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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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SAMP-conjugates are isolated by immunoprecipitationSAMP1 ± ΔGG and SAMP2 ± ΔGG were expressed as N-terminal FLAG-tagged fusions in H. volcanii grown in complex medium (CM) and nitrogen-limiting conditions (− N). Proteins were immunoprecipitated with α-FLAG, boiled and separated by either: a) reducing 12 % SDS-PAGE and analyzed by α-FLAG immunoblot or b) non-reducing 12 % SDS-PAGE and stained for total protein by SYPRO Ruby. Molecular mass standards and range of gel slices excised for MS-analysis are indicated on left. H. volcanii with vector alone served as a negative control in all experiments including MS-analysis of gel slices.
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Figure 4: SAMP-conjugates are isolated by immunoprecipitationSAMP1 ± ΔGG and SAMP2 ± ΔGG were expressed as N-terminal FLAG-tagged fusions in H. volcanii grown in complex medium (CM) and nitrogen-limiting conditions (− N). Proteins were immunoprecipitated with α-FLAG, boiled and separated by either: a) reducing 12 % SDS-PAGE and analyzed by α-FLAG immunoblot or b) non-reducing 12 % SDS-PAGE and stained for total protein by SYPRO Ruby. Molecular mass standards and range of gel slices excised for MS-analysis are indicated on left. H. volcanii with vector alone served as a negative control in all experiments including MS-analysis of gel slices.

Mentions: SAMP-conjugates were purified from H. volcanii cells expressing the FLAG-SAMP fusions by α-FLAG immunoprecipitation (IP) compared to cells expressing the FLAG-SAMP fusions with deletions in their C-terminal diglycine motif (ΔGG) or vector alone (Fig. 4). Unlike most proteins, the vast majority of proteins from haloarchaea are highly acidic and require high salt (> 1 M) for stability and activity20. Non-covalent protein complexes from these ‘salt-loving’ organisms typically dissociate in the low salt and detergent conditions required for IP. Consistent with this, SAMP-conjugates were readily purified by IP from H. volcanii based on α-FLAG immunoblot and SYPRO Ruby stain of these fractions (Fig. 4). The purified SAMP-conjugates were resistant to boiling in the presence of SDS and reducing reagents (Fig. 4a). The results also demonstrated that the C-terminal diglycine motif of SAMP1 and SAMP2 was required for their conjugation to proteins and that IP enhanced the ability to detect a remarkable diversity of SAMP-conjugates present in cells grown under rich and nitrogen-limiting conditions. It should also be noted that the SAMP-conjugate banding patterns were not influenced by addition of reducing reagents. Thus, IP combined with boiling, separation by SDS-PAGE and staining with SYPRO Ruby proved ideal for the isolation of covalently-linked FLAG-SAMP-conjugates (Fig. 4b). Proteins specific for the FLAG-SAMP expressing strains were excised from the gels, digested with trypsin and identified by mass spectrometry (MS). Using this approach, thirty-four SAMP-protein conjugates were identified including those present in cells grown under nutrient rich and nitrogen-limiting conditions (Table 1). Of the proteins identified, all were unique to the strains expressing the FLAG-SAMP fusions compared to cells with vector alone, and two of the conjugates were common to both SAMP1 and SAMP2 (HVO_0558 and HVO_A0230; Table 1). Consistent with their role as small archaeal modifier proteins, SAMP1 and SAMP2 were the only proteins identified in SDS-PAGE gel slices that spanned a wide-range of molecular masses (5 – 125 kDa, Supplementary Table 3).


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)

SAMP-conjugates are isolated by immunoprecipitationSAMP1 ± ΔGG and SAMP2 ± ΔGG were expressed as N-terminal FLAG-tagged fusions in H. volcanii grown in complex medium (CM) and nitrogen-limiting conditions (− N). Proteins were immunoprecipitated with α-FLAG, boiled and separated by either: a) reducing 12 % SDS-PAGE and analyzed by α-FLAG immunoblot or b) non-reducing 12 % SDS-PAGE and stained for total protein by SYPRO Ruby. Molecular mass standards and range of gel slices excised for MS-analysis are indicated on left. H. volcanii with vector alone served as a negative control in all experiments including MS-analysis of gel slices.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2872088&req=5

Figure 4: SAMP-conjugates are isolated by immunoprecipitationSAMP1 ± ΔGG and SAMP2 ± ΔGG were expressed as N-terminal FLAG-tagged fusions in H. volcanii grown in complex medium (CM) and nitrogen-limiting conditions (− N). Proteins were immunoprecipitated with α-FLAG, boiled and separated by either: a) reducing 12 % SDS-PAGE and analyzed by α-FLAG immunoblot or b) non-reducing 12 % SDS-PAGE and stained for total protein by SYPRO Ruby. Molecular mass standards and range of gel slices excised for MS-analysis are indicated on left. H. volcanii with vector alone served as a negative control in all experiments including MS-analysis of gel slices.
Mentions: SAMP-conjugates were purified from H. volcanii cells expressing the FLAG-SAMP fusions by α-FLAG immunoprecipitation (IP) compared to cells expressing the FLAG-SAMP fusions with deletions in their C-terminal diglycine motif (ΔGG) or vector alone (Fig. 4). Unlike most proteins, the vast majority of proteins from haloarchaea are highly acidic and require high salt (> 1 M) for stability and activity20. Non-covalent protein complexes from these ‘salt-loving’ organisms typically dissociate in the low salt and detergent conditions required for IP. Consistent with this, SAMP-conjugates were readily purified by IP from H. volcanii based on α-FLAG immunoblot and SYPRO Ruby stain of these fractions (Fig. 4). The purified SAMP-conjugates were resistant to boiling in the presence of SDS and reducing reagents (Fig. 4a). The results also demonstrated that the C-terminal diglycine motif of SAMP1 and SAMP2 was required for their conjugation to proteins and that IP enhanced the ability to detect a remarkable diversity of SAMP-conjugates present in cells grown under rich and nitrogen-limiting conditions. It should also be noted that the SAMP-conjugate banding patterns were not influenced by addition of reducing reagents. Thus, IP combined with boiling, separation by SDS-PAGE and staining with SYPRO Ruby proved ideal for the isolation of covalently-linked FLAG-SAMP-conjugates (Fig. 4b). Proteins specific for the FLAG-SAMP expressing strains were excised from the gels, digested with trypsin and identified by mass spectrometry (MS). Using this approach, thirty-four SAMP-protein conjugates were identified including those present in cells grown under nutrient rich and nitrogen-limiting conditions (Table 1). Of the proteins identified, all were unique to the strains expressing the FLAG-SAMP fusions compared to cells with vector alone, and two of the conjugates were common to both SAMP1 and SAMP2 (HVO_0558 and HVO_A0230; Table 1). Consistent with their role as small archaeal modifier proteins, SAMP1 and SAMP2 were the only proteins identified in SDS-PAGE gel slices that spanned a wide-range of molecular masses (5 – 125 kDa, Supplementary Table 3).

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