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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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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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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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