In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in -helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed. 1. Introduction Lysine methylation is found in all three domains of life. In bacteria, this posttranslational modification is restricted to a handful of ribosomal and flagellar proteins [1, 2]. In eukaryotes, lysine methylation is also restricted to a subset of proteins and catalysed by highly specific methyltransferases that can generate mono-, di-, and trimethylated lysines (reviewed in [3]). The most well-known examples are the histone proteins, where lysine methylation is carried out by sequence specific SET family methyltransferases using an S-adenosyl methionine (SAM) cofactor. These modifications result in changes in protein？:？protein interactions, chromatin structure and gene expression (reviewed in [4]). A limited number of other eukaryal proteins including notably the large subunit of Rubisco [5] are also subject to lysine methylation, though the function of these modifications is often not known [6]. More recently, proteome-wide studies of lysine methylation in the mouse brain [7] and Saccharomyces cerevisiae [8] have been added to the list of modified proteins. The latter study generated preliminary evidence for 25 monomethylated and 20 dimethylated lysines from a set of 2600 yeast proteins. The methylated proteins tended to have a higher abundance and longer half-life than average and included 11 ribosomal proteins [8]. The euryarchaeon Methanosarcina mazei encodes a clear SET-domain protein that has been shown to methylate a single lysine in the archaeal chromatin protein MC1, suggesting that mechanisms to modulate chromatin by posttranslational modification pre-date the divergence of the archaeal and eukaryal domains
References
[1]
F. N. Chang, “A sensitive method for the quantitative determination of minor bases in ribosomal RNA,” Analytical Biochemistry, vol. 63, no. 2, pp. 371–379, 1975.
[2]
W. K. Paik and S. Kim, “Protein methylation,” Science, vol. 174, no. 4005, pp. 114–119, 1971.
[3]
W. K. Paik, D. C. Paik, and S. Kim, “Historical review: the field of protein methylation,” Trends in Biochemical Sciences, vol. 32, no. 3, pp. 146–152, 2007.
[4]
C. Martin and Y. Zhang, “The diverse functions of histone lysine methylation,” Nature Reviews Molecular Cell Biology, vol. 6, no. 11, pp. 838–849, 2005.
[5]
R. C. Trievel, E. M. Flynn, R. L. Houtz, and J. H. Hurley, “Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT,” Nature Structural Biology, vol. 10, no. 7, pp. 545–552, 2003.
[6]
R. L. Houtz, R. Magnani, N. R. Nayak, and L. M. A. Dirk, “Co- and post-translational modifications in Rubisco: unanswered questions,” Journal of Experimental Botany, vol. 59, no. 7, pp. 1635–1645, 2008.
[7]
H. Iwabata, M. Yoshida, and Y. Komatsu, “Proteomic analysis of organ-specific post-translational lysine-acetylation and -methylation in mice by use of anti-acetyllysine and -methyllysine mouse monoclonal antibodies,” Proteomics, vol. 5, no. 18, pp. 4653–4664, 2005.
[8]
C. N. Pang, E. Gasteiger, and M. R. Wilkins, “Identification of arginine- and lysine-methylation in the proteome of Saccharomyces cerevisiae and its functional implications,” BMC Genomics, vol. 11, article 92, 2010.
[9]
K. L. Manzur and M.-M. Zhou, “An archaeal SET domain protein exhibits distinct lysine methyltransferase activity towards DNA-associated protein MC1-α,” FEBS Letters, vol. 579, no. 17, pp. 3859–3865, 2005.
[10]
B. Maras, V. Consalvi, and V. Consalvi, “The protein sequence of glutamate dehydrogenase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium. Is the presence of N-ε-methyllysine related to thermostability?” European Journal of Biochemistry, vol. 203, no. 1-2, pp. 81–87, 1992.
[11]
B. Maras, S. Valiante, and S. Valiante, “The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium,” Journal of Protein Chemistry, vol. 13, no. 2, pp. 253–259, 1994.
[12]
F. Febbraio, A. Andolfo, and A. Andolfo, “Thermal stability and aggregation of Sulfolobus solfataricus β-glycosidase are dependent upon the N-ε-methylation of specific lysyl residues: critical role of in vivo post-translational modifications,” Journal of Biological Chemistry, vol. 279, no. 11, pp. 10185–10194, 2004.
[13]
H. Baumann, S. Knapp, T. Lundb？ck, R. Ladenstein, and T. H？rd, “Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus,” Nature Structural Biology, vol. 1, no. 11, pp. 808–819, 1994.
[14]
S. Paytubi and M. F. White, “The crenarchaeal DNA damage-inducible transcription factor B paralogue TFB3 is a general activator of transcription,” Molecular Microbiology, vol. 72, no. 6, pp. 1487–1499, 2009.
[15]
A. Shevchenko, M. Wilm, O. Vorm, and M. Mann, “Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels,” Analytical Chemistry, vol. 68, no. 5, pp. 850–858, 1996.
[16]
Y. Korkhin, U. M. Unligil, and U. M. Unligil, “Evolution of complex RNA polymerases: the complete archaeal RNA polymerase structure,” PLoS Biology, vol. 7, no. 5, article e102, 2009.
[17]
A. Hirata, B. J. Klein, and K. S. Murakami, “The X-ray crystal structure of RNA polymerase from Archaea,” Nature, vol. 451, no. 7180, pp. 851–854, 2008.
[18]
S. Paytubi and M. F. White, “The crenarchaeal DNA damage-inducible transcription factor B paralogue TFB3 is a general activator of transcription,” Molecular Microbiology, vol. 72, no. 6, pp. 1487–1499, 2009.
[19]
T. A. Couttas, M. J. Raftery, G. Bernardini, and M. R. Wilkins, “Immonium ion scanning for the discovery of post-translational modifications and its application to histones,” Journal of Proteome Research, vol. 7, no. 7, pp. 2632–2641, 2008.
[20]
D. T. Mackay, C. H. Botting, G. L. Taylor, and M. F. White, “An acetylase with relaxed specificity catalyses protein N-terminal acetylation in Sulfolobus solfataricus,” Molecular Microbiology, vol. 64, no. 6, pp. 1540–1548, 2007.
[21]
G. E. Crooks, G. Hon, J.-M. Chandonia, and S. E. Brenner, “WebLogo: a sequence logo generator,” Genome Research, vol. 14, no. 6, pp. 1188–1190, 2004.
[22]
X. Luo, U. Schwarz-Linek, C. H. Botting, R. Hensel, B. Siebers, and M. F. White, “CC1, a novel crenarchaeal DNA binding protein,” Journal of Bacteriology, vol. 189, no. 2, pp. 403–409, 2007.
[23]
P. A. Kirkland, M. A. Humbard, C. J. Daniels, and J. A. Maupin-Furlow, “Shotgun proteomics of the haloarchaeon haloferax volcanii,” Journal of Proteome Research, vol. 7, no. 11, pp. 5033–5039, 2008.
[24]
Y. Zivanovic, J. Armengaud, and J. Armengaud, “Genome analysis and genome-wide proteomics of Thermococcus gammatolerans, the most radioresistant organism known amongst the Archaea,” Genome Biology, vol. 10, no. 6, article R70, 2009.
[25]
A. M. Lee, J. R. Sevinsky, J. L. Bundy, A. M. Grunden, and J. L. Stephenson Jr., “Proteomics of Pyrococcus furiosus, a hyperthermophilic archaeon refractory to traditional methods,” Journal of Proteome Research, vol. 8, no. 8, pp. 3844–3851, 2009.
[26]
Q. Xia, T. Wang, E. L. Hendrickson, T. J. Lie, M. Hackett, and J. A. Leigh, “Quantitative proteomics of nutrient limitation in the hydrogenotrophic methanogen Methanococcus maripaludis,” BMC Microbiology, vol. 9, article 149, 2009.
[27]
C. Brochier-Armanet, B. Boussau, S. Gribaldo, and P. Forterre, “Mesophilic crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota,” Nature Reviews Microbiology, vol. 6, no. 3, pp. 245–252, 2008.
[28]
E. Hughes, R. M. Burke, and A. J. Doig, “Inhibition of toxicity in the β-amyloid peptide fragment β-(25-35) using N-methylated derivatives. A general strategy to prevent amyloid formation,” Journal of Biological Chemistry, vol. 275, no. 33, pp. 25109–25115, 2000.
[29]
M. ？ebela, T. ？tosová, J. Havli？, N. Wielsch, H. Thomas, Z. Zdráhal, and A. Shevchenko, “Thermostable trypsin conjugates for high-throughput proteomics: synthesis and performance evaluation,” Proteomics, vol. 6, no. 10, pp. 2959–2963, 2006.
[30]
Y. Kim, P. Quartey, and P. Quartey, “Large-scale evaluation of protein reductive methylation for improving protein crystallization,” Nature Methods, vol. 5, no. 10, pp. 853–854, 2008.
