Lysine Methylation Mapping of Crenarchaeal DNA-Directed RNA Polymerases by Collision-Induced and Electron-Transfer Dissociation Mass Spectrometry

Lysine Methylation Mapping of Crenarchaeal DNA-Directed RNAPolymerases by Collision-Induced and Electron-Transfer DissociationMass SpectrometryMikel Azkargorta,† Magdalena N. Wojtas,‡,¶ Nicola G. A. Abrescia,‡,§ and Felix Elortza*,†

†Proteomics Platform, CIC bioGUNE, ProteoRed-ISCIII, CIBERehd, 48160 Derio, Spain‡Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio, Spain§Ikerbasque, Basque Foundation for Science, 48011 Bilbao, Spain

*S Supporting Information

ABSTRACT: Enzymatic machineries fundamental for infor-mation processing (e.g., transcription, replication, translation)in Archaea are simplified versions of their eukaryoticcounterparts. This is clearly noticeable in the conservation ofsequence and structure of corresponding enzymes (see forexample the archaeal DNA-directed RNA polymerase(RNAP)). In Eukarya, post-translational modifications(PTMs) often serve as functional regulatory factors for variousenzymes and complexes. Among the various PTMs, methyl-ation and acetylation have been recently attracting mostattention. Nevertheless, little is known about such PTMs inArchaea, and cross-methodological studies are scarce. We examined methylation and N-terminal acetylation of endogenouslypurified crenarchaeal RNA polymerase from Sulfolobus shibatae (Ssh) and Sulfolobus acidocaldarius (Sac). In-gel and in-solutionprotein digestion methods were combined with collision-induced dissociation (CID) and electron-transfer dissociation (ETD)mass spectrometry analysis. Overall, 20 and 26 methyl-lysines for S. shibatae and S. acidocaldarius were identified, respectively.Furthermore, two N-terminal acetylation sites for each of these organisms were assessed. As a result, we generated a high-confidence data set for the mapping of methylation and acetylation sites in both Sulfolobus species, allowing comparisons with thedata previously obtained for RNAP from Sulfolobus solfataricus (Sso). We confirmed that all observed methyl-lysines are on thesurface of the RNAP.

KEYWORDS: Sulfolobus, RNA polymerase, methylation, acetylation, ETD, CID, post-translational modification

■ INTRODUCTION

Phylogenetic classification based on rRNA sequences placesarchaeal organisms closer to eukaryotic than to bacterial cells.1

Accumulated biochemical, functional, and structural data haveshown that Eukarya and Archaea share essential structural andfunctional protein machinery features. For example, key playersin the eukaryotic genomic DNA replication apparatus arepresent, in a simplified form, in Archaea.2 Thus, these archaealsystems can be used as models for complex eukaryotic cellularprocesses. Recent structural studies of the complete DNA-directed RNA polymerase (RNAP) from Sulfolobus shibatae(Ssh) have shown its striking structural homology with theeukaryotic RNAP II.3,4 However, one of the major differencesbetween archaeal RNAP and eukaryotic RNAPs lies at the levelof regulatory activities. It is still an open question whether post-translational modifications (PTMs) can modulate the activity ofthese archaeal enzymes.Protein methylation is a common protein PTM where a

hydrogen atom is replaced by a methyl group, usually in lysineor arginine residues.5 Histone methylation is the best describedmethylation event; it regulates gene expression and affects

developmental and cellular response processes.6 Histones,however, are not the only eukaryotic proteins that are subjectto this modification.7 A number of non-histone proteinsundergo methylation, which modulates their stability, protein−protein interactions, or transactivational activity. p53, ribosomalprotein Rpl23ab, Dam1, TAF10, TFIID, RuBisCO, andcytochrome c can be lysine-methylated, among others.8

Recently, the non-histone methylome data for eukaryotes hasbeen significantly expanded by application of heavy methylSILAC approach, in combination with extensive fractionationand use of a battery of antibodies or affinity reagents.9,10

Nevertheless, our understanding of non-histone proteinmethylation and its physiological implications is still limited.In contrast with the wealth of information on methylation in

eukaryotes, little is known about this process in Archaea.Proteome-wide analysis in crenarchaeal Sulfolobus solfataricus(Sso) P2 has revealed the existence of a methylated peptide inSso7d protein,11 and analysis of Sso DNA-directed RNA

Received: January 27, 2014Published: March 13, 2014

Article

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© 2014 American Chemical Society 2637 dx.doi.org/10.1021/pr500084p | J. Proteome Res. 2014, 13, 2637−2648

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polymerase has shown extensive methylation in this proteincomplex.12 Interestingly, the existence of a highly conserved,lysine methyltransferase with broad substrate specificity hasbeen recently confirmed in Sulfolobus islandicus.13 It isimportant to remember that archaeal organisms often grow invery hostile environments, e.g., at high temperatures (hyper-thermophiles) and/or under acidic and high ionic strengthconditions (acidophiles and halophiles), and thus theirbiochemistry is of extreme interest. Lysine methylation mightplay an important role in the heat stability of hyperthermophiliccrenarchaeal proteins through the modulation of their pKa,hydrophobicity, and solubility.12,14

Mass spectrometry (MS) has become the analytical tool parexcellence for the identification of proteins and discovery andcharacterization of PTMs.15 Collision-induced dissociation(CID) and electron-transfer dissociation (ETD) are comple-mentary methods for peptide fragmentation and subsequenttandem mass spectrometry analysis and identification. CID ismost effective when working with small, low-charge,unmodified peptides. Instead, ETD is considered to workbetter with high charge-states, generating more extensive seriesof ions than CID and leading to more comprehensive sequenceinformation. This is particularly useful in detecting labilemodifications, such as phosphorylations.16 Thus, using ETD inconjunction with CID can be a very successful approachbecause of differential dissociation characteristics and comple-mentary information these methods provide17−19

In this study, we analyzed methylation pattern ofendogenously purified RNAPs from two archaeal organisms,Ssh and Sulfolobus acidocaldarius (Sac), using CID- and ETD-based mass spectrometry. The sequence coverage for theidentified subunits ranged from 98.1% (for the Rpo3 subunit inSac) to 56.2% (for the Rpo12 subunit in Ssh). Usingconservative conditions for the preliminary screening andmanual validation of the obtained hits, we generated high-confidence methylation data sets for both Ssh and Sac RNAPs.We found 20 sites across 11 subunits for Ssh RNAP and 26

methyl-lysines distributed across 10 subunits for Sac RNAP,adding new information to a previous study of Sso RNAP andstrengthening the evidence and reliability of the methylationdata. We also found two subunits with N-terminal acetylation inboth analyzed species.

■ MATERIALS AND METHODS

Sulfolobus Biomass Production

Biomass production and RNAP purification of S. shibatae wascarried out using a protocol originally devised for the threearchaeal species Sulfolobus acidocaldarius, Sulfolobus shibatae,and Pyrococcus furiosus by Korkhin et al.20 and adapted to ourlaboratory infrastructure.For the present study archaeal (Sac and Ssh) cells were

asynchronously grown. S. shibatae growth media consisted ofSolution A (1.3 g (NH4)2 SO4, 0.28 g KH2PO4, 0.25 g MgSO4,0.07 g CaCl2, and 2 g Bactopeptone (Conda) in 1 L of media;pH 3, adjusted using sulfuric acid; the solution was autoclaved)and Solution B (5 g FeCl3, 0.45 g MnCl2, 1.13 g Na2B4O7,0.055 g ZnSO4, 0.013 g CuCl2, 0.008 g Na2MoO4, 0.008 gVOSO4, and 0.003 g CoSO4 in 100 mL of 1 M HCl filtratedthrough 0.22 μm filter). Complete media (A + B) wereprepared by mixing 1 L of solution A with 0.4 mL of solution Band 10 g of sucrose and prewarmed before adding theinoculum. To start the culture, 10 mL of media (A + B) in a 50

mL Schott bottle was inoculated with a piece of frozen cellpellet. The bottle, with the lid unscrewed, was placed within alarger beaker part-filled with water. The beaker was placed ontoa hot plate at 75 °C. The content of the Schott bottle wasgently stirred and aerated and used as further inoculum oflarger volumes. Once the inoculum reached 3 L, it was used as aseed for 18 L of media. The culture was put in six 5 LErlenmeyer flasks which were placed in a shaker (Innova, NewBrunswick) and agitated at 105 rpm, at 75 °C. The cells werecollected after 4 days and separated by centrifugation at 5500rpm for 20 min in a JLA 8.100 rotor. From these 18 L, weobtained ∼75 g of wet-cell pellet; this was flash-frozen in liquidnitrogen and stored at −80 °C. Our biomass yields werecomparable to those obtained originally by Korkhin et al.20

RNAP Purification

Ssh RNAP was purified to homogeneity, from 100 g of biomass,by ammonium precipitation, followed by anion exchangechromatography (QFF) on a heparin column (HiPrep FF16/10), and polished by gel filtration (HiLoad 16/60 Superdex200) as described previously.20 Highly purified Ssh RNAP (6mg/mL) in buffer containing 50 mM Tris pH 7.8, 25 mMMgCl2, 10% (v/v) glycerol, 10 mM β-mercaptoethanol, and500 mM KCl was aliquoted and flash-frozen in liquid nitrogenfor medium- and long-term storage.Purified Sac RNAP was a generous gift from Yakov Korkhin

and was stored at similar protein concentration as Ssh RNAP in50 mMTris/HC1, pH 7.8, 22 mM NH4CI, 10% (v/v) glycerol,10 mM β-mercaptoethanol, and 300 mM KCl.

Polyacrylamide Gel Electrophoresis and Staining

6X loading buffer was added to RNAP samples to a finalconcentration of 50 mM Tris pH 6.8, 5% glycerol, 1.67%mercaptoethanol, 1.67% SDS, and 0.0062% bromophenol blue.Protein samples were boiled for 5 min and resolved in 12.5%acrylamide gels using a Mini-Protean II electrophoresis cell(Bio-Rad). A constant voltage of 150 V was applied for 45 min.To avoid artifactual modification of acidic residues, the gel wasstained in SimplyBlue Safe Stain (Life Sciences), which doesnot require methanol or acetic acid fixatives or destains. The gelwas washed in Milli-Q water, and protein bands were cut intopieces and subjected to tryptic digestion followed by LC−MS/MS analysis.

Tryptic Digestion

In-Gel Tryptic Digestion. Gel bands were first cut intosmall pieces and washed in Milli-Q water. Reduction andalkylation was achieved by incubation with dithiothreitol (DTT,10 mM in 50 mM ammonium bicarbonate) at 56 °C for 20min, followed by an incubation in iodoacetamide (IA, 50 mMin 50 mM ammonium bicarbonate) for another 20 min, in thedark. Gel pieces were dried and incubated with trypsin (12.5μg/mL, in 50 mM ammonium bicarbonate) for 20 min on ice.After rehydration, the trypsin supernatant was discarded; gelbands were covered with 50 mM ammonium bicarbonate andincubated overnight at 37 °C. After digestion, acidic peptideswere further extracted with TFA 0.1% and dried in a RVC2 25SpeedVac concentrator (Christ). The peptides were resus-pended in 0.1% FA and sonicated for 5 min prior to theiranalysis.

In-Solution Tryptic Digestion. RNAP samples (30 μg)were dried in a RVC2 25 SpeedVac concentrator (Christ) andresuspended in 16 μL of 6 M urea. Protein was reduced andalkylated by incubation in 5 μL of DTT (200 mM, in 50 mM

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ammonium bicarbonate) for 45 min, followed by incubation in4 μL of IA (1 M, in 50 mM ammonium bicarbonate) foranother 45 min, and finally in 20 μL of DTT for 45 min. Allincubations were carried out at 25 °C. Then, the sample wasdiluted to 100 μL by adding 50 mM ammonium bicarbonate,and trypsin (Trypsin Gold, Promega Corporation, Madison,WI, USA) was added to a final trypsin/protein ratio of 1:20.The sample was vortexed and incubated overnight at 37 °C.The peptides were dried in the SpeedVac concentrator,resuspended in 0.1% FA, and sonicated for 5 min. The sampleswere desalted in Zip Tip C18 pipet tips (Millipore) followingthe manufacturer’s protocol, dried in the SpeedVac concen-trator, resuspended in 0.1% FA, and sonicated for 5 min priorto analysis by MS.

NanoLC−MS/MS and Data Analysis

Peptide mixtures obtained from the digestions were separatedby online NanoLC (nLC) and analyzed using electrospraytandem mass spectrometry. Peptide separation was performedon a nanoACQUITY UPLC system (Waters) connected to anLTQ Orbitrap XL ETD mass spectrometer (Thermo Electron,Bremen, Germany). Approximately 500-ng samples wereloaded onto a Symmetry 300 C18 UPLC Trap column, 180μm × 20 mm, 5 μm (Waters). The precolumn was connectedto a BEH130 C18 column, 75 μm × 200 mm, 1.7 μm (Waters)equilibrated in 3% acetonitrile and 0.1% FA. The peptides wereeluted at 300 nL/min using a 30 min linear gradient of 3−50%acetonitrile for in-gel digested samples and a 60 min lineargradient of 3−50% acetonitrile for in-solution digested samples.Samples were loaded directly onto the nanoelectrospray ionsource (Proxeon Biosystems, Odense, Denmark).The mass spectrometer automatically switched between MS

and MS/MS acquisition in DDA mode. Full MS survey spectra(m/z 400−2000) were acquired in the orbitrap with 30000resolution at m/z 400. The five most intense ions weresubjected both to CID and ETD fragmentation in the linear iontrap, in an alternating fashion. Precursors with charge statesequal to or greater than 2 were specifically selected forfragmentation. Collision-energy applied to each peptide wasautomatically normalized as a function of the m/z and chargestate, and charge-state-dependent ETD time was applied.Analyzed peptides were excluded from further analysis during30 s using dynamic exclusion lists.Searches were performed using Mascot Search engine (www.

matrixscience.com, Matrix Science, London, U.K.) on Pro-teome Discoverer v.1.2. software (Thermo Electron, Bremen,Germany). Carbamidomethylation of cysteines was selected asfixed modification, and oxidation of methionines, methylationof lysine and arginine, and protein N-terminal acetylation asvariable modifications. Peptide mass tolerance of 5 ppm and 0.5Da fragment mass tolerance were adopted as search parameters,and 4 missed cleavages were allowed. Spectra were searchedagainst a mixed Sulfolobus + Saccharomyces cerevisiae databaseobtained from UniProt. Sac and Ssh databases were obtainedfrom UniProt/Swiss-Prot (version 2013_07, 540546 entries)and UniProt/TrEMBL (version 2013_07, 39870577 entries)and merged with yeast UniProt/Swiss-Prot database from thesame release. A decoy search was carried out in order toestimate the false discovery rate (FDR) for the samples.Proteins with at least 2 peptides passing the FDR < 5% filterwere considered for further analysis. Only unambiguouslyassigned, rank1 modified peptides with FDR < 5% wereselected in the initial screening. For the final filtering and

selection of the methylated hits, only manually validatedmodified sites present in both experimental replicates wereconsidered.

■ RESULTS

Identification of S. acidocaldarius and S. shibatae RNAPSubunits

RNAPs from both Sac and Ssh were analyzed following dualgel-based and gel-free strategies (Figure 1). Protein subunits

were resolved on an SDS-PAGE gel, and each observed bandwas in-gel digested and analyzed by nLC−MS/MS. Proteinextracts were also directly digested with trypsin in solution andanalyzed by mass spectrometry, using both CID and ETDfragmentation methods. Two independent replicates comingfrom different preparations were run for each procedure.Ssh RNAP yielded 11 prominent bands in the gel-based

approach, whereas RNAP from Sac yielded 9 different bands inthe gel (Figure 2). All 13 different RNAP subunits wereidentified following this approach for both organisms. Some ofthe subunits showed an anomalous electrophoretic mobility inthe gel. This was the case for the newly identified Rpo13,3,21

which resolved with higher than expected molecular weight.Interestingly, discrepancies in the electrophoretic mobility forsome Sac and Ssh subunits were detected (for example, Rpo7).Using the two approaches (gel-based and gel-free), we

obtained an extensive mapping of all 13 subunits composingRNAP. Interestingly, over 90% sequence coverage was obtainedfor 7 of the RNAP subunits from Sac and 3 subunits from SshRNAP. More than 80% sequence coverage was obtained formost of the subunits from both organisms (11 out of 13 for Sac,7 out of 13 for Ssh), confirming that the samples wereextensively analyzed (Figure 3A). In addition to the 13 subunitsof the archaeal RNAPs, some other proteins were identified inboth Sac and Ssh preparations (Supplementary Tables 1 and 2).

Figure 1. Schematic representation of sample preparation and analysisworkflow. RNAP from S. shibatae and S. acidocaldarius was isolatedand processed using in-gel and in-solution approaches. Peptides wereanalyzed in the mass spectrometer using both CID and ETD asfragmentation methods.



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Identification of Methylated Lysines and N-TerminallyAcetylated Proteins in S. acidocaldarius and S. shibataeRNAP

Specific lysine/arginine methylation of peptides and protein N-terminal acetylations were searched using the Mascot searchengine. Only methyl-peptides passing the 5% FDR cutoff andpresent in both replicates were selected. Manual inspection ofthe selected spectra assured that only the most reliable andreproducible results were considered for further analysis. 26peptides mapping to 20 methyl-lysines for Ssh (Table 1, Figure3B) and 33 methyl-peptides mapping to 26 methylation sitesfor Sac RNAP (Table 2, Figure 3B) were selected. We foundtwo N-terminal acetylation sites mapped by four differentpeptides for Ssh (Table 1) and two sites mapped by fourpeptides for Sac (Tables 1 and 2). All of the spectra matchingto the selected peptides are available in Supplementary Figures1 and 2. In addition to these high confidence modified peptidesthat overlapped between replicas, some good-quality peptidespresent in only one replicate were also found for bothorganisms. In order to limit the analysis to only the mostreproducible modifications (with evidence in both replicates),these were not considered for further analysis and discussion.However, since they might reflect true modifications that havebeen left out of our data sets because of the features of DataDependent Acquisition, their information is also provided assupplementary data (Supplementary Figure 3).No reliable arginine methylation was obtained, and data were

also searched against dimethyl-lysine and dimethyl-argininewith no conclusive result (data not shown). The comparison ofthe results obtained for Ssh and Sac with those obtained forSso12 reveals a similar extent of methylation for all threeorganisms, with Sac showing slightly higher methylation levelsthan the others (Figure 3B and C).

Altogether, 66 modified peptides were detected. Wedemonstrated that both fragmentation methods used weresuitable for the identification of modified peptides. The CIDmethod identified 64 of the sites, and 63 peptides were assignedby ETD; there was a large overlap between the identifiedpeptide sets (61 out of 66, 92%). CID supplied three exclusivemodified peptides, and ETD supplied two such peptides(Tables 1 and 2). The commonly identified peptides werefound not only using two different fragmentation methods butalso in different charge states, strengthening the experimentalevidence for the mappings (Figure 4). Moreover, peptides withdifferent numbers of missed cleavages were used for theannotation of the methyl sites. Thus, K519 methylation wasdetected in two different peptides from Ssh Rpo2 (Figure 5).These two peptides were identified using both in-gel and in-solution approaches and successfully fragmented by CID and

Figure 2. Resolution of the RNAP subunits from S. shibatae (Ssh) andS. acidocaldarius (Sac) in SDS-PAGE gel. Methanol- and acetic acid-free SimplyBlue Safe Stain (Life Sciences) was used for gel staining.Mw markers (Mw, central lane) account approximately for thefollowing molecular weights: A = 200 kDa, B = 120 kDa, C = 100kDa, D = 60 kDa, E = 40 kDa, F = 20 kDa, G = 12 kDa, H = 8 kDa.Assignment was based on the number of spectral counts for eachsubunit identified in each of the bands.

Figure 3. Identification of RNAP subunits in the three differentSulfolobus species. (A) Sequence coverage for each of the identifiedsubunits from S. shibatae and S. acidocaldarius. Data from the tworeplicates were merged. (B) Total number of methylation sites andmethylated peptides in S. solfataricus, S. shibatae, and S. acidocaldarius.Data from Botting et al.12 was used for S. solfataricus. Only manuallyvalidated hits present in both Ssh and Sac replicates were consideredfor the comparison. (C) The number of observed modified sites persubunit for S. solfataricus, S. shibatae, and S. acidocaldarius.





Table

1.Identification

andMethylation

ResultsforS.

shibataea

IDinfo

modificatio

ndetection

subunit

entry

seqcov

Mw(K

Da)

no.p

eptid

esmodifsite

peptide

modificatio

nsunmod

gel

sol

CID

ETD

rpo1C

B8Y

B54_S

ULS

H87.85

43.7

47K10

DKSY

LEEK

K2(Methyl)

**

*24

46K20

VKQASN

ILPQ

KK2(Methyl)

**

*53

71rpo1N

B8Y

B53_S

ULS

H85.91

99.6

113

K395

KEL

AST

LAPG

YVVER

K1(Methyl)

**

*79

119

K659

KEIYNEIDR

K1(Methyl)

**

*57

75LE

DVSL

GDDVKKEIYNEIDR

K12(M

ethyl)

**

5387

rpo2

B8Y

B55_S

ULS

H81.87

127.3

140

K490

IVEK

TLY

EMGVVPV

EEVIR

K4(Methyl);M9(Oxidatio

n)*

*51

80

K519

RVTEG

GED

QNEY

LKWSK

K14(M

ethyl)

**

*67

111

VTEG

GED

QNEY

LKK13(M

ethyl)

**

7786

K827

FLQEF

KEL

SPEQ

AK

K6(Methyl)

**

5961

K962

TPIEQ

LQNEILK

K12(M

ethyl)

**

47NA

TPIEQ

LQNEILK

YGYLP

DATEV

TYDGR

K12(M

ethyl)

**

6945

rpo3

B8Y

B56_S

ULS

H97.74

30.1

36N-Term

SINLL

HK

N-Term(Acetyl)

**

4644

SINLL

HKDDK

N-Term(Acetyl)

*47

57SINLL

HKDDKR

N-Term(Acetyl)

*50

54K115

DIKSE

DPS

IVPISG

DIPIVLL

GANQK

K3(Methyl)

**

*103

66K178

VEILG

NCEK

C7(Carbamidom

ethyl);K9(Methyl)

**

47NA

K258

KIEEL

EKK

K7(Methyl)

**

3950

rpo4

B8Y

B58_S

ULS

H95.58

12.8

17N-Term

SSVYIVEE

HYIPYSV

AK

N-Term(Acetyl)

**

103

57

K26

KLL

SDVIK

K8(Methyl)

**

*58

69KLL

SDVIKSG

SSSN

LLQR

K8(Methyl)

**

55113

LLSD

VIKSG

SSSN

LLQR

K7(Methyl)

**

9375

rpo5

B8Y

B60_S

ULS

H76.19

9.7

8HEV

LSID

EAYK

K11(M

ethyl)

**

5237

K30

HEV

LSID

EAYKILK

K11(M

ethyl)

**

*62

76K68

KSQ

LYGEV

VSY

RK1(Methyl)

**

*74

97rpo7

B8Y

B57_S

ULS

H60.56

20.3

26K20

IPPN

EFGKPL

NEIALN

ELR

K8(Methyl)

**

*57

93K130

GIIFG

EKK7(Methyl)

**

*40

36rpo8

B8Y

B59_S

ULS

H69.7

15.1

8K109

IISN

KES

FLK

K5(Methyl)

**

*37

85rpo11

B8Y

B62_S

ULS

H88.04

10.2

8K71

DALL

KAIETVR

K5(Methyl)

*64

66rpo12

B8Y

B64_S

ULS

H56.25

5.6

5K19

TFT

DEQ

LKVLP

GVR

K8(Methyl)

**

*73

72rpo13

B8Y

B65_S

ULS

H79.81

12.1

15K73

LFED

NYKDYEK

K7(Methyl)

**

NA

66rpo6

B8Y

B61_S

ULS

H64.21

10.7

10NA

NA

NA

NA

NA

NA

NA

NA

rpo10

B8Y

B63_S

ULS

H93.94

7.6

11NA

NA

NA

NA

NA

NA

NA

NA

aSearches

wereperformed

usingMascotsearchengine.Subunit:

subunito

fthe

RNAPto

which

theidentified

peptidebelongs.En

try:entrynamefortheproteinintheUniProt

database.Seq

cov:sequence

coverage

obtained

forthatprotein(%

).M

w:m

olecularweighto

fthe

proteinexpressedinkD

a.No.pepts:totalnum

berofpeptides

identified

forthatprotein.Modifsite:the

positio

nofmodified

residuein

theproteinsequence.P

eptid

e:identifi

edpeptidesequence

matchingto

acertainmodificatio

nsite.A

llmodificatio

ncandidates

passed

FDR<5%

filter,werepresentin

tworeplicates,and

werefurther

manually

validated.M

odificatio

n:thetype

ofthemodificatio

n(s)detected

inthepeptide.Unm

od:anasterisk(∗)indicatesthattheunmodified

form

ofthepeptidewasalso

identifi

ed.G

el/Sol:anasterisk

(∗)indicatespositiveidentifi

catio

nforthepeptidein

each

ofthesample-handlingmethods

(in-gel/in-solution).C

ID/ETD:bestscoreobtained

forCID

and/or

ETD,w

henavailable.



Table

2.Identification

andMethylation

ResultsforS.

acidocaldariusa

IDinfo

modificatio

ndetection

subunit

entry

seqcov

Mw(K

da)

no.p

epts

modifsite

peptide

modificatio

nsunmod

gel

sol

CID

ETD

Rpo1C

RPO

A2_

SULA

C90.59

44.4

74K5/K7

MID

EKLK

GYID

KRb

K5(Methyl);K7(Methyl)

**

NA

65K7

LKGYID

KR

K2(Methyl)

**

*36

51K134

TDKDKALD

IAR

K5(Methyl)

**

4162

K350

AAFE

VTVK

K8(Methyl)

**

*59

46AAFE

VTVKHLL

DAAAR

K8(Methyl)

**

7382

Rpo1N

RPO

A1_

SULA

C92.5

99.7

130

K395

DRKEL

ASSITAGYVVER

K3(Methyl)

**

*86

144

KEL

ASSITAGYVVER

K1(Methyl)

**

104

112

K666

IKEG

YSQ

VDEY

IRK2(Methyl)

**

*71

100

K678

KFN

EGQLE

PIPG

RK1(Methyl)

**

*85

73Rpo2

RPO

B_S

ULA

C87.3

126.4

145

K369

DLV

YQLE

KK8(Methyl)

**

*43

24K822

FLQEF

KEL

SPEQ

AKR

K6(Methyl)

**

*46

70Rpo3

RPO

D_S

ULA

C98.11

29.8

39K103

VYLD

VEA

KDQPL

MIYSR

K8(Methyl)

**

6774

K115

DLK

SEDQMITPV

SGAIPIVLL

GSK

K3(Methyl)

**

6154

Rpo4

M1J024_

9CREN

88.6

1316

K23

KYIKEL

IDTGSSSN

LIQK

K4(Methyl)

**

*71

100

YIKEL

IDTGSSSN

LIQK

K3(Methyl)

**

95101

K65

ELEE

IVKR

K7(Methyl)

**

*48

50K110

IIEIIKK

K6(Methyl)

**

4945

K110/K111

IIEIIKK


**

*48

33Rpo5

RPO

H_S

ULA

C86.9

9.4

12K30

HEILQ

LEEA

YKLV

KK11(M

ethyl)

**

*74

119

K68

KSP

FTGES

VTYR

K1(Methyl)

**

*69

67Rpo6

RPO

K_S

ULA

C93.26

10.2

20N-Term

TID

KIN

EIFK

N-Term(Acetyl)

**

*39

26K11

INEIFK

ENWK

K6(Methyl)

**

*55

56

K17/K

20NKLT

KYEIAR


**

*26

67NKLT

KYEIAR

K5(Methyl)

**

4868

Rpo7

RPO

E_SU

LAC

63.39

20.4

32K131

GILIG

EKK7(Methyl)

**

39NA

K180

IEWIN

QEIAK

K10(M

ethyl)

**

*74

59Rpo8

M1J056_

9CREN

92.5

13.9

17K97

ILSK

NGLINSK

K4(Methyl)

**

5272

rpo13

M1IBA6_

9CREN

70.48

12.3

21N-Term

SEDDSK

KEP

EPEE

TEA

EIK

N-Term(Acetyl)

**

6663

SEDDSK

KEP

EPEE

TEA

EIKHEE

ISR

N-Term(Acetyl)

**

7666

N-term/K

20SE

DDSK

KEP

EPEE

TEA

EIKHEE

ISR

N-Term(Acetyl);K19(M

ethyl)

*37

65K67

NEL

SIEE

AK

K9(Methyl)

**

5451

K67/K

68NEL

SIEE

AKK

K9(Methyl);K10(M

ethyl)

**

*50

77NEL

SIEE

AKKMFD

DVAR

K9(Methyl);K10(M

ethyl)

*92

103

NEL

SIEE

AKKMFD

DVAR

K9(Methyl);K10(M

ethyl);M11(O

xidatio

n)*

*35

70K68

KMFD

DVAR

K1(Methyl)

**

6465

KMFD

DVAR

K1(Methyl);M2(Oxidatio

n)*

4260

Rpo11

RPO

L_SU

LAC

82.22

106

NA

NA

NA

NA

NA

NA

NA

NA

Rpo12

RPO

P_SU

LAC

97.92

5.6

10NA

NA

NA

NA

NA

NA

NA

NA

Rpo10

RPO

N_S

ULA

C93.94

7.6

10NA

NA

NA

NA

NA

NA

NA

NA

aSearches

wereperformed

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try:

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olecularweighto

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a.No.pepts:totalnum

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matchingto

acertainmodificatio

nsite.A

llmodificatio

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tworeplicates,and

were



ETD, further strengthening the evidence for this methylationsite.

Comparison of Lysine Methylation in RNAPs from theThree Sulfolobus Species

Overall results for RNAP methylation revealed a similarnumber of methylation sites for each organism, with SacRNAP being slightly more methylated than others (Figure 3B).RNAP subunits of these three Sulfolobus species have a highlevel of sequence identity, with Sac being the least similar. Inparticular, subunits Ssh Rpo8 and Rpo13 have only 43% and39% sequence similarity with Sac (see Supplementary Table 3).The proportion of lysine residues is mostly conserved acrossspecies in subunits Rpo1N, Rpo1C, Rpo2, Rpo3, Rpo7, Rpo10,Rpo12, and Rpo13. In the remaining subunits (Rpo4, Rpo6,Rpo8, and Rpo11), lysine residues are more abundant in Sacthan in the other two species (Supplementary Table 4). Thiseffect is most noticeable for Rpo6 (9% lysines in Sac against the4.2% in Ssh and 2.1% in Sso). Interestingly, this subunit hasonly been reported to be methylated in Sac.Our data on Ssh and Sac RNAPs show that methylation

occurs predominantly on α-helices and loops and only in onecase on an incoming β-strand (Supplementary Figure 4). Thissuggests a structural specificity as it appears that ordered andaccessible parts of the protein, mainly in an α-helical secondarystructure, are more likely to be methylated. The methylationsites in Ssh RNAP were mapped on the available RNAP-DNAbinary complex (PDB ID 4B1O), confirming that the methyl-lysines are located on the surface of this 13 multisubunitenzyme and away from the DNA fragment (Figure 6). Finally,the ratio between methylated and nonmethylated lysinesexposed on the surface is relatively low (∼12%; Figure 6),supporting a selective methylation pattern. These observationsare in agreement with the data reported by Botting et al.,12

suggesting a structure-dependent methylation pattern, andstrengthening the likelihood that the methyltransferase actsafter protein folding.

■ DISCUSSION

We discovered some novel methylation sites in Ssh and SacRNAPs, supporting and expanding the results of previousstudies of archaeal protein methylation. Botting et al. havecharacterized RNAP methylation in Sso12 and found 21 methyl-lysines across 9 subunits of the complex. Our data show theexistence of 20 sites in 11 subunits for Ssh and 26 methyl-lysines distributed across 10 subunits for Sac RNAP (Figure3B). In contrast to the results of large-scale experimentsperformed on mammalian cells,9 there are no reliable reports ofarginine methylation in Archaea, suggesting that lysine is theprevalent residue for archaeal RNAP methylation. We alsofound N-terminal acetylation of Rpo3 and Rpo4 in Ssh andRpo6 and Rpo13 in Sac. N-terminal acetylation of Rpo1N,Rpo2, and Rpo4 has been reported for Sso.12

Use of parallel approaches improves the results of the RNAPmethylation analysis. We used both in-gel and in-solutiondigestion procedures and combined CID and ETD fragmenta-tion methods. This strategy allowed extensive sample analysis(Figure 3A), providing a large number of data for methylationmapping. We found that CID and ETD were equally useful inthe discovery of modified sites. The majority of modifiedpeptides (61 out of 66) were found using both fragmentationmethods. CID contributed three exclusive methyl-peptides, andtwo such peptides were discovered using ETD (Tables 1 andT

able

2.continued

furthermanually

validated.M

odificatio

n:thetype

ofthemodificatio

n(s)detected

inthepeptide.Unm

od:anasterisk(∗)indicatesthattheunmodified

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ofthepeptidewasalso

identified.G

el/Sol:an

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nforthepeptideineach

ofthesample-handlingmethods

(in-gel/in-solution).C

ID/ETD:bestscoreobtained

forCID

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ETD,w

henavailable.bThe

peptide

MID

EKLK

GYID

KRwas

identified

inboth

replicates,b

utwith

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thereplicates.T

hebestscorewas

selected

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thenon-oxidized

form

.



2). The large overlap between the results of thesecomplementary fragmentation techniques strengthened theevidence necessary for the mapping of methylation sites. Thisoverlap supplies additional corroboration of the results, notonly because different fragmentation methods were used butalso because the identification was performed using differentpeptide species (different m/z, Figure 4). Peptides withdifferent numbers of missed cleavages (Figure 5) were alsoused for some of the assignments. Applying stringent

conditions for the selection of modified peptides (peptideFDR cutoff at 5%, methyl-peptides present in two replicates,and manual validation of the hits obtained) also helped us tolay down solid foundations for reliable modification assign-ments.The total numbers of methyl sites were similar for the three

organisms compared here, but some of the RNAP subunitsseem to be differentially methylated in various Sulfolobusspecies. Rpo1C and Rpo8 were found to be methylated only in

Figure 4. Representative spectra for the identification of modified peptides with different charge-states obtained using CID and ETD.kELASTLAPGYVVER (K1me) from S. shibatae Rpo1N was identified with z = 2 by CID (A) and with z = 3 (B) by ETD.




Sac and Ssh, whereas evidence for Rpo11 and Rpo12methylation was found in Ssh and Sso, but not in Sac. Rpo6methylation was found only in Sac. Interestingly, a well-fragmented peptide from Sac carrying a methylation at Rpo10K33 was identified in one of our replicates (VMGGEDPE-kVLTELGVNR, M2-Oxidation, K9-Methyl). A nonmisscleavedpeptide carrying the same methylation was identified in theother replicate (VMGGEDPEK, M2-Oxidation, K9-Methyl),but fragmentation information was limited (SupplementaryFigure 5). In addition, a peptide carrying K33 methylation wasidentified in one of the Ssh replicates (Supplementary Figure

3). These assignments were left out of our selected data sets,although they present some evidence for Rpo10 methylation.For the first time, we provide evidence for Rpo1C, Rpo6, and

Rpo8 methylation (these modifications have not been reportedfor Sso). Rpo1C was extensively methylated in Sac, where 4methyl-lysines were detected. Two Rpo1C lysine methylationsites, different from those in Sac, were identified in Ssh. Theselysines are conserved between species, but the surroundingsequences are quite different in most of the cases (Figure 7,Supplementary Figure 4). Rpo6, however, is methylated only inSac (two methyl-peptides mapping to three methylation sites)and is also N-terminally acetylated (one single peptide). It is

Figure 5. Representative spectra for the identification of modified peptides with different number of missed cleavages. K519 methylation in S.shibatae Rpo2 was identified in two different peptides: (A) VTEGGEDQNEYLkWSK, K13me, no missed cleavages and (B) RVTEGGEDQNEY-LkWSK, K14me, 2 missed cleavages.




noteworthy that Rpo6 in Sac has the greatest proportion oflysines (Supplementary Table 4). Only one of these lysines isconserved in Ssh, where it is not methylated. Finally, singlemethylation sites were found in Rpo8 subunits of Sac and Ssh.They were located at K97 in Sac and K109 in Ssh, both at thestart of a β-strand (Figure 7). These lysines are conservedacross all three species, but their modification seems to beorganism-specific; no conclusive evidence for their methylationwas found in Sso.

Thus, different methylation patterns are found in differentSulfolobus RNAPs, even if the lysines are conserved. We cannotbe sure whether this reflects insufficient sensitivity of detectionmethods or real in vivo differences. Not much is known aboutthe Sulfolobus methylation process, but the available informa-tion suggests low sequence specificity.12,13 Archaeal methyltransferase lacks the typical substrate-recognition and bindingdomain and, as a result, can methylate lysines in a sequence-unspecific way, as has been reported by Botting et al.12 and Chu

Figure 6. Mapping of methyl sites to the structure of S. shibatae RNAP. The RNAP complex appears with a bound DNA molecule (green:nontemplate strand; cyan: template strand). Methylated sites identified for S. shibatae are shown in red and unmodified sites in yellow. Allmethylation sites map to the surface of the protein, suggesting that the methylation happens after complex formation.

Figure 7. Sequence alignment of S. shibatae, S. solfataricus, and S. acidocaldarius RNAP subunits. Observed methyl-lysines are marked below thesequence: green circle, S. shibatae; cyan square, S. solfataricus; and magenta triangle, S. solfataricus. Secondary structure assignment extracted from thestructure of S. shibatae (PDB ID 4AYB) is depicted above the alignment using ESPRIPT software.31





et al.13 Currently available data suggest that the specificity ofmethylation pattern, rather than being determined by aminoacid sequence, might be guided by the local secondary structureand accessibility of the residue to be modified. This supportsthe idea of a certain structural specificity that may helpunderstand the differences in the methylation patternsdescribed.Lysine methylation in archaeal organisms has been directly

linked to protein stabilization.14,22,23 Methylation of lysinesincreases the pKa of the side chains, changing the hydropathy ofproteins and strengthening ionic interactions, and thereforecontributing to the side-chain stability.12 Lysine methylationmaps onto the surfaces of the three RNAP complexes,suggesting that the modifications happen after the assemblyof subunits into a full-complement RNAP enzyme. Bremang etal. have found that in mammalian HeLaS3 cells methylatedresidues are over-represented in proteins that form complexes.9

Their results suggest that methylation may play a role in theformation or stabilization of complexes. This modificationmight also be involved in the formation or stabilization ofarchaeal protein complexes. Methylation of certain sites mightstrengthen the protein structure through chemical modifica-tions; it might also improve the protein stability by competingfor that site with modifications that compromise proteinsurvival, such as ubiquitination. This mechanism has beenproposed for the stabilization of some eukaryan proteins,23 anda similar relationship between methylation and SAMPylationcould be proposed for archaeal organisms.24

Recent studies of methylation of other molecular machinessuch as ribosomes from Saccharomyces cerevisiae have revealedthe existence of both surface and buried methylation sites, thelatter being involved in the regulation of the interactionsbetween ribosomal proteins and rRNA.25 However, since noburied methyl-lysines were found in our study, this does notseem to apply to archaeal organisms. This observation impliesthat these PTMs are not involved in the interactions with theDNA or RNA bound by the complex. Similarly, one could alsospeculate that, in addition to its probable stabilizing role, lysinemethylation on the RNAP surface might serve as a rudimentalregulatory mechanism modulating RNA binding during its exitfrom the enzyme. Biophysical studies have shown that nascentmRNA binds to Rpo4/7;19 methylation sites on the stalkRpo4/7 domain might guide the exit of mRNA from theRNAP. In Eukarya, this putative crude regulatory mechanismwould have been superseded by the more sophisticatedregulatory system.We also identified some functionally related proteins,

purified together with the archaeal RNA subunits (seeSupplementary Tables 1 and 2). Heat stabilization-relatedthermosome subunits alpha (P46219), beta (P28488), andgamma (Q9HH21)26,27 were among the most abundant co-purified proteins in Ssh RNAP extracts. DNA/RNA-bindingproteins involved in transcription regulation, such as Alba(Q4J973 and P60848 UniProt entries)28,29 and TBP (TATAbox-binding protein, inferred from homology in UniProtM1IYI2),30 were also identified together with RNAP subunits.We also found some uncharacterized proteins such as single-stranded DNA-binding protein (ORF Name SacRo-n12I_04725, predicted protein, M1J131), uncharacterizedprotein M1IBK9, and the probable exosome complexexonuclease 1 (ECX1, Q4JB27), which should be highlightedas putative interactors with RNAP complexes. Interestingly,modified peptides for some of these putative interactors were

also identified in our approach (Supplementary Figure 3),showing that not only RNAP proteins may be modified andsuggesting additional regulation levels for their interaction.Further experiments should be performed to assess thesignificance of the proteins co-purified with the RNAP. Thepossibility that methylation might be a signal for therecruitment of specific proteins and cofactors, modulatingprotein degradation or protein activity, should not beoverlooked.

■ CONCLUSIONTo summarize, we performed in-solution and in-gel digestion ofRNAP from Ssh and Sac and combined CID and ETDfragmentation methods in mass spectrometry analysis tocharacterize methylation and N-terminal acetylation of theRNAP component subunits. We identified 20 methyl-lysines forSsh and 26 for Sac RNAP. Moreover, two N-terminalacetylation sites were found for each of the analyzed species.When we mapped the obtained data to the three-dimensionalstructure of the RNAP-DNA complex from Ssh, it becameapparent that all of the methyl-lysines were on the surface, farfrom the bound DNA. We hypothesize that these particularPTMs in archaeal RNAP would fulfill additional roles to that ofincreasing the stability of the enzyme in harsh environments,such as the modulation of the RNAP binding to othercomponents of the transcriptional machinery; however, furtherstudies are needed to test this hypothesis.

■ ASSOCIATED CONTENT*S Supporting Information

Supplementary tables and figures as described in the text. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*Tel: +34 94 406 13 15. Fax: +34 94 657 25 02. E-mail:[email protected] Address¶M.N.W.: EMBL-Grenoble, France.Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Yakov Korkhin for the gift of purified S.acidocaldarius RNAP and Stephen D. Bell for S. shibatae cells.Marina Ondiviela and Pietro Roversi are also thanked for helpduring S. shibatae biomass production, and Ibon Iloro andIraide Escobes for sample preparation and useful discussion.This work is supported by the CIC bioGUNE (to MA andMNW) and the Spanish Ministerio de Economia yCompetitividad BFU2012-33947 (to NGAA).

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Lysine Methylation Mapping of Crenarchaeal DNA-Directed RNA Polymerases by Collision-Induced and Electron-Transfer Dissociation Mass Spectrometry