate and [3H]glucosamine were analyzed by SDS-PAGE, the 70,000 component (accounting for approximately 30% of the 35S and 35% of the 3H of the total sulfated glycoprotein) co-migrated with five major proteins with apparent molecular weights (Mrs) of 70,000, 66,000, 58,000, 51,000, and 48,000, which together accounted for about 57% of the total tissue protein. All five proteins cross-reacted with an antibody against human sole keratin, indicating that they are cytokeratin polypeptides of the corneal epithelium. Amino acid analysis demonstrated that they had high contents of glycine, serine, glutamic acid, leucine, and aspartic acid. Two-dimensional tryptic peptide maps indicated that they were all different. Analysis of radiolabeled materials released by alkaline borohydride treatment of the sulfated glycoproteins which were synthesized in the presence and absence of tunicamycin and co-purified with the five cytokeratin polypeptides, revealed that they contained both N- and O-glycosidically linked sulfated oligosaccharides. All the results obtained in the present study indicate that the five sulfated glycoproteins are similar, if not identical, to the cytokeratin polypeptides. This is consistent with the result in the accompanying paper that these sulfated glycoproteins are localized intracellularly.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.     

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.