Mar 25, 2015 - is energy-dependent, and the addition of ATP is spe- cifically required. ... Here we present data which demonstrate for the first time the cell-free fatty ... Metabolic Labeling of VSV-infected BHK Cells-Metabolic labeling and preparation .... number of G protein molecules are supposed to be in a compartment ...
Vol. 262, No. 9, Issue of March 25, pp. 42974302.1987 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American h i e @ of Biological Chemists, Inc.
Cell-free Fatty Acylation of Microsomal Integrated and Detergentsolubilized Glycoprotein of VesicularStomatitis Virus* (Received for publication, October 20, 1986)
Dietrich MackSQ, Michael Berger, Michael F. G. Schmidt, and Joachim KruppaSlI From the SInstitut fur Physiologische Chemie, Abteilung Mobkularbiologie, Universitat Hamburg, 0-2000 Hamburg and the Institut fur Virologie,Justus-Liebig-Universitat, 0-6300 Giessen, Federal Republicof Germany
An enzymatic activity associated with intracellular arations as acceptors (Berger and Schmidt,1984a, 1985; Adam membrane fractions of Merwin plasma cell tumor 11, et al., 1984; Slomiany et al., 1984; Towler and Glaser, 1986). baby hamster kidney, and chicken embryo fibroblast So far, attempts to purify acyltransferases to homogeneity cells and bovine kidney has been characterized whichhave been unsuccessful. There is an urgentneed to develop a covalently links fatty acids onto the G protein ofvesic- way of acylating endogenous polypeptidesin order to establish ular stomatitis virus. Exogenous G protein extracted a system for the purificationof palmitoylating andmyristoyfrom native vesicular stomatitisvirus particles can be lating enzymes (Riendeau and Guertin,1986). acylated in vitro only after it hasbeenpreviously To meet thischallenge, we have investigated the conditions deacylated. The fatty acids transferred in vitro are of cell-free acylation using the G protein of vesicular stomasensitive to treatmentwith hydroxylamine, indicating titis virusas an acceptor. There aretwo cell-associated forms an ester linkage. Cell-free acyl transfer was also obof the G protein of VSV,’ termed G1 and G, (Knipe et al., served with endogenous G proteinpresentinmembrane fractions prepared from vesicular stomatitis vi- 1977). The GI species corresponds to the core glycosylated rus-infected cells. In this case, the fatty acids become form of the G protein. Since the high mannose oligosacchalinked to a G protein species (GI) which is not termi- rides are transferred cotranslationally, the GIspecies can be labeled by a short pulse with [35S]methionine (Rothman and nally glycosylated and therefore has not entered the trans-Golgi compartment. The sameG protein species Lodish, 1977; Kruppa, 1979). G1 has a higher electrophoretic also becomes acylated in infected cells during short mobility in SDS-PAGE than Gz and is the kinetic precursor pulses with radioactive palmitic acid. Acylation of the of the GPspecies. Apparently, thecause of the electrophoretic G protein in vitro with free palmitic or myristic acid differencebetween the twospecies is the presence of N is energy-dependent, and the addition ofATP is spe- acetylneuraminic acid on the Gz form. The G, species can be cifically required. Other nucleoside triphosphates can- selectively labeled by a [35S]methioninepulse followed by a not substitute for ATP in the activation of free acyl chase of40-50 min. Trimming of the initially transferred chains. Alternatively, activated fatty acids linked ina GlcNAczMan9Glcs residues of the G1 form occurs during the high energy thioester bond to coenzyme A, e.g. [‘“C] passage of the G protein from the endoplasmic reticulum via palmitoyl-CoA, are suitable lipid donors in the in vitro the Golgi to the plasma membrane. Conversion of the Gl to acylation reactions. Palmitic acid transfer onto G pro- the G, species, giving riset o GlcNAc5Man3Ga13NeuAc3oligotein shows the typical characteristics of an enzyme- saccharide side chains (Reading et al., 1978), is finished prior catalyzed reaction. to the appearance of the G, form on the cell surface where this species becomesintegrated into the budding virus particle (Knipe et al., 1977). The fatty acylation of proteins occurs in many different Here we present data which demonstrate for the first time cell types of widely differing phylogenetic origins (for review, the cell-free fatty acylation of the G protein of VSV, thereby see Schmidt, 1983). Acylation has been connected with imutilizing either exogenous G protein solubilized by detergent portant cellular functions such as membrane anchorage and treatment from viral particles (G2) or endogenous G protein growth regulation (Klockmann and Deppert,1983; Schultz et integrated in microsomal membranes from infected cells (Gl) al., 1985), morphogenesis (Schlesinger and Malfer,1982; Bo- as acceptor polypeptides. The conditions of cell-free transfer lanowski et al., 1984), receptor assembly (Olson et d., 1984), of palmitic acid onto these differentially glycosylated G proprotease protection (Slomiany et al., 1983), and membrane tein species are compared. fusion (Lambrecht and Schmidt,1986). EXPERIMENTALPROCEDURES The transfer of acyl chains onto polypeptides has been Growth and Purification of Virus- The San Juan strain of VSV studied in several cell-free systems by utilizing either various (serotype Indiana) was grown and purified as described previously isolated proteins or peptides orreticulocyte membrane prep- (Graeve et al., 1986). Monolayers of BHK cells were infected with a * This work was supported in part by the Deutsche Forschungsgemeinschaft through SFB 47 and a grant (to J. K.) and by a shortterm fellowship for the collaboration in Giessen from the BoehringerIngelheim Fonds Foundation for Basic Research in Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 Recipient of a followship from the Boehringer-Ingelheim Fonds, Foundation for Basic Research in Medicine. ll To whom correspondence should be addressed.
multiplicity of infection of 0.1 plaque-forming unit/cell. After 18 h of infection, the medium was collected and the cell debris was removed by centrifugation at 10,000 X g. Virus particles were harvested by pelleting at 120,000 X g for 1 h and further purified by density gradient centrifugation at 155,000 X g for 2 h at 4 “C using 10-45% Na+,K+-tartrategradients. The virus band was collected and diluted with 2 volumes of water. Viral particles were recovered by sedimentation at100,000 X g for 1 h at 4 “C, resuspended in 20mM Tris-HC1, The abbreviations used are: VSV, vesicular stomatitis virus; BHK, baby hamster kidney; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; MPC11, Merwin plasma cell tumor 11.
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pH 7.5, at a concentrationof 5-10 mg of viral protein/ml, and stored in small aliquots a t -70 "C. Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Metabolic Labelingof VSV-infected BHK Cells-Metabolic labeling and preparation of cytoplasmic extracts of VSV-infected BHK cells were as described (Garreis-Wabnitz and Kruppa, 1984). Confluent monolayers of BHK cells were grown in Joklik's minimal essential medium and supplemented with 5% (v/v) newborn calf serum (Boehringer Mannheim). Cells were washed and inoculated with VSV at a multiplicity of infection of 40 plaque-forming units/cell. After 30 min of adsorption at room temperature, the inoculum was removed and 10 ml of complete medium was added. The infection was allowed to proceed for 4.5 h a t 37 "C. For labeling with [35S]methionine,monolayers were washed once with prewarmed methionine-free medium and labeled by addition of 50 pCi of [35S]methioninein 3 ml of methionine-free medium for 10 min a t 37 "C. Thereafter,either cells were chased with medium containing nonradioactive methionine for 50 min or cytoplasmic extracts were prepared as described below. For labeling with [3H]palmitic acid, cells were washed with prewarmed serum-free medium and labeled with 400 pCi of [3H]palmitic acid in 3 mlof the same medium for 2 min at 37 "C. Medium was removed, and cells were washed twice with ice-cold phosphate-buffered saline. Cells were scraped off, collected by centrifugation, and lysed in a buffer (15 mMKC1, 1.5 mMMgC12, and 10 mM Tris-HC1, pH 7.4) containing 1%Triton X-100. Nuclei were removed by centrifugation at 800 X g for 5 min, and cytoplasmic extracts were stored at -20 "C. Preparation of Microsomal Membranes Containing Protein Acyltransferase Activity-Microsomal membranes from mouse myeloma cell line MPC 11 were prepared as described by Berger and Schmidt (1984a) with minor modifications. Briefly, MPC 11 cells growing in suspension in roller bottles in Joklik's minimal essential medium (Flow Laboratories, Inc.) were supplemented with 2% (v/v) fetal calf serum and 5.5% (v/v) horse serum (Boehringer Mannheim). At a density of 0.4 X lo6 to 1.0 X lo6cells/ml, they were rapidly cooled on ice, collected by centrifugation, and washed with ice-cold phosphatebuffered saline. After swelling on ice for 20 min in 20 mM Tris-HC1, pH 7.5, cells were homogenized by 15-20 strokes with a tight-fitting Dounce homogenizer. After addition of 0.1 volume of 100 mM NaCl, 30 mM MgClz and three additional strokes, nuclei were removed by centrifugation at 800 X g for 5 min. The postnuclear supernatant was centrifuged a t 10,000 X g for 10 min. The resulting postmitochondrial supernatant was again centrifuged at 100,000 X g for 1h in a Beckman Ti-60 rotor. The microsomal pellet was resuspended in TNE buffer (20 mM Tris-HC1, pH 7.5, 50 mM NaCl, 5 mM EDTA) at a concentration of about 20-40 mg of protein/ml and stored in small aliquots a t -70 "C. Microsomes from VSV-infected MPC 11 cells were prepared as follows. Cells were collected by centrifugation, washed with serum-free medium, and suspended a t a density of lo7cells/ml in this medium. Cells were inoculated with VSV using a multiplicity of infection of 40 plaque-forming units/cell and incubated at room temperature for 30 min, after which the culture was diluted with medium containing serum to a density of 1.5 X lo6 cells/ml and incubated at 37 "C for 4.5 h. Thereafter, cells were rapidly cooled to 0 "C using ice cubes of phosphate-buffered saline, and microsomal membranes were prepared as described above. Microsomal membranes from bovine kidney, BHK, and chicken embryo fibroblast cells were prepared as described previously (Berger and Schmidt, 1984a). Cell-free Acyhtwn of G Protein of VSV-In uitro acylation reactions routinely contained about 250 pg of microsomal protein, 1 mM ATP, and 25 pCi of[3H]palmiticacid in TNE buffer in afinal volume of 250 pl.In vitro reactions for the acylation of endogenous G protein in microsomes from VSV-infected cells contained no further additions. Reactions testing the reacylation of exogenous, deacylated G protein contained 25 pl ofNonidet P-4O-solubilized, deacylated VSV (about 100-130pgof viral protein), giving a final Nonidet P-40 concentration of less than 0.1%. The reaction mixtures were incubated at 28 "C for 45 min. Incubations were terminated by the addition of 1ml of chloroform/methanol(l:2, v/v). For the biochemical characterization of the in vitro transfer of [3H]palmitic acid on endogenous and exogenous VSV G protein, parameters of this reaction protocol were modified as described in detail in the respective figure legends. Deacyhtion and Solubilization of Exogenous Acceptor ProteinPurified VSV (5-10 mg of viral protein/ml) was solubilized with 1%
Nonidet P-40 for 15 min at room temperature. 2.5 M hydroxylamine, pH 7.0, was added to give a final concentration of hydroxylamine of 0.54 M. Deacylation of the viral G protein proceeded for 4 h at room temperature. Aliquots of the deacylated viral acceptor protein were utilized in in vitro acylation reactions. VSV preparations which were labeled with [3H]palmitic acid in vivowere used as controls. The conditions described above resulted in a complete release of fatty acids bound to the G protein. Polyacryhmide Gel Electrophoresis-Protein precipitates were solubilized in sample buffer containing 4% SDS, 160 mM Tris-HC1, pH 6.8, and 5% (v/v) 8-mercaptoethanol by heating to 96 "C for 5 min. Proteins were separated on discontinuous 10% SDS-polyacrylamide gels as described by Laemmli (1970). Gels were fixed and stained by Coomassie Blue in 10% acetic acid, 40% methanol and destained in the same solvent. Gelswere prepared for fluorography using 1 M sodium salicylate according to Chamberlain (1979), dried, and exposed on Cronex 4 x-ray film (Du Pont) at -70 "C. For quantitative evaluation, fluorograms were scanned using an Ortec densitometer as described (Kruppa, 1983). The peak areas were determined, and relative intensities were calculated by setting the maximal intensity of the respective experiment as 100%. Materials-Nonidet P-40 and TritonX-100 were from Sigma.[35S] Methionine (specific activity 1190 Ci/mmol) and [1-"Clpalmitoyl coenzyme A (specific activity 60 mCi/mmol) were purchased from Amersham Corp. [9,10-3H)Palmitic acid (specific activity 30 Ci/ mmol) and [9,10-3H]myristic acid (specific activity 12.9 Ci/mmol) were obtained from New England Nuclear. RESULTS
Cell-free Acylation of Exogenous and Endogenous G Protein of VSV-Initial experiments set outto reacylate the G protein of VSVin purified viral particles. Microsomes from uninfected MPC 11 cells were used as source of protein acyltransferase. The exogenous viral acceptor was prepared by solubilizingthe lipid envelope of the virus with Nonidet P-40. A complete release of fatty acids bound to G protein was achieved by hydroxylamine (cf. "Experimental Procedures"; Magee et al., 1984). Incubation of the microsomal membranes with the detergent-treated glycoprotein resulted in a transfer of [3H] palmitic acid onto the exogenous, deacylated G protein. This transfer was completely dependent on the presence of ATP (Fig. 1, lanes 2 and 3 ) .Only verylittle transferof [3H]palmitic acid occurred onto exogenous, native VSG G protein (Fig. 1, lanes 4 and 5 ) . Native G proteinwas prepared similarly to the deacylated acceptor but treated with Tris-HC1 buffer instead of hydroxylamine as described in the legend of Fig. 1. Treatment with Tris-HC1 does not lead to a release of fatty acids bound to the G protein (Magee et al., 1984). The exogenous, deacylated VSV G protein differs in its oligosaccharide moieties from the newly synthesized acceptor which is acylated in uiuo. Transfer of palmitic acid onto viral proteins is believed to occur in cis-Golgi vesiclesor in the late endoplasmic reticulum (Schmidt and Schlesinger, 1980; Dunphy et al., 1981; Quinn et al.,1983;Berger and Schmidt,1985). In these compartments, preliminary trimming of the high mannose oligosaccharides of the G protein occurs, but the processing of the oligosaccharides continues subsequently during the passage of the G protein through the trans-Golgi region en route to theplasma membrane where the glycoprotein accumulates and becomes integrated into budding virus particles (Kornfeld and Kornfeld, 1985). During the intracellular transport of the G protein from its site of synthesis in the endoplasmic reticulum to the plasma membrane, a small number of Gprotein molecules are supposed to be in a compartment justprior to thesite of fatty acid transfer. Thus, microsomal membrane preparations of VSV-infected cells should contain the native acceptor molecules for the in vitro acylation reaction. To test this hypothesis, we prepared microsomal membranes from VSV-infected MPC 11 cells as described under
Cell-freeAcylation of VSV G Protein A
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FIG.1. Cell-free acylationof exogenous, deacylatedand endogenous G protein of VSV with [3H]palmitic acid. Proteins were separated by SDS-PAGE. A fluorogram of the gel is shown after microsomal 5 weeks of exposure. Lanes 2-5 contained 250pgof protein from uninfected MPC 11 cells. Lanes 2 and 3 also contained about 100 pg of exogenous, deacylated viral proteins, and lanes 4 and 5 contained an equal amount of exogenous, nondeacylated viral proteins which were treated in parallel as described under “Experimental Procedures” except that 2 M Tris-HC1, pH 6.8, was used in place of hydroxylamine. Lanes 6 and 7contained 250 pg of microsomal protein from VSV-infected MPC 11cells. In vitro acylation reactions were carried out as described under “Experimental Procedures” except that in lanes 3, 5, and 7 ATP was omitted from the reaction mixtures. Lane 8 contains a cellular extract of VSV-infected BHK cells labeled in vivo for 2 min with [3H]palmitic acid. Lane 1 contains VSV marker proteins.
FIG.2. In vivo labeling of G protein with [SH]palmitic acid in VSV-infected BHK cells. VSV-infected BHK cells were labeled with [3H]palmitic acid and [35S]methionineas described under “Experimental Procedures.” Cytoplasmic extracts were prepared, and proteins were separated by SDS-PAGE. A fluorogram of the gel is shown after 4 days of exposure. VSV-infected cells were labeled with [35S]methioninefor 10 min (lane2) or for 10 min and then chased for an additional 50 min (lane 3) or with [3H]palmitic acid for 2 min (lane4 ) . Lane 1 contains [3H]leucine-labeledVSV proteins as markers.
“Experimental Procedures.” Incubation of these microsomes for in vitro acylation without any detergents resulted in an ATP-dependent transfer of [3H]palmitic acid onto the G protein of VSV (Fig. 1, lanes 6 and 7). The question then is whether the endogenous G protein of the microsomal membranes, which can be labeled with [3H]palmitic acid in vitro, represents the same G protein species which is normally acylated in vivo. To ascertain the primary acyl acceptor, we labeled VSV-infected BHK cells with a 2-min pulse of [3H] palmitic acid(Fig. 2, lane 4). A comparison of G protein labeled either with a 10-min pulse of [35S]methionine(GI; Fig. 2, lane 2) or a 10-min pulse and 50-min chase (G2; Fig.2, lane 3) clearly indicated that in vivo the GI species is acylated first with [3H]palmiticacid. Since the endogenous G protein in the microsomal membrane fraction which has been acylated with [3H]palmiticacid in vitro co-migrates on SDS-polyacrylamide gels with the GI protein which has been pulse-labeled with [3H]palmiticacid in vivo (Fig. 1, lanes 6 and 8),we concluded that in vivo and in vitro [3H]palmitic acid is first transferred onto the GI form of the G protein. [3H]Palmitic acid seemed to be covalently bound after the transfer to theexogenous as well as theendogenous G protein substrates. The labeled G protein was resistant to extraction with organic solvents, heating to 96 “C in sample buffer, reduction by P-mercaptoethanol, and SDS-PAGE conditions. After in vitro transfer, the labeled palmitic acid was bound to the G protein moiety in an ester-like linkage. Treatment of gels containing separated G protein bands with 1 M hydroxylamine, pH 6.8, for 4 h gave rise to a substantial release of labeled fatty acid from exogenousand endogenous G protein, whereas an identical gel which was treated with 1 M TrisHC1, pH 6.8, for 4 h did not lose any radioactivity (Fig. 3).
These hydrolytic conditions did not result in a degradation or elution of viral polypeptides from the gels (Fig.3), as has been controlled by [3H]leucine-labeledVSV marker proteins. Since VSV has a broad host range and fatty acid acylation of the G protein has been reported for cell lines of several eucaryotes (Schmidt and Schlesinger, 1979,1980; Dunphy et al., 1981; Rose et al., 1984), we tested in vitro microsomes of chicken embryo fibroblasts, bovine kidney,and BHK cellsfor the presence of protein acyltransferase activity using exogenous, deacylated VSV G protein as an acceptor (Fig. 4,lanes 1 3 ) . Different activities of the protein acyltransferase were observed for the respective microsomal preparations. In vitro transfer of acyl chains onto exogenous, deacylated G protein could be demonstrated also by using [3H]myristi~ acid together with ATP or [‘4C]palmitoyl-CoA (Fig.4,lanes 6 and 7). The other radioactively labeled polypeptides detected on this gel could represent endogenous acyl proteins, which are uncharacterized at thepresent time (Riendeau and Guertin, 1986). Reaction Conditions for Cell-free Transfer of pH]Palmitic Acid onto Exogenous and Endogenous G Protein of VSVTransfer of [3H]palmitic acid was strictly dependent on the presence of ATP (Fig. 1, lanes 3, 5, and 7). It became detectable at concentrations greater than 250 p~ ATP and reached a maximum at 1mM ATP (data not shown). Transfer of [3H]palmitic acid onto exogenous, deacylated G protein increased with increasing amounts of microsomes and reached a plateau at 250 pg of microsomal protein (Fig. 5B). Transfer also depended on the amount of deacylated VSV acceptor proteins, reaching a plateau at 75 pg of the viral protein (Fig.. 5A). [3H]Palmitic acid was transferred in the cell-free system enzymatically because the microsomes
Cell-free Acylation of VSV G Protein
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FIG.5. Conditions of cell-free [SH]palmitic acid transfer onto exogenous, deacylated and endogenous VSV G protein.
M -
Several parameters of standard in vitro acylation reactions were varied as described below. After separation of proteins by SDS-PAGE, fluorograms were quantitatively evaluated as described under “EXFIG.3. Deacylation by hydroxylamine of in vitro acylated perimental Procedures.” A, standard cell-free acylation assays conG protein of VSV. Exogenous, deacylated VSV G protein and taining increasing amounts of deacylated acceptor protein were inendogenous G protein were acylated i n vitro in reactions as described cubated while keeping the final concentration of Nonidet P-40 conin the legend ofFig. 1. Corresponding aliquots were separated by stant. B, standard cell-free acylation reactions containing about 130 SDS-PAGE. Replicate gels were treated with 1 M hydroxylamine, pH pg of deacylated viral acceptor protein were incubated with increasing 6.8, or 1 M Tris-HC1, pH 6.8, for 4 h. After one wash with water, gels amounts of microsomal proteins of uninfected MPC 11 cells. C, were fixedand treatedfor fluorography using dimethyl sulfoxide/2,5- varying amounts of microsomes of VSV-infected cells containing diphenyloxazoleas described by Laskey (1980).A fluorogram of those endogenous G protein were incubated in standard in vitro acylation gels is shown after 5 weeks of exposure. Lanes 1 and 4, [3H]leucine- assays. D,temperature dependence of cell-free acylation of exogenous, labeled VSV markerproteins; lanes 2 and 5, in vitro reacylated deacylated VSV G protein (0)and endogenous G protein in microexogenous G protein; lanes 3 and 6, in vitro acylated endogenous G somes of VSV-infected cells (@. Standard cell-free acylation reactions protein. as described under “Experimental Procedures” were incubated a t varying temperatures.
1 2 3 4 5 6 7
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FIG.4. I n vitro acylation of exogenous, deacylated G protein using microsomes from different species or different acyl donors. I n vitro acylation reactions contained about 120 pg of deacylated VSV protein and 200 pg of microsomal protein prepared from chicken embryo fibroblast cells ( l a n e I ) , bovine kidney ( l a n e 2), and BHK cells (lanes 3-7). Each reaction contained 2 m M ATP and 25 pCiof [3H]palmitic acid (lanes 1-5) or 50 pCi of [3H]myristic acid ( l a n e 6). No ATP was added to the reaction containing 0.5 pCi of [“C]palmitoyl-CoA ( l a n e 7). Microsomes were boiled for 5 min prior to incubation ( l a n e 5), or thereaction mixture was incubated at 0 “C ( l a n e 4). Proteins were separated by SDS-PAGE. A fluorogram of the gel is shown after 5 weeks of exposure.
lost their acyltransferase activity after boiling, and transfer was also blocked at 0 “C (Fig. 4, lanes 4 and 5 ) . These latter characteristics of the acyl transfer reactions were identical for microsomes from uninfected or VSV-infected MPC 11 cells (data not shown). The in vitro transfer of [3H]palmiticacid on the endogenous
G protein of VSV-infected microsomal membranes was also dependent on the concentration of microsomal protein. Acylation was first detectable at a concentration of 125 pg of microsomes (data not shown) and reached a maximum at 250-500pgof microsomal protein (Fig. 5C). Beyond this concentration, transfer of [3H]palmitic aciddeclined. The decrease of labeling may result from the dilution of the labeled fatty acid in increasing amounts of endogenous lipids and, in addition, most probably by an endogenous inhibitory activity in the microsomal preparation. This effective lossof label for the protein acylation would decrease the specific radioactivity in the G protein. The temperature dependence of the cell-free transfer of [3H]palmiticacid was comparedfor exogenous, deacylated G protein and endogenous G protein in microsomes from infected cells. For both acceptors, transfer of labeled palmitic acid was detectable between 23 and 37 “C with an optimal temperature of 28 “C for the exogenous acceptor and 32 “C for the endogenous acceptor (Fig. 50). Nucleoside Triphosphate Activation of PHlPalmitic Acid for AcylationReaction-Although previous experiments suggested palmitoyl-CoA as the immediate lipid precursor for fatty acid transfer onto acyl proteins in vivo and in vitro (Berger and Schmidt, 1984a, 1984b; Slomiany et al., 1984)) one cannot exclude that other mechanisms for the activation of fatty acids for the transfer ontoacyl proteins may exist.In analogy to the activation of fatty acids by the acyl-CoA synthetase, where an acyl-AMP intermediate is formed, the protein acyltransferase.might transfer fattyacids via an acylnucleotide intermediate directly onto acyl proteins (Groot et
of Acylation Cell-free al., 1976). Fatty acids can also be activated by enzymes using G T P instead of ATP. Furthermore, it is known that other nucleoside triphosphates are used for the activaton of acyl groups in lipid biosynthesis (Bell and Coleman, 1980). We therefore tested whether [3H]palmitic acid can be transferred onto exogenous, deacylated and endogenousG protein in the presense of 1 mM ATP, CTP, GTP, or UTP, respectively. Transfer of [3H]palmitic acid onbothacceptorsis considerably decreased in the presence of CTP, GTP, and UTP ascompared to ATP. Nevertheless,very low labeling is observed using GTP and UTP, whereas almost no radioactivity is detected using CTP (data not shown). Presently, it is not clear whether these nucleotides are involved directly in the activation of the long chain fatty acid or are used form to ATP. DISCUSSION
We report here the ATP-dependent transfer of [3H]palmitic acid onto exogenous, deacylated G protein using microsomes from uninfected MPC 11 cells as source of protein acyltransferase as well as onto endogenous G protein integrated in microsomes from VSV-infected MPC 11 cells. Acyl transfer was highly specific since exogenous G protein extracted from purified viral particles could only serve as an acceptorwhen its protein-bound fatty acidswere released by treatment with hydroxylamine. Only very low amounts of [3H]palmitic acid could be transferred to the exogenous, nondeacylated G protein (Fig. 1, lanes 2-5). This observation suggests that the G protein in mature virus particles is almost completely modified by covalently bound fatty acids. The observed cell-free transfer of [3H]palmitic acid onto the G protein resembles fatty acid transfer i n vivo because the GI speciesbecomes specifically acylated invitro whenendogenousG protein contained inmicrosomal membranes from VSV-infected cells was used as an acceptor (Fig. 1, lanes 6-8). The Gl species is also labeled in VSV-infected BHK cells i n vivo after very short pulses of [3H]palmiticacid (Fig. 2). During intracellular transport, the G1 species is modified by trimming and processing to theG, species. The GP speciesof the viral glycoprotein has acquired neuraminicacid on its oligosaccharide side chains by passing through theGolgi compartment on itsway to the plasma membrane where it becomes integrated in the budding virus particle (Knipe et al., 1977). Taken together, the results indicate that the viral integrated G2 protein can only be acylated i n vitro after release of the already bound fatty acids. Consistent with this interpretation is the fact that in cell-free systems only the GI but not the GZ species of the G protein of intracellular membrane vesicles can be acylated. From these observations, one would assume that also in the in vitro transfer reaction [3H]palmitic acid is linked to the correct acylation site. The specific ester linkage of [3H]palmitic acid transferred i n vitro to exogenous and endogenous G protein is further confirmed by the sensitivity of these bonds to hydroxylamine (Fig. 3; Magee et al., 1984; Schmidt and Lambrecht, 1985). The biochemical conditions for the transferof [3H]palmitic acid are very similar for the exogenous, deacylated and the endogenous G protein with respect to 1) ATP dependence,2) optimum concentrationof microsomal proteins presentin the of incubation. reaction, and3) dependence on the temperature The minor difference in the optimum temperature for the acyl transfer onto these acceptorsi n vitro might be explained by the presence of detergent in the incubations containing the exogenous G protein or by slight conformational differences between the terminally glycosylated G protein of viral particles or the core glycosylated GI species of microsomes
VSV G Protein
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from infected cells (Kornfeld and Kornfeld,1985). The transfer of [3H]palmiticacid onto G protein i n vitro is ATP-dependent, i.e. energy-dependent (Fig. 1, lanes 3, 5, and 7). Substitution of ATP by CTP, GTP, and UTP dramatically decreased the transfer of [3H]palmiticacid onto both G protein acceptors. The low yield observed in the presence of G T P and UTP can reflect activation of palmitic acid by a GTPdependent acyl-CoA synthetase or by regeneration of ATP from endogenous ADP by nucleoside-diphosphate kinase (Groot et al., 1976). Since the G protein of VSV may i n vitro be acylated independently from ATP using [14C]palmitoylCoA as lipid donor,thisismost probably theimmediate precursor for transfer of palmitic acid onto the G protein. This fact is in agreement with observation the that palmitoylCoA is one of the predominantlipids labeled after very short pulses with [3H]palmiticacid i n vitro and i n vivo (Berger and Schmidt, 1984a, 1984b). The biochemical conditions for the i n vitro transfer of palmitic acid on exogenous,deacylated El glycoprotein of Semliki Forest virus and Gonprotein of VSV are very similar withrespecttoATP dependence, concentrations of viral acceptor proteins and microsomal protein, and temperature of incubation (Berger and Schmidt,1984a). Indirect evidence has been obtained for the linkages of fatty acids to the El glycoprotein and the G protein. Ester bonds to serine have been suggested for the El glycoprotein (Schmidt and Lambrecht, 1985), whereas a thioester bond toa cysteine residue in thecytoplasmic domain hasbeen proposed forthe G protein (Rose et al., 1984; Magee et al., 1984). If this is thecase and a single protein acyltransferasemodifies both of these proteins, this enzyme has to be able to form acyl linkages via ester and thioester bonds,respectively. Adams and Rose (1985) produced several deletion mutants inthetransmembranedomain of the G protein by sitedirected mutagenesis of the cDNA clone of the G protein. These deletions were located 6-10 amino acids proximal to the putative acylation site in the cytoplasmic domain of the the G protein. G protein molecules having a deletion of more than 12 out of 20 amino acids of the transmembrane domain were not labeled by [3H]palmitic acid after expression of the corresponding cDNAs, although these proteins had a transmembrane orientation and were properly glycosylated. Unfortunately,thesemutants which lacked acylation were also impaired in intracellular transport and accumulated in the endoplasmic reticulum or Golgi apparatus (Adams and Rose, 1985). It is difficult tointerpretthese negativeresults.A conformational change in the protein effected by the deletion of part of the transmembrane domain might have impaired the intracellur transport of the respective molecule and thus preclude that it ever reaches the site where the protein acyltransferase is active. Therefore, the studyof acyl transfer i n vitro onto peptides prepared by fragmentation of the G protein or onto synthetic peptides may help to elucidate the structural requirements for peptides to serve as acceptors for the protein acyltransferase. Acknowledgments-The technical assistance of Eva Kro11 is gratefully acknowledged. We thank Dr. G. Koch for his continuous support and Dr. G. Koch and Dr. R. hell for critical reading of the manuscript. REFERENCES Adams, G. A., and Rose, J. K. (1985) Cell 4 1 , 1007-1015 Adam, M., Rodriguez, A., Turhide, C., Larrick, J., Meighen, E., and Johnston, R. M. (1984) J. Biol. Chem. 2 5 9 , 15460-15463 Bell, R. M.,and Coleman, R. A. (1980) Annu. Reu. Biochem. 49,459487 Berger, M., and Schmidt, M. F. G. (1984a) J.Biol. Chem. 259,72457252
4302
ofAcylation Cell-free
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