We present here a new method for inhibiting protein acetylation in a rabbit reticulocyte cell-free protein- synthesizing system. This procedure utilizes S-acetonyl.
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 256. No. 15. Iswe of August 10, pp. 8149-8155, 1981 Printed in U.S.A.
NH2-terminal Acetylationof Dictyostelium discoideum Actin in a Cellfree Protein-synthesizing System* (Received for publication, February 9, 1981, and in revised form, April 14, 1981)
Peter Rubenstein, Patricia Smith, Jon Deuchler,and Kent Redman From the Department of Biochemistry, College of Medicine, Uniuersity of Zowa, Zowa City, Iowa 52242
W e p r e s e n there a new method for inhibitingprotein caused an increase in the nonacetylated form at the expense acetylation in a rabbit reticulocyte cell-free proteinof the maturespecies (4). synthesizing system.This procedure utilizes S-acetonyl Actin is one of a very few examples of a eukaryoticcytosolic coenzyme A, a nonreactive acetyl-coA analogue, as an protein inwhich the acetyl moiety is attached to anN”-amino inhibitor of the NHz-terminalprotein acetyltransferase group of an acidic amino acid, either aspartic or glutamic acid, in this lysate. With this procedure, we can make, in depending on the source of the actin (7-10). For other acetvitro, Dictyostelium discoideum actin whichis 85%non- ylated proteins, the most common acetylated NHZ-terminal acetylated but fully translated. amino acids are alanine, threonine, serine, glycine, and meWith the fully translated but nonacetylated actin as thionine (11). a substrate, the actin canbe almost completely acetyla- Actin is unusual in another respect concerning processing ted post-translationallyin an acetyl-CoA-dependent at the NH2 terminus. Eukaryotic proteins are initiated with system after the actin has left t h e ribosome. Using methionine (11, 12). In many cases, the methionine is then formylated and nonformylated [35S]Met-tRNAfMetas a source of label and in conjunction with detailed peptide cleaved by a protease to leave the second amino acid as the mapping experiments with trypsin and thermolysin, new NH:! terminus. In these cases, however, for methionine the in vitro acetylation is shown to occur at t h e NH2 cleavage to occur, the second amino acid must be small and terminus of the newly synthesized actin.Furthermore, uncharged, such as alanine, serine, threonine, or glycine (13). the initiator methionine residue, contrary to expecta- Since the second amino acid coded for by the actin gene is tion, is not cleaved off but remainsstable for at least 50 aspartic or glutamic acid, methionine cleavage for this protein would not be expected. The original coded amino acid semin. Thus, in the acetylating reticulocyte lysate system, the primary complete translation product in actin syn- quence for D. discoideum actin is Met-Asp-Gly-Glu-Asp (9). thesis is Ac-Met-Asp and not Ac-Asp. Following processing, it becomes acetyl-Asp-Gly-Glu-Asp (7, 10). In comparison, bovine cardiac troponin C has the NH,terminal sequence acetyl-Met-Asp (14). Even thoughits NH,terminal dipeptide is identical with actin’s, its methionine is Actin, a major contractileproteinin both muscle and not cleaved, the usual result for such a situation. nonmuscle cells, contains two post-translational modificaBecause of this unusual behavior of actin, we undertook a tions. One is an NHs-terminal acyl group shown to be an detailed examination of the events occurring during the synacetyl residue where examined in detail.The second is a single thesis and processing of the NHp-terminal of D. discoideum 3-methylhistidine. Little is known either about the enzymes actin in a rabbit reticulocyte cell-free protein-synthesizing carrying out these modifications or their functional imporsystem. In this paper, we reportan improved method of tance. Previous in vivo studies of actin biosynthesis revealed inhibiting endogenous protein acetylation in vitro based on the presence of rapidly labeled but nonaccumulating actin use of a nonreactive acetylcoenzyme analog, S-acetonyl-CoA species in cultured rat skeletal muscle cells (2), Drosophila (15). Using this procedure, we have achieved the synthesis of melanogaster (3), Dictyostelium discoideum (4), and chick 8540% nonacetylated actin which we can subsequently acebrain ( 5 ) .These species differed from their mature countertylate in an acetyl-CoA-dependent reaction. We show that parts by a single positive charge leading to speculation that nonacetylatedactin is not more susceptible to proteolytic these rapidly labeled species were nonacetylated forms of the degradation in the reticulocyte lysate than is the acetylated mature actins. With restrictive labeling conditions using species. We demonstrate unequivocally that thein vitro acet[3H]acetate, we demonstrated for D. discoideium that the ylation occurs at the NH2terminus of the actin polypeptide. rapidly labeled species was nonacetylated while the mature Finally, we demonstrate that, in the reticulocyte lysate sysform was acetylated andthatthese two forms remained tem, after a 50-min incubation, the newly synthesized actin constant through the D. discoideum life cycle (4). Furtherstill possesses a methionineat theNH2 terminus and thatthis more, we showed that translation of D.discoideum RNA in a methionine is most probably acetylated. mRNA-dependent rabbit reticulocyte lysate translation system produced an acetylated form of actin. Partial inhibition EXPERIMENTALPROCEDURES of endogenous acetylation by the procedure of Palmiter (6) * This work was supported by Grant GM-24702 (to P. R.)from the National Institutes of Health. A preliminary account of this work was presented at the American Society of Biological Chemists meeting in New Orleans, June, 1980 (1).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.
Materials-Materials for isoelectric and gel electrophoresis were those described by O’Farrell (16). ~-[”‘SS]Methionine(loo0 Ci/mmol) was obtained from Amersham. Acetyl-coA, pig heart citrate synthetase, cis-oxaloacetic acid, rabbit liver tRNA, creatine phosphokinase, hemin, and CoA were obtained from Sigma. Acetonyl-CoA was prepared according to the method of Rubenstein and Dryer (15). mRNAdependent rabbit reticulocyte lysates werepreparedaccording to Pelham and Jackson (17). D. discoideum whole cell RNA was obtained from cells of the Ax-3 strain grown in HL-5 medium by the
8149
8 150
Acetylation
Actin
procedure of Ullrich et al. (18). XR-2 x-ray film was obtained from Eastman Kodak, and cellulose thin layer plates, 100 pm thick, were purchased from Eastman. Tosyl phenylalanyl cholormethyl ketonetreated trypsin and microccocal nuclease were purchased from Worthington Biochemicals, and thermolysin was purchased from Boehringer. All other chemicals were reagent grade. Preparation of [s5SJMet-tRNAfM" a n d N-F~rmyl-[~'SJMettRNAfMC'-[''5SS]Met-tRNAr"P' was prepared according to the procedures of Stanley (19) and Levy and Baglioni (20). In short, rabbit liver tRNA was charged with [""Slmethionine (1000 Ci/mmol) by a soluble Escherichia coli extract. In this system, only the initiator tRNA is charged. To make the formyl derivative, 0.25 mM calcium leucovorin was included in the reaction mixture. Cell-free Actin Synthesis-The basic reaction mixture usedfor synthesis of D. discoideum actin contained 35 p1 of mRNA-dependent rabbit reticulocyte lysate supplemented with the requisite energy generating system, 19 unlabeled amino acids, excluding methionine, 30 pCi of L-[""Slmethionine (1000 Ci/mmol), and 15 pg of whole cell D. discoideum RNA in a total volume of 50 pl. Incubations were carried out for 30-45 min a t 25 "C. When the ['"S]Met-tRNArM" or its formylated derivative was used, 25 pCi of the charged tRNA was used/50-p1 assay. Also included was 75 PM unlabeled methionine to ensure that any methionine hydrolyzed from the tRNAiw" could not be added to tRNA.,""' by the lysate-charging enzymes. Upon completion of the reaction, samples were prepared for isoelectric focusing and subjected to two-dimensional gel analysis using isoelectric focusing in a pH 5-7 gradient in the first dimension and sodium dodecyl sulfate-gel electrophoresis in the second (16). Following fixing and drying of the gels, fluorograms were generated using 1 M sodium salicylate as the fluor and preflashed film (21-23). These were then quantitated by densitometric analysis of the actin region of the autoradiogram using a Transidyne General RFT-I1 scanning densitometer a t 550 nm. Enhanced Blockage of NH2-terminal Protein Acetylation-To 50 pl of a complete reticulocyte translation mixture minus mRNA was added 1 mM oxaloacetate, pH 7.5, freshly prepared, and 35 units/ml of citrate synthetase (6).The synthetase was made by centrifuging an ammonium sulfate suspension of the enzyme a t 50,000 X g for 5 min in a Beckman Airfuge, carefully removing the ammonium sulfate supernatant solution, and dissolving the pellet in distilled water. The reaction mixture was incubated at 25 "C for 7 min. Then 75 p~ Sacetonyl-CoA (15) was added with the mRNA, and the translation reaction was allowed to occur for the desired period of time. Isolation of D. discoideum Actin Labeled in Vivo or in Vitro with [35SJMethionine or Cysteine-D. discoideum actin was labeled in vivo with [""Slmethionine or [%]cysteine and isolated as described by Simpson and Spudich (24) with the following modifications. Labeled amino acid was useda t a level of 12 mCi/l. The growth medium included the amino acid mixture of Franke and Kessin (25) except that the methionine or cysteine was reduced by 75% depending on which label was used. The remainder of the medium contained the sugar, phosphate, and Vi of the yeastextract specified for HL-5 medium (26). For in vitro-synthesized actin, the standard5O-pl cell-free reaction mixture was scaled up to 1 ml. After a 55-min incubation, the mixture was made 10%in formamide, 2 mM in CaCI2, and applied to a 0 . 5 4 DNAse-I-Sepharose column. The column was washed andeluted with buffered 40%formamide as described by Zechel (27).The eluted material, following addition of 300 pgof unlabeled D. discoideum actin (28), was diluted 1:l with 10 mM Tris-HC1, pH 8.0, containing 0.2 mM MgATP and 2 mM mercaptoethanol and applied to a 0.5-ml DEAE-cellulose column equilibrated in the same buffer. The column was washed with 5 ml of the same buffer containing in addition 0.1 M KCI. The radioactive actin was eluted with the buffer containing 0.6 M KCI. The eluted material was then dialyzed extensively against H20 and lyophilized to dryness. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this material and subsequent fluorography resulted in a single labeled polypeptide co-migrating with authentic actin. Peptide MappingStudies-Either in vivo or in vitro labeled purified actin was dialyzed extensively against H2O to remove all traces of CI-. The protein was then lyopholized to dryness, dissolved in 97% formic acid and performic acid oxidized according to Hirs (29). The performic acid was removed by lyophilization, the protein was dissolvedin 0.1 M NHnHCO:,, pH 8.0, and tosyl phenylalanyl chloromethyl ketone-treated trypsin was added a t a protein:enzyme ratio of 201. The digestion was carried out for 150 min at room temperature, and the reaction mixture was quenched by freezing and lyophilized to
dryness. The residue was dissolved in pH 6.5 pyridine acetate buffer (pyridine:acetic acidHZ0, 25:1:225), applied to a cellulose thin layer plate, and electrophoresed for 75 min in the same buffer a t 400 V using orange G as a marker. An autoradiogram was made and the desired radioactive peptide elutedfrom the cellulose with the pyridine acetate (7). For thermolysin digestion, the labeled tryptic peptide was dissolved in 15 pl of 10 mM NaHCO:,, pH 8.0, containing 2 mM CaCI2.Thermolysin, 2 pg in 10 pl of the same buffer, was added, and digestion was carried out for 2 h a t 30 OC. The reaction mixture was applied directly to a cellulose thin layer plate, and electrophoresis was carried out as described above. Thin LayerElectrophoresis-Electrophoresis of peptides was carried out on 100-pm thick cellulose layers using the apparatusdescribed by Kempe et al. (30). RESULTS
Inhibition ofActin Acetylation-In order to studythe NH2terminal acetylation of D.discoideum actin in vitro, we required as a substrate actinwhich was 85-9076 nonacetylated. Otherwise, background levels against which acetylation could be measured would betoo high. Initial attempts toblock actin acetylation in vitro using the Palmiter procedure (6) were largely unsuccessful. A t best, we achievecd with this method 50% levels of nonacetylated actin, and in most cases we achieved 25-3076 (Fig. l b ) . To circumvent this problem, we tried an acetyl-coAanalog, S-acetonyl-CoA, asan acetylation inhibitor (Fig. 2). This compound contains a nonreactive thioether moiety as opposed to the usual thioester group, and it was found to be a good competitive inhibitor of three acetyl-CoA-requiring enzymes
FIG. 1. Enhanced inhibition ofrabbit reticulocyte lysate protein acetylation with S-acetonyl-CoA. Proteins were synthesized in a mRNA-dependent rabbit reticulocyte lysate containing ["SS]methionine under the direction of D. discoideum whole cell RNA as described under "Experimental Procedures." In all four panels: A, acetylated actin;X,nonacetylated actin; Y , a 25,000-dalton acetylated protein; 2,position of its nonacetylated form. Panel a, endogenous acetylation was uninhibited;panel b, oxaloacetate ( 1 mM) and citrate synthetase (35 units/ml) were added to the lysate a t room temperature for 7 min prior to RNA addition; panel c, S-acetonyl-CoA (100 p ~ was ) added to the lysate with the RNA; panel d, the lysate was treated with citrate synthetase andoxaloacetate as described in panel b. Then 75 p~ S-acetonyl-CoA was added with the RNA.Actin regions of autoradiograms of the gels are shown.
Actin Acetylation
8151
0 c~A-s-cH~-C-CH~ FIG. 2. Structural formula of S-acetonyl-CoA.
X
A
j
x
A
FIG. 4. Post-translational acetylationof D. discoideium actin lysate. Nonacetylated actin, labeled with in the rabbit reticulocyte
FIG. 3. Dependence of actin acetylation inhibition on increasing concentrationsof S-acetonyl-CoA. A mRNA-dependent rabbit reticulocyte lysate was pretreated with oxaloacetate and citrate synthetase as previously described. Acetonyl-CoA was added at the concentrations shown with D.discoideurn RNA. Following a 30-min incubation at 25 "C, the relative amounts of acetylated and nonacetylated actin in each point were determined as a function of the concentration of added acetonyl-CoA.
[".S]methionine, was synthesized for 30 min in an enhanced acetylation-blocked reticulocyte lysate as described under "Experimental Procedures." Cycloheximide (100 pg/ml) was introduced to block further translation. The reaction mixture was divided into two aliquots. To one, 2 mM acetyl-coA was added and to the other, an equivalent amount of water was added. The samples were incubated for an additional 30 min. Two-dimensional gels of the samples were made and used to generate autoradiograms. a, no acetyl-coA was added; b, 2 mM acetyl-coA was added. The actin ( A ) regions of the gels and their densitometric tracings are shown.
(15). However, S-acetonyl-CoA was no more effective at a concentration of 100 FM in blocking endogenous actin acetylation than was the citrate synthetase-oxaloacetate method (Fig. IC). Wethen attempted to use the two proceduresin tandem. The mRNA-dependent reticulocyte lysate was f i i t treated as described by Palwith citrate synthetase and oxaloacetate miter (6). Then S-acetonyl CoA, 75 p ~ was , added to the lysate with the RNA. Useof this combined procedure resulted in the synthesis of 85% nonacetylatedactin (Fig. Id). An examination in greater detailof the dependenceof acetylation inhibition onthepresence of acetonyl-CoArevealed that approximately 7 PM acetonyl-CoA was required for50% inhibition (Fig. 3). We continued touse 50-75 p~ acetonyl-CoA in 0 15 30 45 60 our lysates to ensure thatinhibition of acetylation would be TIME (MIN.) maximal since some lysate preparations contained more acFIG. 5. Time course of actin acetylation. Nonacetylated D. etyl-coA than others. discoideurn actin was synthesized in oitro for 30 min. Cycloheximide, Enzymatic Acetylation of Nonacetylated Actin-We next 100 pg/ml, was added to block further translation and 1.6 mM acetylsought to determine whether fully translated but nonacety- CoA was introduced. Acetylation was allowed to proceed at 25 "C. lated actin could be subsequently acetylated. Actin was syn- Aliquots were withdrawn a t various times and assayed for the extent of actin acetylation as described earlier. thesized for 30 min in an acetylation-inhibited lysate (see "Experimental Procedures"). Cycloheximide was then added 2 mM acetyl-coA was show here that acetylation topreventfurthertranslation,and is NH2-terminal using two typesof introduced. After an additional 30-min incubation, samples experimental procedures. were withdrawn for two-dimensional gel analysis. A parallel The first involves theuse of formyl-["S]Met-tRNAfMe' control was also carried out inwhich acetyl-coA was not where f stands for the initiator tRNA species. When mamadded. The results shown in Fig. 4, when compared with the malian tRNA is charged with anE. coli extract,only the met control, demonstrate that acetylation of fully translated but initiator tRNA ischarged. Formylmethionyl-tRNAfMe' can be nonacetylatedactindoes occur. Furthermore,thecontrol made with the same procedure by including formyltetrahyshows that the nonacetylated actin isselectively not degraded drofolicacidin thecharging mixture. Formyl-[%]Metby proteases over the time of the experiment. A time course tRNAfMe' is often used in eukaryotic protein synthesis studies for the acetylation reaction is shown in Fig. 5. since introduction of fMet on the endof a polypeptide chain ActinSynthesisinthePresence of F~rmyl-[~"S]Met- prevents subsequent removalof the methionine where it othtRNA["' a n d [3"S]tRNA,Me'-Within the cell, actin is acet- erwise would normally occur (31).Label incorporated in such ylated only at itsNH2 terminus. Althoughwe had shown that a situation would remain confined to the NH2 terminus of the the difference between acetylated and nonacetylated actin polypeptide. made in vitro is a single positive charge we had no evidence If actin acetylation in vitro is normally NH2-terminal and that acetylation observed in the reticulocyte lysate occurred if the end is blocked with m e t , acetylation would be inhibited at the NH2 terminus and not at an internal lysine (4). We either in the absence or presence of the acetylation inhibition
8152
Acetylation
Actin n.
f.
+ -- +
0
1
-
FIG. 6. Use of f-[3sS]Met-tRNAr"'L and [3SS]Met-tRNAfM"in in o i t m actin synthesis. Proteins were synthesized in the presence of either of these two labeled tHNAs as a sole source of isotope as ) included described in thetext. Unlabeled methionine (75 p ~ was in all assays. a, diagram of the actin region of a gel obtained when ["'S]Met is used in an acetylating system; h), result obtained in the same system when acetylation is blocked; c, expected result if fMettRNAfw" is used and acetylation normally observed is NHp-terminal (The same result would be obtained whether or not the acetylation inhibition system is used.); d, expected result iffMet-tHNAr"" is used, acetylation usually occurs at an internal lysine, and acetylation is allowed; e, sameas d except acetylation inhibition system is imposed; fi result actually obtained when f[''SS]Met-tRNAiM"is used under acetylating conditions; g, same as f except acetyaltion is inhibited; h, ["'S]Met-tRNAfW"is used and acetylation is allowed; i, same as h but acetylation is inhibited.
system. The gel pattern expected is shown in Fig. 6c. If acetylation occursat an internallysine and the NHn-terminal is already blocked with a formyl group, the resulting labeled actin will move to an even more acidic position than would acetylated but nonformylated actin(Fig. 6d).Introduction of the acetonyl-CoA system shouldnow cause the actin to return to theoriginal position occupied by either the singly acetylated or formylated actin since theywould both have the same FIG. 7. The initiator methionineis cleaved from many polycharge (Fig. 6e). When such an experiment was performed, lysate system if the methionine is the results shownin Fig. 6, f and g,were obtained. Using the peptides in the reticulocyte Two parallel translations containing equal amounts nonformylated. unlabeled lysate actin as a marker for nonacetylated D.disof nonformylated ( a )or formylated ( b ) [:"S]Met-tKNArw"'were carcoideum actin, it is clear that the fMet actin co-migrates ried with out. Equal amounts of each translation mixture were applied to normally acetylatedactin.Furthermore,the acetonyl-CoA two-dimensional gels, and autoradiograms shown here were exposed inhibition system has no effect on the actin's mobility. Thus for identical times. the acetylation usually observed in vitro is probably NHnterminal. Stability of the Initiator Methionine Residue of Actin in was now found in the nonacetylated actin position (Fig. 6, h Vitro-Since methionineis removed fromtheactin NH2 and i ) . The NH2-terminal methionine is stable in actin over terminus in vivo, we wished to determine whether Metcleav- the 50-min incubation used in our experiment. Furthermore, this methionine apparently is the site of the acetylation obage occurred in vitro as well. We repeated the experiment described above using [ " ' S S ] M ~ ~ - ~ R N A ' asTa~ 'sole source of served in vitro andnottheaspartic acidwhichoccupies label. The absence of the formyl groupshouldmakethe position 2 of the primary translation product. Methionine is terminal methionine accessible to the methionine amidopep- being cleaved from a number of peptides in the reticulocyte tidase known to be present in these lysates (31).T o prevent lysate as is evidenced by comparing two-dimensional gels of incorporation of methionine into internal methionine positions the f ["S]Met-tRNAr'" and the [""S]Met-tRNAfM" translation products (Fig. 7). If methionine cleavage were not occurin the actinas a result of hydrolysis of the [:"S]Met-tRNA;"' and charging of the tRNA,"' with the free [""Slmethionine, ring, the two gels would be identical. 75 PM unlabeled methionine was included in each translation. NHn-terminal Tryptic Peptides of Acetylated and NonaWhen [""Slmethionine equal in amount and specific activity cetylated Actin Synthesized in Vitro-We wished to gather to the [:''SS]Met-tRNArM" used was put into a control lysate, additional structural evidence concerning the site of in vitro no label was detected in the actin over the exposure period actin acetylation and the retention of the NH2-terminal meused in generating autoradiogramsin this experiment. thionine residue. For this purpose, we made useof the known When the ["'SS]M~~-~RNAT~" was used under acetylating trypsin and thermolysin cleavage sites in actin reported by conditions, a heavily labeled actin polypeptide appeared in Vandekerckhove and Weber (7). The structure of the NH2the acetylated actin position. If the acetylation inhibition terminal tryptic peptide of D.discoideurn actin made in vivo system was imposed, the actin remained heavily labeled but is shown in Fig. 8. A single methionine is present at position
Actin Acetylation
A. B.
8153
B1-ASP-GLY-GLU-ASP-VAL~GLN-ALA-LEU~VAL-ILE-ASP-ASN-GLV~SER-GLY-*MEl-CVS-LYS Ac-WET-ASP-GLV-GLU-ASP-VAL-GLN-ALA
*
FIG. 8. NHs-terminal tryptic peptide of D. dismideum actin. The structureis based on the data of Vandekerckhove and Weber (7, IO) and Firtel et al. (9). A single thermolysin cleavage siteexists between Ala, and Leu". N, denotes the amino thermolysin peptide and C, thecarboxylpeptide.Thenumbersindicatethenegative
+
.
* *
*
chargespresentonbothpeptides at pH 6.5 followingthermolysin cleavage of the performic acid oxidized tryptic peptide. A, the NHzterminal tryptic peptide of actin synthesized in vivo; B, the NH2terminaltrypticpeptide of acetylatedactinmade in vitro if the initiator methionine residue is not removed.
+
T Nt
TO
T T
Ct
0 S
c B
S
FIG. 9. [36S]Methioninecontaining acidic tryptic peptides of D. dismideum actin synthesized in vitro. A, acetylationwas permitted in the lysate;B, acetylation was inhibited in the lysate;S, [wS]methionine-labeled NHZ-terminal tryptic peptide of D. discoideum actin synthesized in vivo; T,position of the NHZ-terminal position of the tryptic peptide of acetylated actin made in vitro; T,,, nonacetylated NHz-terminal tryptic peptide. Electrophoresis was ried out as described in the text. An autoradiogram of the thin layer plate is shown. 16, and an unique thermolysin cleavage site exists at Ala, to give an amino portion (Nt peptide) and a carboxyl portion (C, peptide) of the tryptic peptide. The acetylated NH2-terminal tryptic peptide was isolated after electrophoresis at pH 6.5 as a single highly acidic labeled peptide co-migrating with orange G, containing either [:%]cysteine or [%]methionine, depending on the label used (7). It was clearly distinguishable from the COOH-terminal cysteine sulfonic acid-phenylalanine dipeptide. This NH2-terminal tryptic peptide wasused as a standard in the following experiments. Actin was made in vitro in the presence of ['sS]methionine under acetylating or nonacetylating conditions and purified as described under "Experimental Procedures." The methioninecontaining tryptic peptides were subjected to electorphoresis at pH 6.5 (Fig. 9). The autoradio'gram shows that, in the acetylating system, the NH2-terminal tryptic peptide of actin made in vivo co-migrates with the NH2-terminal tryptic peptide of acetylated actin made in vitro. When acetylation is inhibited, a single new NH2-terminal peptide of lower mobility corresponding to one less charge appears. These results conf i i our finding with the f[""S]Met-tRNAfM"that in vitro acetylation is NH2-terminal. Thermolysin Digestion of the Isolated [35S]Methioninelabeled NH2-tenninal Tryptic Peptidesof Actin Synthesized in Vitro-Thermolysin cleavage of the isolated NH2-terminal
=t
0-
FIG. 10. "hemolysin digestion of [3sS]methionine-labeled NHAerminal tryptic peptides of acetylated and nonacetylated D. dismideum actin synthesized in vitro. Experimental details are given in the text. Electrophoretograms were run at pH 6.5. Left A, NH2-terminal tryptic peptide of actinsynthesized in vitro and labeled uniformly with ["Slmethionine; B, thermolysin digest of the in vitro NH2-terminal tryptic peptide; C, thermolysin digest of the NH2-terminal tryptic peptide of actin synthesized in vivo and uniformly labeled with [%]methionine; D,NH2-terminal tryptic peptide of D.discoideum actin synthesized in vivo and labeled with [""s]methionine; T,position of the tryptic peptide; C,; carboxyl thermol(The digestionwas ysinpeptide; Nt, aminothermolysinpeptide. carried out in 20 mM NaHCO:, buffer. An autoradiogram is shown.) carRight, the digestion was carried outin Tris buffer which accounts for the multiplicity of the NI and N,,, peptides. Densitometric scans of the autoradiogram of the thin layer plate are shown. 0, origin; A, acetylated tryptic peptide is the substrate; B, nonacetylated tryptic peptide is the substrate. Both tryptic peptides give rise to the same C , peptide.
tryptic peptide made in vivo should result in a single labeled C, peptide of lower mobility than thestarting material and an unlabeled N, peptide of higher mobility. If the NH2-terminal tryptic peptide of actin made in vitro contains an NH2-termind methionine, both the N, and Ct peptides should be labeled. The results are shown in Fig. 10. The isolated NH2terminal tryptic peptides of acetylated actin and nonacetylated actin made in vitro and of actin made in vivo all give rise to theidentical labeled C,peptide. This result shows that both the acetylated and nonacetylated tryptic peptides arise from the actin NH2 terminus. Both the acetylated and nonacetylated tryptic peptides produce a labeled thermolysin Nt peptide, proving methionine is present at the NH2 terminus of the actin synthesized in vitro. The difference in mobilityof these peptides shows again that acetylation is NH2-terminal. When Tris-HC1 buffer was used inthe thermolysin digestion the N, peptide moved as a doublet. Prevention of acetylation caused both members of the doublet to move with a lower mobility. This result would beexpected if the multiplicity was due to an alterationof some other amino acid in N,such as a deamidation of Glns which would increase the charge of the affected peptide fraction by one charge.
8154
Actin Acetylation
'
AcCoA ASP-PR
Ac-ASP-PR
FIG. 11. Proposed biosynthetic pathway for B.discoideum actin. PR, remainder of the actin polypeptide. In scheme 2- - -, postulated but not as yet proven reactions.
NET-ASP-PR A$oA
Ac-MET-ASP-PR -
---
-
i
- 3 ASP-PR - - 2 * i, Ac-ASP-PR
Ac-MET
ktmL2 DISCUSSION
When we attemptedto use the Palmiter procedure to completely block endogenous actin acetylation in the rabbit reticulocyte system, we found it largely ineffective. Aswe have demonstrated, this problem could be overcome by using thethioether analogue of acetyl-coA, S-acetonyl-CoA, in addition tocitratesynthetaseand oxaloacetate. By this method, the actin synthesized under the direction of total cell RNA was 85-90% nonacetylated. Our procedure allows the presence of acetylation to be assessed usingsmall amounts of RNA and is particularly valuable in observing acetylation when using crude RNA mixtures to direct protein translation. With this enhanced acetylation block, we produced enough actin touse as anacetylation substrate, and we demonstrated that fully translated but nonacetylated D.discoideum actin could be acetylated by an enzyme present in the reticulocyte lysate. Previously, Strous et al. (32) and Pestana and Pitot (33) reported that nascent polypeptide chains were NH2-terminally acetylated early in translation while still attached to the ribosome. Therefore, considering our results, two acetylation pathways may be possible for actin: co-translationally while attached to theribosome and post-translationally after release of the completed polypeptide chain from the ribosome surface. Whether this post-translational NH2-terminal acetylation pathway exists for all acetylatable proteins is not certain at this point and requires further experimentation. The results obtained when we usedformyl-["'SIMettRNAfMe'in the translation system indicate that the actin acetylation we see in vitro is NH2-terminal .The stable formylated actin migrated to the same place on the gel as did monoacetylated actin. Had acetylation occurred at an internal lysine, the labeled formylated actin would have migrated to an even more acidic position under conditions where acetylation was allowed and returned to its original position if acetylation were inhibited. Therefore, to confirm this result, we utilized detailed peptide mapping studies of actin synthesized in the presence of ["'Slmethionine so all methionines were labeled. We showed that the NH,-terminal tryptic peptide of in vitro acetylated actin co-electrophoresed with the acetylated in vivo actin NHn-terminal tryptic peptide. If acetylation was inhibited in vitro, a new labeled peptide of lower mobility corresponding to one more positive charged appeared. This new peptide was derived from the NHz-terminal tryptic peptide. Both in vitro tryptic peptides and the in uiuo peptide gave rise to the same labeled thermolysin peptide, representing amino acids 8-18 of the NH2-terminal tryptic peptide. Most interesting was our finding that actin, synthesized in the presence of ["'S]Met-tRNA~Me',retained its NHP-terminal methionine for 50 min whether the actin was acetylated or not. Control experiments showed that this methionine was
only at the NH2 terminus and did not arise from insertion of the label into internal methionine positions. This result was confirmed by peptide mapping experiments with thermolysin using as a substrate theNH2-terminaltryptic peptide of actin labeled at all methionine positions with 36S.When the NH2terminal tryptic peptide from in uitro-synthesized acetylated actin was treated with thermolysin, two labeled peptides were seen. One of these, on the basis of mobility, was identified as the NHz-terminal part of the tryptic peptide. When the same tryptic peptide from nonacetylated actin was used, this new N, peptide showed a decreased mobility consistent with its having a nonacetylated amino group at the NH2 terminus, Finally quantitation of the label in the Nt andC, halves of the NH,-terminal tryptic pegtide showed that essentially every new actin polypeptide made had this relatively stable NH2terminal methionine even though the methionine is missing when bulk actin is isolated from whole cells. The reticulocyte lysate system can remove the initiator methionine from a number of proteins made in vitro (12, 31, 34). For ovalbumin, which ultimately i s NH2-terminallyacetylated, the initiator methionine is removed after 19-21 amino acids have been polymerized, and acetylation occurs after 45 residues have been joined. In the case of globin, also NH2terminally acetylated, the methionine is removed when the new polypeptide chain is 15-20 residues long. Our results confirm the removal of methionine from many proteins in this system. Three additional proteins, a-crystallin, adenylate cyclase, and troponin C, end in Ac-Met-Asp or Ac-Met-Glu (11). In all cases, the Ac-Met is found whenthe proteins are isolated in uiuo. The Ac-Met is retained as well when a-crystallin is synthesized in vitro. Thus, actin in the reticulocyte lysate system is synthesized initially as a protein like a-crystallin with astable Ac-Met-Asp end. We must yet determine whether, in uiuo, the NH2-terminalactin Ac-Met moiety is as stable as it is in the reticulocyte lysate. We first believed that actin processing proceeded at the NH2 terminus according to scheme I shown in Fig. 11. Our evidence indicates the presence of a rapidly formed and stable acetylmethionine on the NH2 terminus of newly synthesized and completely translated actin. Based on this evidence, we propose that scheme 2 in Fig. 11 is the actual pathway by which synthesis of D. discoideum actin may occur. A protein from beef liver has been described which will remove acetylmethionine from the NH2 terminus of an oligopeptide (35). However, it is inactive with respect to large polypeptides on which it hasbeen tested. Acknowledgments-We thank Dr. David Soll, Department of Zoology, University of Iowa, for providing us with the Dictyostelium discoideum. We also thankSteven Ellis for helping us with the preparation of the aminoacyl-tRNA synthetases.
Acetylation
Actin
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