protein methylation pea chloroplasts' · proteinsofwheatgerm.blacketal....

Plant Physiol. (1990) 93, 1235-12400032-0889/90/93/1 235/06/$01 .00/0

Received for publication January 16, 1990Accepted April 3, 1990

Protein Methylation in Pea Chloroplasts'

Kevin J. Niemi*, Julius Adler, and Bruce R. SelmanDepartment of Biochemistry (K.J.N., J.A., B.R.S.) and Department of Genetics (J.A.), University of

Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT

The methylation of chloroplast proteins has been investigatedby incubating intact pea (Pisum sativum) chloroplasts with (3H-methyl]-S-adenosylmethionine. Incubation in the light increasesthe amount of methylation in both the thylakoid and stromalfractions. Numerous thylakoid proteins serve as substrates forthe methyltransfer reactions. Three of these thylakoid proteinsare methylated to a significantly greater extent in the light thanin the dark. One is a polypepfide with a molecular mass of 64 kD,a second has an Mr of 48 kD, and the third has a molecular massof less than 10 kD. The primary stromal polypeptide methylatedis the large subunit of ribulose bisphosphate carboxylase/oxy-genase. One other stromal polypeptide, having a molecular massof 24 kD, is also methylated much more in the light than in thedark. Two distinct types of protein methylation occur. One methyl-linkage is stable to basic conditions whereas a second type isbase labile. The base-stable linkage is indicative of N-methylationof amino acid residues while base-lability is suggestive of car-boxymethylation of amino acid residues. Labeling in the lightincreases the percentage of methylation that is base labile in thethylakoid fraction while no difference is observed in the amountof base-labile methylations in light-labeled and dark-labeledstromal proteins. Also suggestive of carboxymethylation is thedetection of volatile [3Hjmethyl radioactivity which increases dur-ing the labeling period and is greater in chloroplasts labeled inthe light as opposed to being labeled in the dark; this implies invivo tumover of the [3H]methyl group.

Many types of posttranslational modifications of proteinsoccur in both prokaryotic and eukaryotic organisms. Exam-ples include phosphorylation, glycosylation, and methylation.

Protein methylation in prokaryotes is exemplified by Es-cherichia coli and Halobacterium halobium. In E. coli chemo-taxis, attractant or repellent chemicals interact with cyto-plasmic-membrane receptor proteins that signal to the flagellato create the desired movement ofthe bacterium either towardor away from the chemicals; then this signal is terminated byglutamate-carboxyl methylation of these receptor proteins inthe case of attractants and demethylation in the case ofrepellents (7, 14, 15, 22, 29, 31). The methylating agent isAdoMet2 (23), and the demethylation product is methanol(14, 31), which is excreted (28). In H. halobium phototaxis,

'Supported by National Science Foundation grant BNS-8804849to J. A. and National Institutes of Health grant GM 31384 to B. R. S.

2Abbreviations: AdoMet, S-adenosylmethionine; RuBPCase, ri-bulose bisphosphate carboxylase/oxygenase; SAH, S-adenosylhomo-cysteine.

cytoplasmic-membrane proteins are methylated in attractantvisible light and demethylated in repellent ultraviolet light (1,4, 21, 24).

Protein methylation in plants has not been thoroughlycharacterized. Gupta et al. ( 11) reported a protein methylaseinvolved in the methylation of arginine residues in histoneproteins ofwheat germ. Black et al. (5) described methylationof proteins in freshly broken spinach chloroplasts and con-cluded that both light-dependent and light-independent meth-ylation of chloroplast proteins occurs. These authors con-cluded that at high concentrations of AdoMet methylationappears to be nonspecific and that possibly it has a similarfunction to that found in the human erythrocyte (2), namelyto serve as a repair mechanism for racemized aspartic acidresidues. At low concentrations of AdoMet, however, theyconcluded that the methyl-transfer reactions appear to bemore specific with respect to their substrates. The only iden-tified substrates for posttranslational methylation in chloro-plasts are the large and small subunits of RuBPCase as de-scribed by Black et al. (5) and Houtz et al. (12). The role ofspecific protein methylation in plants is not known.

This project was developed with the endosymbiont theoryin mind. This theory proposes that eukaryotic organelles arederived from ancient prokaryotes (20). Modem prokaryotessuch as those described above utilize protein glutamate-car-boxyl methylation in their chemotactic and phototactic re-sponses. Our investigation was initiated to examine whethersimilar methylation reactions occur in the chloroplast. Wewanted to determine the extent of protein methylation in peachloroplasts and to find out whether protein methylation isinvolved in their light-responsive events.

MATERIALS AND METHODS

Plant Material

Pea (Pisum sativum) plants were grown under a 12 hphotoperiod in a 1:1, vermiculite:perlite mixture in a temper-ature-controlled, 25°C/18°C day/night, growth chamber.Light levels were approximately 300 to 400 gE m-2 s-' atplant height.

Chloroplast Isolation

Chloroplasts were isolated by the following modified pro-cedure of Bartlett et al. (3). Pea leaves 14 to 24 d old wereground in ice-cold grinding buffer consisting of 50 mm Hepes/KOH (pH 7.5), 330 mm sorbitol, 2 mm K-EDTA, 1 mMMgC92, 1 mM MnCl2, and 5 mM isoascorbate for 5 to 10 s in

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a Waring blender. The slurry was filtered through four layersof Miracloth. Chloroplasts were pelleted by centrifugation for3 min at 3,000g. The pellet was suspended in 1 to 2 mL ofgrinding buffer. This suspension was placed on a preformed(40 min at 35,000g) linear, 15 to 80% Percoll gradient andcentrifuged for 10 min at 8,000g, and the rotor was allowedto stop without braking. The lower band in the gradient wasremoved by pipetting and diluted with three volumes of ice-cold resuspension buffer consisting of 66 mm Tricine/KOH(pH 8.3), 200 mM KCI, and 6.6 mM MgC92. The suspensionwas centrifuged for 6 min at 1,500g and again allowed to stopwithout braking. The pellet was dispersed in resuspensionbuffer. Isolation procedures were performed at 4°C.The Chl concentration was determined spectrophotomet-

rically by suspension in 80% acetone using the extinctioncoefficients of Lichtenthaler (16). Chloroplast intactness wasassessed by ferricyanide reduction and phase contrast micros-copy.

Labeling Conditions

Chloroplasts, at 0.3 to 1.0 mg Chl/mL resuspension buffer,were preincubated in either the light or dark (foil-wrappedtubes) for 15 min at 25°C in 15 mL Pyrex tubes in a clear-bottom temperature-controlled circulating bath. The tubeswere 24 cm above a General Electric 400 W high-intensitydischarge lamp. After the preincubation period, 0.3 gM [3H]-methyl]S-adenosyl-L-methionine, (55-85 Ci/mMol, Dupont)was added for 5 to 30 min. The contents were then transferredto 1.5 mL polypropylene microcentrifuge tubes and fraction-ated as described below.

Fractionation

The radioactive chloroplasts were sedimented in a micro-fuge and resuspended in 10 mM NaPPi/HCl (pH 6.5), andplaced on ice for 15 min to rupture the chloroplasts. Thyla-koid membranes were pelleted by centrifugation for 5 min ina microfuge at 10,000g, resuspended in 10 mM NaPPi/HCl(pH 6.5), and repelleted. The two supernatants were combinedfor the soluble, stromal fraction. Thylakoid membranes werewashed three more times with 10 mM NaPPI/HCl (pH 6.5),with pelleting each time for 10 min at 10,000g. The finalresuspensions of thylakoid membranes were in 0.2 mL of 10mM NaPPi/HCl (pH 6.5). Then the thylakoid and the stromalfractions were both precipitated with either 5 volumes of-20°C acetone or with the addition ofTCA to 5%, put on icefor '0 min, and then pelleted for 10 min at 10,000g. Tofurther reduce the unincorporated background radiation, thepellets were resuspended in 0.2 mL of 100 mm NaPPi/HCl(pH 6.5) and precipitated with either acetone or TCA asdescribed above. The pellets were solubilized in 10% SDS (w/v). Aliquots were counted for radioactivity in Readysafe scin-tillation fluid in a Packard Tri-Carb 460C. Protein was deter-mined by the Lowry method as modified by Markwell withuse of bovine serum albumin as a standard (17).

Inhibitor Studies

The blockage of photosynthetic electron transport was ac-complished by including 5 ,M DCMU during the labelingreactions.

The inhibition of methylation by SAH was assessed byincluding varying concentrations ofSAH from 5 nm to 5 mMduring incubation of chloroplasts with the methyl-labeledAdoMet. Following incubation the samples were treated asdescribed above.

Volatile Radioactivity Produced by Alkaline Hydrolysiswith Heating

In a microdistillation technique (26), base was added (0.1mL of 1 M Na2CO3) to 13 x 100 mm test tubes that containedan aliquot of the labeled sample. H20 replaced the base incontrol tubes. The tubes were capped with serum caps andheated to 80C. The volatile radioactive components werevented via a syringe needle and tygon tubing into a scintilla-tion vial containing 10 mL of Readysafe scintillation fluid.After 30 min and 45 min, 1 mL cold methanol chases wereadded with a syringe to fully purge the volatile radioactivityfrom the test tubes into the scintillation fluid. After 60 minthe scintillation vials were counted for radioactivity. Countswere corrected for methanol quenching (using the samplechannels ratio method) and the efficiency of the microdistil-lation technique. The efficiency of microdistillation (69%)was determined using '4C-labeled methanol.

Volatile Radioactivity Produced Without AlkalineHydrolysis and Without Heating

The Fetters et al. (10) modification of the vapor equilibra-tion method of Chelsky et al. (8) was employed. Aliquots ofthe labeling mixture were removed at time points, TCAprecipitated (final concentration 5%), and centrifuged for 10min at 16,000g. The supernatant was transferred to a poly-propylene microcentrifuge tube, 0.05 mL of20% (w/v) phos-photungstic acid and 10 umol unlabeled AdoMet were added,the tube was closed, and the mixture was incubated overnightat 4°C to precipitate AdoMet, which was then removed bycentrifugation for 60 min at 16,000g at 40C. The supernatantwas assayed for volatile [3H]methyl radioactivity by putting itin a polypropylene microcentrifuge tube and placing the opentube into a scintillation vial containing 10 mL of Readysafescintillation fluid. The scintillation vial was sealed and setaside at room temperature for 4 to 48 h. The scintillationvials were then counted (without removing the microcentri-fuge tube from within) in a liquid scintillation counter. Thecounts detected are indicative of volatile [3H]methyl groups.

SDS-PAGE/Fluorography

Fractions containing equal amounts of radioactivity wereanalyzed on polyacrylamide gels (7.5 to 15% linear gradientLaemmli gels with a 4% stacking gel).

Fluorography was performed using DMSO and PPO. Thegels, sealed between cellophane sheets and dried, were exposedat -80°C to preflashed Kodak X-Omat AR film for a numberof weeks. After the exposure period, the gels were rehydratedin 10% acetic acid, the cellophane was removed, and the PPOwas removed from the gel by soaking in DMSO for at least24 h. The gels were then stained with Coomassie brilliant blue

1 236 NIEMI ET AL.

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PROTEIN METHYLATION IN PEA CHLOROPLASTS

in methanol:H20:acetic acid (4.5:4.5:1, v/v) and destainedwith H20:methanol:acetic acid:glycerol (5.15:4:0.75:0.1, v/v).

RESULTS

Light-Enhanced Methylation

TCA-insoluble compounds became methylated when intactpea chloroplasts were incubated with [3H-methyl]S-adenosyl-methionine (Table I). Stromal-fraction compounds were

much more heavily methylated than thylakoid-fraction com-pounds. The methylation of both fractions increased in thelight albeit at different rates. For example, after a 5 minexposure to light, the increase in the incorporation of methyllabel into the stromal fraction amounted to approximately25% whereas the thylakoid fraction had only slightly higheramounts of label than when labeled under dark conditions.After an additional 10 min of labeling, the methylation of thestromal fraction did not increase. In contrast, the additional10 min exposure to light further increased the differencebetween the light- and dark-labeled thylakoid fraction (light,142% increase; dark, 54% increase). The level of incorpora-tion of label into the thylakoid fraction after 15 min in thelight was about 70% above the dark-labeled amount. Black etal. (5) reported similar light effects on the methylation levelof proteins in broken spinach chloroplasts.

Labeled Polypeptides

Exhaustive pronase treatment of these TCA-precipitableproducts renders all radioactivity TCA-soluble and not visibleon a fluorograph (data not shown), so the observed radioac-tivity is indeed in protein.

Figure 1 shows the polypeptides obtained from thylakoidand stromal fractions. The Coomassie-stained polypeptideprofile of the thylakoid and stromal fractions is shown inpanel A of Figure 1. A comparison of these polypeptideprofiles (panel A) and the fluorographed (radioactive) meth-

Table I. Amount of Methyl Label Transferred from AdoMet to TCA-Insoluble Compounds in Intact Pea Chloroplasts

Pea chloroplasts were isolated, incubated with [3H-methyl]AdoMetfor either 5 min or 15 min, in either light or dark conditions, andfractionated into a stromal and a thylakoid fraction as described in"Materials and Methods." Aliquots of each fraction were counted forradioactivity and protein content was determined for each aliquot (n= 6, ± 1 SD; on average 1 pmol of label transferred equalled 60,800cpm).

Label TransferredTreatment

Stromal Thylakoid

pmol- mg-1 protein

5 Minute labeling periodLight 30.7 ± 1.96 0.301 ± 0.008Dark 24.8 ± 1.54 0.280 ± 0.038

15 Minute labeling periodLight 28.6 ± 5.29 0.730 ± 0.098Dark 22.2 ± 1.87 0.431 ± 0.113

ylation profiles (panel B) clearly shows that the polypeptidesare not methylated in proportion to their abundance. Thereare numerous examples of polypeptides that are undermethy-lated relative to their abundance, e.g. thylakoid polypeptidewith a molecular mass of 25 kD, the most prevalent Coo-massie-stained polypeptide in lane 1 ofA gave no evidence ofmethylation in the corresponding fluorogram, lane 1 of B.There are also examples of polypeptides not readily evidentin the Coomassie-stained lanes of panel A but very evident inthe fluorogram B, e.g. the polypeptide with Mr around 45 kDin the thylakoid-fraction lane 1. Similar examples are alsoevident in the stromal fraction. It is clear that the methylationlevel is not proportional to the amount of a polypeptidepresent in the chloroplast.Ofinterest in the thylakoid fraction are several polypeptides

that are methylated more in the light than in the dark (Fig. 1,panel B, closed arrowheads; lane 1, light versus lane 3, dark).The molecular masses of these polypeptides are about 64, 48,and <10 kD. The inclusion ofDCMU during the incubationof chloroplasts with AdoMet in the light results in decreasedmethylation of these three polypeptides (Fig. 1, panel B, lanes5 and 7).The bulk of the label in the stromal fraction appears in a

polypeptide with an apparent molecular mass of 50 to 55 kD(Fig. 1, panel B, lanes 2 and 4). This polypeptide is methylatedin both light- and dark-labeling conditions. This methylatedpolypeptide is most likely the large subunit of RuBPCase.Houtz et al. (12) reported the methylation of specific lysineresidues in the N-terminal region of the large subunit inseveral other species. The stromal fraction also contains apolypeptide ofmolecular mass about 24 kD that is methylatedmore in the light than in the dark (Fig. 1, panel B, openarrowhead; lane 2, light versus lane 4, dark). The identity ofthis polypeptide is not known. Several other stromal polypep-tides are also methylated.The methylation reactions are enzymatic in that the inclu-

sion of SAH, an inhibitor of methyltransferase enzymes (6),during the labeling incubations drastically reduced theamount of methylation. Micromolar concentrations of SAHwere sufficient to cause a 50% reduction and millimolarconcentrations totally inhibited transfer from methyl-labeledAdoMet to chloroplast protein (data not shown).

Base-Labile versus Base-Stable Methylation

Methyl groups transferred to chloroplast proteins frommethyl-labeled AdoMet were found in both base-labile andbase-stable linkages. As shown in Table II, about 5 to 20% ofthe methyl groups incorporated into chloroplast protein werebase labile. The remainder of the label was not hydrolyzedunder these basic conditions. In the thylakoid fraction light-labeled samples had a higher proportion ofbase-labile linkagesthan dark-labeled samples. In contrast the light conditionsduring labeling had no effect on the percentage of base-labileand base-stable linkages in the stromal fraction.

Volatile Radioactivity Production

Figure 2 presents data indicating the production ofa volatile[3H]methyl radioactive compound during the labeling period.

1 237




The amount of volatile radioactivity increases over the dura-tion of the labeling period when the chloroplasts are labeledin the light. There is also a slight lag in the period of timewhen the [3H]methyl volatile products are detected; volatile[3H]methyl is not detected at ten minutes but rises steadilyafter 20 and 30 min of labeling. The amount of [3H]methylvolatile radioactivity produced by chloroplasts incubated inthe dark is only slightly above the background level of volatileradioactivity.

DISCUSSION

Several of our findings indicate a light-responsive role forprotein methylation in the chloroplast. First, several polypep-tides are methylated to a greater degree in the light. Second,there is an increase in the amount of base-labile linkages inthylakoid protein when chloroplasts are labeled in the light.Third, chloroplasts labeled in the light show an increasingamount of volatile [3H]methyl radioactivity. This volatile

Table II. Volatile [3H]Methyl Radioactivity Produced by AlkalineHydrolysis with Heating of Transferred [3H]Methyl Label inChloroplast Fractions

Intact pea chloroplasts were labeled with [3H-methyl]AdoMet inthe light or dark and fractionated as described in "Materials andMethods." The TCA-insoluble aliquots were resuspended and aknown amount of radioactivity (average 30,000 cpm) was microdis-tilled under basic conditions (pH 12) at 800C for 1 h as described in"Materials and Methods." Values are mean of two experiments ± 1SD.

Base-Labile LinkagesLabeling Condition

Stromal Thylakoid

Light 18.3 ± 1.3 9.6 ± 2.5Dark 17.8±1.5 5.8±0.1

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iL:) (; MUtt + + +* ± i'+Figure 1. Methylation profile of pea chloroplast polypeptides. Intact pea chloroplasts were incubated with [3H-methyl]AdoMet for 15 min,fractionated, separated by a 7.5 to 15% linear gradient SDS-PAGE gel, and fluorographed as described in "Materials and Methods." A,Coomassie-stained polypeptide profile; B, fluorograph of [3H]methylated polypeptides. Fraction: t, thylakoid; s, stromal. Light: +, labeled in light;-, labeled in dark. DCMU: +, inclusion of 5 jM DCMU during labeling; -, no DCMU added. Arrowheads denote position of polypeptides that aremethylated to a greater degree in light; (p'), thylakoid fraction; (o), stromal fraction. Equal cpm were loaded in each lane. Lanes containing molwt standards are indicated by diamonds (C). The apparent mol wt of standards is shown at the left. The positions of the standards on fluorogramwere identified with a marking pen. Standards were from a Bio-Rad prestained SDS-PAGE standards kit (130 kD, phosphorylase b; 75 kD, BSA;50 kD, ovalbumin; 39 kD, carbonic anhydrase; 27 kD, soybean trypsin inhibitor; 17 kD, lysozyme).

1 238 NIEMI ET AL.

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Figure 2. Volatile [3H]methyl radioactivity produced at pH 8.3 withoutheating from chloroplasts incubated with [3H-methyl]AdoMet. Ali-quots of the labeling mixture were removed at the time pointsindicated. These aliquots were then analyzed for volatile [3H]methylgroups as described in "Materials and Methods." Data points are themean of two replicates ± 1 SD; this experiment was repeated threetimes with similar results.

radioactivity produced by pea chloroplasts may well be meth-anol, and this would be indicative of in situ turnover ofcarboxymethylesters.At least five amino-acid residues of proteins have been

reported to be acceptors of the methyl group from AdoMet.The methyl esters of glutamate and also aspartate are baselabile (13, 27). Hydrolysis of aspartate and glutamate meth-ylesters produces methanol. Base-stable methyl-accepting sitesare arginine (30), histidine (18), and lysine (12, 19). We foundboth base-labile and base-stable linkages; under our labelingconditions 20% or less of the incorporated methyl groups are

base labile. Black et al. (5) reported that 60 to 70% of thelabel incorporated into spinach chloroplast proteins was baselabile. This discrepancy in the amounts of base-labile andbase-stable linkages in spinach and pea chloroplast proteinsmay result from the lack of methylated lysine residues in theN-terminal region of spinach RuBPCase (12), from the differ-ent methods used to measure the amount of base-labile andbase-stable linkages, or from the different labeling and wash-ing methods used.At least two classes of protein carboxyl methyltransferase

enzymes have been characterized (9). The carboxymethyla-tion of glutamate residues in prokaryotes involves class Icarboxylmethyltransferase enzymes. These have to date beenfound only in prokaryotes and are involved in their tacticresponses. Class II protein carboxylmethyltransferase enzymesare thought to identify D-aspartyl residues in protein for repairto the L-form in erythrocytes (9) and are ubiquitous in bothprokaryotes and eukaryotes. The predominant class of meth-yltransferase activity in broken spinach chloroplasts was re-

ported by Black et al. (5) to be class II carboxylmethyltrans-ferase activity. The methylation pattern in chloroplast poly-peptides which indicates a light/dark difference could arise

from either carboxyl- or N-methylations. This is under inves-tigation.The chemotactic system of E. coli and the phototactic

system of H. halobium both involve methylation of mem-brane proteins (see Introduction). It has been demonstratedthat the turnover of the methyl group on esters of glutamateresidues via a methylesterase (25) produces methanol ( 14, 3 1).This methyl group turnover is involved in the adaptationresponse of the organism to the external stimulus. Someproperties of the methylesters involved in the tactic responsein prokaryotes are base-labile methylation of glutamate resi-dues in protein and production of volatile methanol by en-zymatic hydrolysis of the methylesters, which is influenced byexternal stimuli. We have reason to believe that chloroplastmethylation fits several of these criteria. Our volatile [3H]methyl detection may arise from either aspartate- or gluta-mate-ester hydrolysis, or both. However, the detection ofgreater amounts of volatile [3H]methyl radioactivity due tothe labeling condition, i.e. light versus dark, and the obser-vation of light-dependent protein methylations makes gluta-mate carboxymethylation reasonable to suspect.

In conclusion, we present evidence for the post-translationalmethylation of many chloroplast polypeptides. Several of thepolypeptides are methylated to a greater extent in the lightthan in the dark. More numerous are polypeptides methylatedequally in both light and dark. We present data supportingtwo distinct types of methyl-linkages in the proteins of intactpea chloroplasts. One methyl-linkage produces volatile radio-activity and is base labile, properties similar to carboxymeth-ylated proteins involved in the chemotactic and phototacticresponses of prokaryotes. The other methyl-linkage is basestable and apparently involves N-methylations of amino acidresidues.

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