Release of fatty acids from virus glycoproteins by hydroxylamine

The fatty acids bound to the glycoproteins of Sindbis and vesicular stomatitis viruses can be released by treating the protein with 1 M hydroxylamine at pH 8.0, but the rates of release vary greatly among the three proteins. The most labile fatty acyl bonds were in the Sindbis virus PE2/E2 proteins and the most stable were in the E1 protein. Some of the fatty acids in Sindbis virus glycoproteins were reduced to the alcohol after treatment with sodium borohydride, indicating that protein-bound fatty acids could be in thiolester linkage. Sindbis virus PE2/E2 has several cysteine residues near the carboxy terminus, a region of the protein postulated to be localized on the inside (cytoplasmic face) of the bilayer, and protease digestion of microsomal membranes containing E2 protein removed a small portion of this cytoplasmic tail as well as significant amounts of the fatty acid. For the vesicular stomatitis virus G protein, the sensitivity of fatty acid hydrolysis appeared to depend on the conformation of the protein and a significant fraction of G protein was converted to a disulfide-linked dimer by hydroxylamine. These data implicate cysteinyl groups on these proteins as sites involved in fatty acid acylation.     

ATP- and coenzyme A-dependent fatty acid incorporation into proteins of cell-free extracts from mouse tissues

The incorporation of tritiated fatty acids into proteins has been studied in cell-free extracts from mouse tissues. Incubation of heart extracts with [3H]tetradecanoic or [3H]palmitic acid in the presence of ATP and CoA resulted in the time-dependent and selective labeling of proteins (Mr = 60,000, 47,000, 42,000, 31,000, 16,000, and 13,000) which could be detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Two polypeptides (Mr = 47,000 and 42,000) reached a maximum in fatty acid incorporation very rapidly and were mainly localized in the membrane subcellular fractions of the extract. These proteins underwent transient labeling with [3H] tetradecanoyl-CoA, the maximum incorporation being obtained within 1 min. The fatty acid-labeled proteins from tissue extracts had the same properties as other proteins known to be acylated in intact cells, i.e. the acyl moiety was resistant to delipidation with organic solvents but could be hydrolyzed by treatment with neutral hydroxylamine. Screening of different tissues showed that extracts from liver and kidney also catalyze the ATP- and CoA-dependent formation of a similar group of fatty acid-acylated proteins. The results provide evidence for a group of proteins in mammalian tissues which selectively incorporate fatty acids in vitro and should be of value for further studies on the biosynthesis of acylated proteins.     

Fluorescence anisotropy of a fatty acid covalently linked in vivo to the glycoprotein of vesicular stomatitis virus

The covalently-attached fatty acid of the membrane glycoprotein (G) of vesicular stomatitis virus was fluorescently labeled biologically by isolating vesicular stomatitis virus from infected baby hamster kidney clone 21 cells that had been grown in the presence of 16(9-anthroyloxy)palmitate. The fluorescent labeling was specific for the G protein; the other viral membrane protein, the matrix (M) protein, was not labeled. Steady state fluorescence anisotropy of the 16(9-anthroyloxy)palmitate-labeled G protein reconstituted into dipalmitoylphosphatidylcholine vesicles indicated that the fatty acid attached to G protein is located in a dipalmitoylphosphatidylcholine domain that does not undergo the gel to liquid-crystalline phase transition.     

Synthesis and assembly of membrane glycoproteins. Membrane anchoring COOH-terminal domain of vesicular stomatitis virus envelope glycoprotein G contains fatty acids

Two polypeptides associated with the envelope of vesicular stomatitis virus are obtained by exhaustive proteolytic digestion of the virion. Analysis of the tryptic peptides and determination of the partial amino acid sequence show that the larger membrane-anchoring peptide is derived from the hydrophobic COOH terminus of the viral transmembrane glycoprotein G. The smaller peptide is, however, derived from the nonglycosylated matrix protein M. Analysis of the membrane-anchoring peptide fragments obtained from virus labeled with [3H]palmitic acid shows that the larger peptide fragment contained all the fatty acid present in G, suggesting that the fatty acids in conjunction with the hydrophobic domain may be involved in the binding of G protein to the membrane.     

Characterization of a protein fatty acylesterase present in microsomal membranes of diverse origin

A microsomal activity of baby hamster kidney cells which cleaves ester-type bound fatty acids from acyl proteins in vitro has been characterized. This activity is also present in microsomal membranes from pig liver, calf kidney, and human mucous cells. Cell free deacylation is described for the Semliki Forest virus acyl proteins E1 and E2 and the precursor of E2 designated p62. Acyl chain cleavage operates with both exogenous and endogenous viral acyl protein substrates. The in vitro cleavage requires microsomes solubilized by detergents of which various kinds are equally effective (Nonidet P-40, Tween 20, sodium deoxycholate, Triton X-100, or octyl-beta-D-glucoside). If microsomes are boiled for 15 min prior to the incubation, deacylation is abolished completely and no radioactivity is released from the palmitoylated acyl proteins during incubation with either detergents or microsomes alone. No changes in the molecular structure of the deacylated Semliki Forest virus proteins were detected, and the cleavage product was identified as free fatty acid. Deacylation is time- and temperature-dependent and can be enhanced by increasing the concentration of microsomal protein in the incubation mixture. It is completely inhibited under acidic conditions (pH 5) and at low temperature (4 degrees C). Deacylation also occurs in the presence of EDTA and bivalent cations such as Mg2+, Mn2+, and Ca2+ which influence the reaction marginally. On the other hand, fatty acid release is drastically reduced with a mixture of Co2+, Zn2+, and Hg2+ ions. The activity is not identical with protein fatty acyltransferase operating in the reverse direction, since a partially purified preparation of this acyltransferase failed to cleave fatty acids from fatty acylated substrate proteins. Taken together, these data lead us to postulate an enzymatic activity which cleaves fatty acids from ester-type fatty acylated proteins, and we propose to designate this enzyme a protein fatty acylesterase.     

Intersubunit transfer of fatty acyl groups during fatty acid reduction

Fatty acid reduction in Photobacterium phosphoreum is catalyzed in a coupled reaction by two enzymes: acyl-protein synthetase, which activates fatty acids (+ATP), and a reductase, which reduces activated fatty acids (+NADPH) to aldehyde. Although the synthetase and reductase can be acylated with fatty acid (+ATP) and acyl-CoA, respectively, evidence for acyl transfer between these proteins has not yet been obtained. Experimental conditions have now been developed to increase significantly (5-30-fold) the level of protein acylation so that 0.4-0.8 mol of fatty acyl groups are incorporated per mole of the synthetase or reductase subunit. The acylated reductase polypeptide migrated faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the unlabeled polypeptide, with a direct 1 to 1 correspondence between the moles of acyl group incorporated and the moles of polypeptide migrating at this new position. The presence of 2-mercaptoethanol or NADPH, but not NADP, substantially decreased labeling of the reductase enzyme, and kinetic studies demonstrated that the rate of covalent incorporation of the acyl group was 3-5 times slower than its subsequent reduction with NADPH to aldehyde. When mixtures of the synthetase and reductase polypeptides were incubated with [3H] tetradecanoic acid (+ATP) or [3H]tetradecanoyl-CoA, both polypeptides were acylated to high levels, with the labeling again being decreased by 2-mercaptoethanol or NADPH. These results have demonstrated that acylation of the reductase represents an intermediate and rate-limiting step in fatty acid reduction. Moreover, the activated acyl groups are transferred in a reversible reaction between the synthetase and reductase proteins in the enzyme mechanism.     

Identification of acyl donors and acceptor proteins for fatty acid acylation in BHK cells infected with Semliki Forest virus

The modification of viral glycoproteins through the covalent attachment of fatty acids was studied in baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV). Comparative pulse-chase experiments with [3H]palmitic acid and [35S]methionine revealed that a precursor polypeptide, designated p62, of the structural SFV glycoprotein and E1 serve as the primary acceptors of acyl chains. Acylation of p62 occurs immediately prior to its proteolytical cleavage to E2 and E3 emphasizing the post-translational and specific nature of this hydrophobic modification. To trace the acyl donor(s) for protein acylation the covalent attachment of fatty acids to p62 was studied after extremely short labeling periods with [3H]palmitic acid and correlated to the metabolism of the exogenous tritiated fatty acid. The shortest possible labeling time, a 10 s pulse with [3H]palmitic acid, was sufficient to acylate SFV p62. Analysis of the labeled lipids extracted from the same cells revealed that palmitoyl-CoA and phosphatidic acid showed the highest specific radioactivity among the tritiated lipid species. Out of these lipid species palmitoyl-CoA was identified as the functional acyl donor lipid in a cell-free system for the acylation of polypeptides.     

Identification of covalently bound fatty acids on acylated proteins immobilized on nitrocellulose paper

A general method for identification of fatty acids covalently bound to acylated proteins following their electrophoretic transfer onto nitrocellulose paper is described. As demonstrated for [3H]palmitoylated RAS1 protein of Saccharomyces cerevisiae and the acylated acyl carrier protein of Spirodela oligorrhiza, this procedure alleviates the need for elution of proteins from polyacrylamide gel slices. Fatty acid ligands of such proteins are hydrolyzed directly from their immobilized state on the nitrocellulose paper, then derivatized with p-nitrophenacyl bromide, and finally resolved by reversed-phase high-performance liquid chromatography. The amount of acylated protein required for identification of acyl groups is minimized compared to that required for more conventional approaches by coupling a radioactive flow detector with the HPLC system.     

Protein acylation in normoxic and ischemic/reperfused cardiac tissue.

In addition to a prominent role in tissue energy conversion, fatty acids are involved in signal transduction and modulation of cellular protein localization and function. The latter is accomplished by acylation of specific cellular proteins. In the present study the amount of fatty acyl moieties covalently bound to cardiac proteins and the effect of myocardial ischemia and reperfusion on the degree and relative fatty acyl composition of cardiac proteins have been investigated in isolated rat hearts. In the normoxic heart about 0.32% of the cellular fatty acyl pool is covalently bound to proteins. Approximately 90% of these fatty acyl chains are thio-esterified, whereas a relatively minor part is attached to cardiac proteins through amide linkage. Thio-esterified fatty acyl chains are derived from palmitic, stearic, oleic, linoleic, arachidonic and docosahexaenoic acid. In contrast, amide linked protein acylation shows a preference for myristic acyl chains. Acute ischemia and reperfusion inflicted upon the isolated rat heart did enhance significantly the content of (unesterified) fatty acids, but did neither affect the degree of protein acylation nor the relative fatty acyl composition of acylated proteins in cardiac tissue.     

An evaluation of the contribution of membrane lipids to protection against ultraviolet radiation

Using dioleoylphosphatidylcholine liposomes incorporating various fatty acids and neutral lipids, we have examined the ability of such lipids to provide protection of Escherichia coli and vesicular stomatitis virus (VSV) against the lethal effect of ultraviolet (254 nm) radiation. While the presence of varying amounts of saturated (palmitic) or polyunsaturated (arachidonic) fatty acids or the lipid antioxidant, alpha-tocopherol, had little effect on killing by ultraviolet radiation, considerable radioprotection was observed with beta-carotene, retinal and vitamin K-1 at final concentrations of 1 mg/ml. In another approach, vesicular stomatitis virus grown under conditions in which its envelope fatty acid composition was substantially modified, showed little change in its sensitivity to inactivation by ultraviolet radiation. The results provide strong evidence for a radioprotective role of certain, relatively rare natural lipid components with conjugated polyene systems, but not of the more ubiquitous and abundant membrane fatty acids.     

Transport of the vesicular stomatitis glycoprotein to trans Golgi membranes in a cell-free system

Terminal steps in the transport of the vesicular stomatitis virus glycoprotein (G protein) in the Golgi stack have been reconstituted in a cell-free system. Incorporation of sialic acid into the oligosaccharide chains of G protein was used to monitor transport into the trans Golgi compartment. Transport-coupled sialylation required cytosol, ATP, an N-ethylmaleimide-sensitive factor extractable from Golgi membranes, and long chain acyl coenzyme A. The G protein receiving sialic acid in the cell-free system begins its in vitro transport bearing galactose residues acquired in vivo. Earlier reports (Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984a) Cell 39, 405-416) documented that transport of G protein into the medial (GlcNAc Transferase-containing) compartment is reconstituted under the same conditions. On the basis of the results reported here, it now appears that a more complete set of transport operations of the Golgi stack may be simultaneously reconstituted.     

Fatty Acid Acylation of Rat Brain Myelin Proteolipid Protein In Vitro: Identification of the Lipid Donor

The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.     

Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein.

The glycoprotein (G) of vesicular stomatitis virus (VSV) binds 1–2 moles of fatty acid per mole of protein. The fatty acids cannot be released by repeated extractions of the protein with organic solvents, nor can they be released by denaturing the protein with ionic or nonionic detergents. Pronase digestion of G yields an organic extractable fragment that contains bound fatty acid. The fatty acid is quantitatively released from this fragment and from intact G by mild alkali treatment in methanol and is identified by gas-liquid and thin-layer chromatography as, predominantly, the methyl ester of palmitic acid. Insignificant amounts of phosphate are found in G, thus ruling out the presence of bound phospholipid. Chicken embryo fibroblast pre-labeled with ³H-palmitate and then infected with VSV for 4 hr show the presence of ³H label in G but not in other viral structural proteins. The ³H label is present only in the fatty acid moiety of the protein. Much smaller amounts of ³H fatty acid are bound to G protein formed by the VSV mutant ts045 grown at the nonpermissive temperature, and no ³H fatty acid is bound to G synthesized at 37°C in cells pretreated with tunicamycin, an inhibitor of glycosylation. However, infection with the VSV-Orsay strain at 30°C in the presence of tunicamycin allows for production of VSV particles with nonglycosylated G (Gibson, Schlesinger and Kornfeld, 1979), and this G has the same proportion of the fatty acid as does the normal glycosylated G. These data indicate that fatty acids become covalently attached to the G polypeptide chain during maturation of the protein—perhaps as the glycoprotein moves to the cell's plasma membrane.     

Circular dichroism studies on a synthetic peptide corresponding to the membrane-spanning region of vesicular stomatitis virus G protein and its fatty acyl derivative

The conformations of synthetic peptides Lys-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-OCH3 and Lys(epsilon-palmitoyl)-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-O CH3, which constitute a part of the membrane-spanning region of the vesicular stomatitis virus G protein, have been studied by circular dichroism (CD) spectroscopy. Secondary structural features are observed for both peptides in trifluoroethanol, methanol, aqueous mixtures of trifluoroethanol and methanol and in a micellar environment. In trifluoroethanol, the CD spectra indicate the presence of a helical conformation, whereas in aqueous mixtures of organic solvents, both helical and beta-conformations are observed. While fatty acid acylation does not directly modulate peptide conformation, it promotes self-association of the acylated peptide and association with micelles. In a micellar environment, the acylated peptide adopts an alpha-helical conformation.     

Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation

Cerulenin, an antibiotic that inhibits de novo fatty acid and cholesterol biosynthesis, effectively inhibited the formation and release of virus particles from chicken embryo fibroblasts infected with Sindbis or vesicular stomatitis virus (VSV). When added for 1 h at 3 h postinfection, the antibiotic blocked VSV particle production by 80 to 90% and inhibited incorporation of [3H]palmitic acid into the VSV glycoprotein by an equivalent amount. The effect of this antibiotic on virus protein and RNA biosynthesis was significantly less than that on fatty acid acylation. Nonacylated virus glycoproteins accumulated inside and on the surface of cerulenin-treated cells. These data indicate that fatty acid acylation is not essential for intracellular transport of these membrane proteins, but it may have an important role in the interaction of glycoproteins with membranes during virus assembly and budding.     

In vitro acylation of the transferrin receptor

In vitro fatty acylation of the transferrin receptor with [3H]tetradecanoate or [3H]tetradecanoyl-CoA has been demonstrated for isolated sheep reticulocyte plasma membranes. Although less than 5% of the receptor was labeled in vitro, the acylated protein could be readily observed after sodium dodecyl sulfate-gel electrophoresis. The acylated transferrin receptor in the reticulocyte membrane was specifically precipitated with a monoclonal antibody and was absent from mature red cell membranes. Incorporation of fatty acid was dependent on ATP, and fatty acid was 5-10 times less effective as an acyl donor than the acyl-CoA derivative, pointing out the strong potential of this reagent for in vitro acylation of membrane proteins. During in vitro maturation of reticulocytes, the receptor is released in vesicles into the incubation medium. Using reticulocytes labeled with [3H]tetradecanoate, it can be shown that the 3H-labeled receptor is transferred from the cells to the vesicles without loss of acyl groups, suggesting that the vesiculation process does not involve deacylation.     

Cotranslational attachment of fatty acids to nascent peptides in gastric mucus glycoprotein

Using gastric mucous cells which are involved exclusively in the synthesis of secretory O-glycosidic glycoprotein (mucin), the relationship between protein core synthesis and its acylation with fatty acids was investigated. Labeling of the cells with [3H]palmitic acid and [35S]methionine followed by isolation of peptidyl-tRNA and release of nascent peptides, indicated that these peptides contain covalently bound fatty acids. The high performance thin layer chromatography, SDS-gel electrophoresis, and radioactivity scanning revealed that the preparation contained three fractions labeled with palmitate (Mr 15,000-3,600) and two (Mr 1,500 and less) without this label. Based on these data and the nascent peptides amino acid analysis, we conclude that the protein core of the O-glycosidic glycoprotein is acylated with fatty acids during translation, when the peptide chain is longer than 21 amino acid residues.     

Evidence for the regulation of exocytic transport by protein phosphorylation

We investigated the effects of the protein phosphatase inhibitors okadaic acid and microcystin-LR upon transport of newly synthesized proteins through the exocytic pathway. Treatment of CHO cells with 1 microM okadaic acid rapidly inhibited movement of a marker protein (vesicular stomatitis virus G protein) from the endoplasmic reticulum to the Golgi compartment. Both okadaic acid and microcystin-LR also inhibited transport in an in vitro assay reconstituting movement to the Golgi compartment, at concentrations equivalent to those required to inhibit phosphorylase phosphatase activity. Inhibition both in vivo and in vitro could be antagonized by protein kinase inhibitors, suggesting that protein phosphorylation was directly responsible for this effect. An early stage in the transport reaction associated with vesicle formation or targeting was inhibited by protein phosphorylation, which could be reversed by fractions enriched in protein phosphatase 2A. Protein kinase antagonists did not inhibit transport between sequential compartments of the exocytic pathway in vitro, suggesting that protein phosphorylation is not itself required for vesicular transport. During mitosis, vesicular transport is inhibited simultaneous to the activation of maturation-promoting factor. It is proposed that the inhibition caused by okadaic acid and microcystin-LR involves a similar mechanism to that responsible for the mitotic arrest of vesicular transport.     

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.     

Role of cell membrane composition in receptor-mediated internalization of vesicular stomatitis virus in human HEp-2 cells

The role of essential fatty acids in membrane functions related to receptor-mediated endocytosis of vesicular stomatitis virus (VSV) was investigated using a human laryngeal carcinoma cell line (HEp-2) grown in chemically defined serum-free medium (DM) to deplete their essential fatty acid contents. VSV replicated much less effectively in HEp-2 cells grown in DM as compared to serum containing complete medium (CM). Observed reduction in the rate of virus multiplication was, at least in part, due to reduced virus penetration which was monitored using VSV labeled with nitroxyl free radicals as electron spin probe. Surface proteins of VSV were labeled with maleimide spin-label, and succinimide spin-label. Ni2+ was used as a broadening agent to identify the spin-label signals from viruses inside the cell. HEp-2 cells and mouse leukemia cell line L1210 treated with 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) cadaverine, an agent previously shown to inhibit the uptake of VSV in vitro, was used as a positive control in some experiments. VSV penetrated less effectively in both DM-grown cells and in CM-grown cells in the presence of dansylcadaverine. Similar results were obtained by monitoring the uptake of 125I-labeled VSV. When HEp-2 cells grown for several generations in DM were incubated with 10% fetal calf serum for 16 h, the cells supported virus replication to a similar extent as the cells grown in CM. In contrast, addition of arachidonic acid restored VSV growth only partially. Continued growth of HEp-2 cells in DM resulted in a shift in fatty acyl chain composition of phospholipids. The results indicate a finite role for essential fatty acids in receptor-mediated internalization of virus particles.