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

Humbard MA, Miranda HV, Lim JM, Krause DJ, Pritz JR, Zhou G, Chen S, Wells L, Maupin-Furlow JA - Nature (2010)

Bottom Line: This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes.The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway.The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, USA.

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 beta-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 epsilon-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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MS/MS spectra of SAMP2-conjugate sitesSAMP2-modification of: (a) HVO_2328 K90 based on mass difference between b2-22 and b2-23 ions and loss of Gly1-Gly2 at 1238.46 m/z from the b2-23 ion derived from the triply charged precursor ion. (b) HVO_0025 K162 based on mass difference between both ion series derived from the doubly charged precursor ion: (i) y4 and y5 ions and loss of Gly1-Gly2 at 618.21 and 560.11 m/z, (ii) b7 and b8 ions and loss of Gly1-Gly2 at 976.06 m/z. (c - d) HVO_1727 K63 and K53. SAMP2 C-terminal diglycine (-Gly1-Gly2). Other MS/MS spectra, Supplementary Figure 2.
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Figure 6: MS/MS spectra of SAMP2-conjugate sitesSAMP2-modification of: (a) HVO_2328 K90 based on mass difference between b2-22 and b2-23 ions and loss of Gly1-Gly2 at 1238.46 m/z from the b2-23 ion derived from the triply charged precursor ion. (b) HVO_0025 K162 based on mass difference between both ion series derived from the doubly charged precursor ion: (i) y4 and y5 ions and loss of Gly1-Gly2 at 618.21 and 560.11 m/z, (ii) b7 and b8 ions and loss of Gly1-Gly2 at 976.06 m/z. (c - d) HVO_1727 K63 and K53. SAMP2 C-terminal diglycine (-Gly1-Gly2). Other MS/MS spectra, Supplementary Figure 2.

Mentions: To enhance MS-coverage and map the sites of SAMPylation, FLAG-SAMP2-conjugates were purified by α-FLAG in liquid phase for analysis of trypsinized peptides by reversed phase liquid chromatography coupled with tandem mass spectrometry (RP-LC-MS/MS) using a data dependent MS/MS scan mode and parent mass list method. Unlike SAMP1, which has a limited number of C-terminal trypsin cleavage sites, SAMP2 has a lysine at position 64. Thus, if an isopeptide bond is formed between the C-terminal carboxylate of SAMP2 and an amino group of the substrate protein, SAMP2 will leave a ‘GG-footprint’ on the target site after trypsinization. Using this approach, eleven sites of SAMP2 modification were mapped by collision-induced dissociation (CID) based MS/MS (Table 2). The sites were based on the mass differences between the y and b ion series containing the SAMP2-derived GG-footprint on lysine residues (Fig. 6 and Supplementary Fig. 2). The SAMPylated peptides were detected from doubly- to quadruply-charged molecular ions and mapped by more than one peptide on the same protein.


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

Humbard MA, Miranda HV, Lim JM, Krause DJ, Pritz JR, Zhou G, Chen S, Wells L, Maupin-Furlow JA - Nature (2010)

MS/MS spectra of SAMP2-conjugate sitesSAMP2-modification of: (a) HVO_2328 K90 based on mass difference between b2-22 and b2-23 ions and loss of Gly1-Gly2 at 1238.46 m/z from the b2-23 ion derived from the triply charged precursor ion. (b) HVO_0025 K162 based on mass difference between both ion series derived from the doubly charged precursor ion: (i) y4 and y5 ions and loss of Gly1-Gly2 at 618.21 and 560.11 m/z, (ii) b7 and b8 ions and loss of Gly1-Gly2 at 976.06 m/z. (c - d) HVO_1727 K63 and K53. SAMP2 C-terminal diglycine (-Gly1-Gly2). Other MS/MS spectra, Supplementary Figure 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2872088&req=5

Figure 6: MS/MS spectra of SAMP2-conjugate sitesSAMP2-modification of: (a) HVO_2328 K90 based on mass difference between b2-22 and b2-23 ions and loss of Gly1-Gly2 at 1238.46 m/z from the b2-23 ion derived from the triply charged precursor ion. (b) HVO_0025 K162 based on mass difference between both ion series derived from the doubly charged precursor ion: (i) y4 and y5 ions and loss of Gly1-Gly2 at 618.21 and 560.11 m/z, (ii) b7 and b8 ions and loss of Gly1-Gly2 at 976.06 m/z. (c - d) HVO_1727 K63 and K53. SAMP2 C-terminal diglycine (-Gly1-Gly2). Other MS/MS spectra, Supplementary Figure 2.
Mentions: To enhance MS-coverage and map the sites of SAMPylation, FLAG-SAMP2-conjugates were purified by α-FLAG in liquid phase for analysis of trypsinized peptides by reversed phase liquid chromatography coupled with tandem mass spectrometry (RP-LC-MS/MS) using a data dependent MS/MS scan mode and parent mass list method. Unlike SAMP1, which has a limited number of C-terminal trypsin cleavage sites, SAMP2 has a lysine at position 64. Thus, if an isopeptide bond is formed between the C-terminal carboxylate of SAMP2 and an amino group of the substrate protein, SAMP2 will leave a ‘GG-footprint’ on the target site after trypsinization. Using this approach, eleven sites of SAMP2 modification were mapped by collision-induced dissociation (CID) based MS/MS (Table 2). The sites were based on the mass differences between the y and b ion series containing the SAMP2-derived GG-footprint on lysine residues (Fig. 6 and Supplementary Fig. 2). The SAMPylated peptides were detected from doubly- to quadruply-charged molecular ions and mapped by more than one peptide on the same protein.

Bottom Line: This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes.The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway.The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, USA.

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 beta-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 epsilon-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.

Show MeSH
Related in: MedlinePlus