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Ubiquitin-like small archaeal modifier proteins (SAMPs) in Haloferax volcanii

Abstract

Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a β-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, that form protein conjugates in the archaeon Haloferax volcanii. The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the ε-amino group of lysines from a number of protein targets and Lys 58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.

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Figure 1: Multiple amino acid sequence alignment of the C termini of Ub, Urm1 and PUP to select diglycine motif proteins of H. volcanii.
Figure 2: SAMP1 and SAMP2 are differentially conjugated to proteins and influenced by nitrogen-limitation.
Figure 3: SAMP-conjugates are altered by proteasomal gene knockout.
Figure 4: SAMP-conjugates are isolated by immunoprecipitation.
Figure 5: SAMP and SAMP-conjugates are related to sulphur-activation and ubiquitination pathways.
Figure 6: MS/MS spectra of SAMP2-conjugate sites.

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References

  1. Hochstrasser, M. Origin and function of ubiquitin-like proteins. Nature 458, 422–429 (2009)

    Article  ADS  CAS  Google Scholar 

  2. Pickart, C. M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610–616 (2004)

    Article  CAS  Google Scholar 

  3. Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009)

    Article  CAS  Google Scholar 

  4. Ciechanover, A. & Ben-Saadon, R. N-terminal ubiquitination: more protein substrates join in. Trends Cell Biol. 14, 103–106 (2004)

    Article  CAS  Google Scholar 

  5. Burns, K. E., Liu, W. T., Boshoff, H. I., Dorrestein, P. C. & Barry, C. E. Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein. J. Biol. Chem. 284, 3069–3075 (2009)

    Article  CAS  Google Scholar 

  6. Pearce, M. J., Mintseris, J., Ferreyra, J., Gygi, S. P. & Darwin, K. H. Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis . Science 322, 1104–1107 (2008)

    Article  ADS  CAS  Google Scholar 

  7. Striebel, F. et al. Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes. Nature Struct. Mol. Biol. 16, 647–651 (2009)

    Article  CAS  Google Scholar 

  8. Liao, S. et al. Pup, a prokaryotic ubiquitin-like protein, is an intrinsically disordered protein. Biochem. J. 422, 207–215 (2009)

    Article  CAS  Google Scholar 

  9. Chen, X. et al. Prokaryotic ubiquitin-like protein pup is intrinsically disordered. J. Mol. Biol. 392, 208–217 (2009)

    Article  CAS  Google Scholar 

  10. Iyer, L. M., Burroughs, A. M. & Aravind, L. The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like β-grasp domains. Genome Biol. 7, R60 (2006)

    Article  Google Scholar 

  11. Burroughs, A. M., Balaji, S., Iyer, L. M. & Aravind, L. A novel superfamily containing the β-grasp fold involved in binding diverse soluble ligands. Biol. Direct 2, 4 (2007)

    Article  Google Scholar 

  12. Burroughs, A. M., Iyer, L. M. & Aravind, L. Natural history of the E1-like superfamily: Implication for adenylation, sulfur transfer, and ubiquitin conjugation. Proteins 75, 895–910 (2009)

    Article  CAS  Google Scholar 

  13. Kessler, D. Enzymatic activation of sulfur for incorporation into biomolecules in prokaryotes. FEMS Microbiol. Rev. 30, 825–840 (2006)

    Article  CAS  Google Scholar 

  14. Yao, T. & Cohen, R. E. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002)

    Article  ADS  CAS  Google Scholar 

  15. Verma, R. et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615 (2002)

    Article  ADS  CAS  Google Scholar 

  16. Cope, G. A. et al. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298, 608–611 (2002)

    Article  ADS  CAS  Google Scholar 

  17. Zhou, G., Kowalczyk, D., Humbard, M. A., Rohatgi, S. & Maupin-Furlow, J. A. Proteasomal components required for cell growth and stress responses in the haloarchaeon Haloferax volcanii . J. Bacteriol. 190, 8096–8105 (2008)

    Article  CAS  Google Scholar 

  18. Kaczowka, S. J. & Maupin-Furlow, J. A. Subunit topology of two 20S proteasomes from Haloferax volcanii . J. Bacteriol. 185, 165–174 (2003)

    Article  CAS  Google Scholar 

  19. Reuter, C. J., Kaczowka, S. J. & Maupin-Furlow, J. A. Differential regulation of the PanA and PanB proteasome-activating nucleotidase and 20S proteasomal proteins of the haloarchaeon Haloferax volcanii . J. Bacteriol. 186, 7763–7772 (2004)

    Article  CAS  Google Scholar 

  20. Albuquerque, C. P. et al. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol. Cell. Proteomics 7, 1389–1396 (2008)

    Article  CAS  Google Scholar 

  21. Leidel, S. et al. Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA. Nature 458, 228–232 (2009)

    Article  ADS  CAS  Google Scholar 

  22. Schlieker, C. D., Van der Veen, A. G., Damon, J. R., Spooner, E. & Ploegh, H. L. A functional proteomics approach links the ubiquitin-related modifier Urm1 to a tRNA modification pathway. Proc. Natl Acad. Sci. USA 105, 18255–18260 (2008)

    Article  ADS  CAS  Google Scholar 

  23. Furukawa, K., Mizushima, N., Noda, T. & Ohsumi, Y. A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes. J. Biol. Chem. 275, 7462–7465 (2000)

    Article  CAS  Google Scholar 

  24. Schmitz, J. et al. The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins. Biochemistry 47, 6479–6489 (2008)

    Article  CAS  Google Scholar 

  25. Noma, A., Sakaguchi, Y. & Suzuki, T. Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions. Nucleic Acids Res. 37, 1335–1352 (2009)

    Article  CAS  Google Scholar 

  26. Gonen, H. et al. Protein synthesis elongation factor EF-1α is essential for ubiquitin-dependent degradation of certain Nα-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu. Proc. Natl Acad. Sci. USA 91, 7648–7652 (1994)

    Article  ADS  CAS  Google Scholar 

  27. Gonen, H., Dickman, D., Schwartz, A. L. & Ciechanover, A. Protein synthesis elongation factor EF-1α is an isopeptidase essential for ubiquitin-dependent degradation of certain proteolytic substrates. Adv. Exp. Med. Biol. 389, 209–219 (1996)

    Article  CAS  Google Scholar 

  28. Humbard, M. A., Stevens, S. M. & Maupin-Furlow, J. A. Posttranslational modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii . J. Bacteriol. 188, 7521–7530 (2006)

    Article  CAS  Google Scholar 

  29. Maupin-Furlow, J. A., Wilson, H. L., Kaczowka, S. J. & Ou, M. S. Proteasomes in the archaea: from structure to function. Front. Biosci. 5, d837–d865 (2000)

    CAS  PubMed  Google Scholar 

  30. Goehring, A. S., Rivers, D. M. & Sprague, G. F. Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p. Eukaryot. Cell 2, 930–936 (2003)

    Article  CAS  Google Scholar 

  31. Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. 'Protein Modifications: Beyond the Usual Suspects' review series. EMBO Rep. 9, 536–542 (2008)

    Article  CAS  Google Scholar 

  32. Schulman, B. A. & Harper, J. W. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nature Rev. Mol. Cell Biol. 10, 319–331 (2009)

    Article  CAS  Google Scholar 

  33. Hoeller, D. et al. E3-independent monoubiquitination of ubiquitin-binding proteins. Mol. Cell 26, 891–898 (2007)

    Article  CAS  Google Scholar 

  34. Tarasov, V. Y. et al. A small protein from the bopbrp intergenic region of Halobacterium salinarum contains a zinc finger motif and regulates bop and crtB1 transcription. Mol. Microbiol. 67, 772–780 (2008)

    Article  CAS  Google Scholar 

  35. Borden, K. L. RING fingers and B-boxes: zinc-binding protein-protein interaction domains. Biochem. Cell Biol. 76, 351–358 (1998)

    Article  CAS  Google Scholar 

  36. Kirkland, P. A., Reuter, C. J. & Maupin-Furlow, J. A. Effect of proteasome inhibitor clasto-lactacystin-β-lactone on the proteome of the haloarchaeon Haloferax volcanii . Microbiology 153, 2271–2280 (2007)

    Article  CAS  Google Scholar 

  37. Kirkland, P. A., Gil, M. A., Karadzic, I. M. & Maupin-Furlow, J. A. Genetic and proteomic analyses of a proteasome-activating nucleotidase a mutant of the haloarchaeon Haloferax volcanii . J. Bacteriol. 190, 193–205 (2008)

    Article  CAS  Google Scholar 

  38. Leimkuhler, S., Freuer, A., Araujo, J. A., Rajagopalan, K. V. & Mendel, R. R. Mechanistic studies of human molybdopterin synthase reaction and characterization of mutants identified in group B patients of molybdenum cofactor deficiency. J. Biol. Chem. 278, 26127–26134 (2003)

    Article  Google Scholar 

  39. Matthies, A., Rajagopalan, K. V., Mendel, R. R. & Leimkuhler, S. Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans. Proc. Natl Acad. Sci. USA 101, 5946–5951 (2004)

    Article  ADS  CAS  Google Scholar 

  40. McLuskey, K., Harrison, J. A., Schuttelkopf, A. W., Boxer, D. H. & Hunter, W. N. Insight into the role of Escherichia coli MobB in molybdenum cofactor biosynthesis based on the high resolution crystal structure. J. Biol. Chem. 278, 23706–23713 (2003)

    Article  CAS  Google Scholar 

  41. Colnaghi, R., Cassinelli, G., Drummond, M., Forlani, F. & Pagani, S. Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition. FEBS Lett. 500, 153–156 (2001)

    Article  CAS  Google Scholar 

  42. Spallarossa, A. et al. The “rhodanese” fold and catalytic mechanism of 3-mercaptopyruvate sulfurtransferases: crystal structure of SseA from Escherichia coli . J. Mol. Biol. 335, 583–593 (2004)

    Article  CAS  Google Scholar 

  43. Dyall-Smith, M. The Halohandbook: Protocols for Halobacterial Genetics. (2008)

    Google Scholar 

  44. Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the staff at UF ICBR including C. Diaz and R. Zheng for MS and S. Shanker for DNA sequencing. Thanks to M. Terns and F. Aydemir for advice on purification of SAMP conjugates from anti-Flag agarose, N. Furlow for plasmid DNA preparations and J. Foster for other advice. We thank also T. Allers, M. Mevarech, M. Dyall-Smith and M. Danson labs for H. volcanii strains and plasmids. This work was funded in part by NIH 1S10 RR025418-01 to SC, Integrated Technology Resource for Biomedical Glycomics at UGA (supported by NIH/NCRR P41 RR018502, L.W. senior investigator) and NIH R01 GM057498 and DOE DE-FG02-05ER15650 to J.A.M.-F.

Author Contributions J.A.M.-F., M.A.H., D.J.K., J.R.P. and H.V.M. performed cloning and immunoblot experiments. M.A.H. and H.V.M. purified SAMP conjugates by anti-Flag immunoprecipitation and chromatography. G.Z. transformed H. volcanii and prepared media. S.C. directed the identification of SAMP conjugates by MS. J.-M.L. and L.W. mapped the SAMP-conjugate sites by CID-based MS/MS. J.A.M.-F., L.W., M.A.H. and J.-M.L. interpreted the data. J.A.M.-F.planned the studies and wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Julie A. Maupin-Furlow.

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Humbard, M., Miranda, H., Lim, JM. et al. Ubiquitin-like small archaeal modifier proteins (SAMPs) in Haloferax volcanii. Nature 463, 54–60 (2010). https://doi.org/10.1038/nature08659

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