SAMP3 Tandem Mass Spectrometry Data

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Title:
SAMP3 Tandem Mass Spectrometry Data
Series Title:
Archaeal ubiquitin-like SAMP3 is Isopeptide-linked to Proteins by a UbaA-dependent Mechanism
Physical Description:
Dataset
Creator:
Miranda, Hugo
Publisher:
The American Society for Biochemistry and Molecular Biology
Place of Publication:
Molecular and Cellular Proteomics
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Genre:
dataset   ( sobekcm )

Notes

Abstract:
SAMP1/2 are ubiquitin-like proteins that function as protein modifiers and are required for the production of sulfur-containing biomolecules in the archaeon Haloferax volcanii. Here we report a novel small archaeal modifier protein (named SAMP3), with a β-grasp fold and C-terminal diglycine motif characteristic of ubiquitin, that is functional in protein conjugation in Hfx. volcanii. SAMP3 conjugates were dependent on the ubiquitin activating E1 enzyme homolog of archaea (UbaA) for synthesis and were cleaved by the JAMM/MPN+ domain metalloprotease HvJAMM1. Twenty-three proteins (28 lysine residues) were found to be isopeptide-linked to the C-terminal carboxylate of SAMP3, and 331 proteins were reproducibly found associated with SAMP3 in a UbaA-dependent manner based on tandem mass spectrometry (MS/MS) analysis. The molybdopterin (MPT) synthase large subunit homolog MoaE, found samp3ylated at conserved active site lysine residues by MS/MS, was also shown to be covalently bound to SAMP3 by immunoprecipitation and tandem affinity purifications. HvJAMM1 was demonstrated to catalyze the cleavage of SAMP3 from MoaE, suggesting a mechanism of controlling MPT synthase activity. The levels of samp3ylated proteins and samp3 transcripts were found to be increased by the addition of DMSO to aerobically growing cells. Thus, we propose a model in which samp3ylation is covalent, reversible and controls the activity of enzymes such as MPT synthase. Sampylation of MPT synthase may govern the levels of molybdenum cofactor (MoCo) available and, thus, facilitate the scavenging of oxygen prior to the transition to respiration with MoCo-containing terminal reductases that use alternative electron acceptors such as DMSO. Overall, our study of SAMP3 provides a new insight into the diversity of functional ubiquitin-like protein modifiers and the network of Ubl protein targets in Archaea.
Acquisition:
Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Hugo Miranda.
Publication Status:
Published

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Source Institution:
University of Florida Institutional Repository
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution.
System ID:
IR00003529:00001

Full Text

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Table 5 3 Relative representat ion (RR) of COG groupings of proteins found to co purify with SAMP3 in the presence of UbaA compared to the proteins deduced from genome sequence a a F unctional categories of clusters of orthologous genes (COGs) ( http://www.ncbi.nlm.nih.gov/COG ), as updated for Archaea by Wolf et al. (Wolf et al ., 2012) Gen #, COG groupings of proteins deduced from the Hfx. volcanii DS2 genome sequence (Hartman et al. 2010) according to Wolf et al (Wolf et al ., 2012) Fr Gen, fraction of the total number of deduced proteins classified. Expect S3, COG distribution of SAMP3 A90K associated proteins enriched in wild type (compared to ) predicted based on the number of SAMP3 A90K associated p roteins identified b y MS that were classified to COGs ( 330 proteins) compared to Fr Gen. Actual S3 # and Fr Actual S3, COG distribution and fraction of the total number of classified proteins identified by MS to be SAMP3 A90K associated, respectively. RR S3/Gen, relative rati o of fractions of proteins identified by MS of SAMP3 samples (S3) and deduced from genome sequence ( Gen ), indicated as S3/ Gen if S3 > Gen (S3 over represented), and as Gen /S3 if Gen > S3 (S3 under represented) G, G test on RR of S3 compared to Gen ( where designates p < 0.0 01 ** designates p < 0.00 0 1 ) COG description COG Gen # Fr Gen Exp ect S3 Actual S3 # Fr Actual S3 RR S3/ Gen G Information storage and processing Translation, ribosomal structure and biogenesis J 172 0.04 5 14.9 35 0.10 6 + 2.3 6 21.03 ** Transcription K 264 0.06 9 22.8 21 0.064 1 .08 0.15 Replication, recombination and repair L 296 0.077 25.4 21 0.064 1. 21 0.88 Cellular processes and signaling Cell cycle control, cell division, chrom. partitioning D 21 0.00 6 2.0 4 0.012 + 2.02 1.60 Defense mechanisms V 70 0.018 5 .9 5 0.01 5 1. 19 0.16 Signal transduction mechanisms T 162 0.042 13.9 3 0.0 09 4.62 12.91* Cell wall/membrane/envelope biogenesis M 97 0.025 8.3 12 0.03 6 + 1.45 1.54 Cell motility N 56 0.01 5 5.0 1 0 .003 4.95 4.75 Intracell. t rafficking, secretion & vesicular transport U 20 0.005 1.7 0 0 inf. 3.31 Posttransl. Mod., protein turnover, chaperones O 114 0.030 9.9 17 0.052 + 1.72 4.34 Metabolism Energy production and conversion C 221 0.05 8 19.1 27 0.082 + 1.41 3.06 Carbohydrate transport and metabolism G 154 0.040 13.2 17 0.052 + 1. 29 1.05 Amino acid transport and metabolism E 360 0.09 4 31.0 48 0.145 + 1.55 8.93 Nucleotide transport and metabolism F 79 0.02 1 6.9 5 0.015 1.39 0.61 Coenzyme transport and metabolism H 128 0.033 10.9 2 5 0.076 + 2.30 13.96* Lipid transport and metabolism I 82 0.021 6.9 22 0.067 + 3.1 7 21.40 ** Inorganic ion transport and metabolism P 177 0.046 15.2 13 0.039 1.17 0.34 Secondary metabol biosyn. transp. & catabolism Q 53 0.01 4 4.6 8 0.024 + 1.73 2.06 Poorly characterized General function prediction only R 416 0.108 35.6 21 0.064 1.70 7.78 Unknown S 900 0.234 77.2 25 0.076 3.09 58.13 ** Total classified 3842 330 unclassified 442 1 Total 4284 331



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Table 5 4 Samp3ylation sites identified via LC MS/MS proteomic analysis. Normalized spectral counts d Locus tag GI number Protein / homolog description Mod. Site c S3 C R S3/C COG e SAMP1/2 associated /modified f HVO_0078 292654255 HemC porphobilinogen deaminase K233 (3x) 32.0 <1.0 32.0 H HVO_0206 292654386 Alanine -tRNA ligase alaS1 K517 (2x) 71.7 5.5 13.0 J HVO_0337 292654517 G lutaredoxin like protein K19 (1x) 6.3 <1.0 6.3 O HVO_0359 a 292654537 T ranslation elongation factor EF K99 (1x) 125.0 31 4.0 J SAMP1 associated, HVO_0359 protein accumulates after CLBL treatment HVO_0455 292654632 CctB thermosome subunit 2 K280 (1x) 57.7 <1.0 57.7 O HVO_0736 292654899 DUF302 superfamily protein K57 (2x) 20.3 <1.0 20.3 S SAMP2 associated HVO_0835 292654998 Acetyl CoA C acyltransferase acaB1 K330 (3x) 55.7 4.5 12.4 I HVO_0860 292655023 SufB FeS assembly protein K115 (3x) K316 (3x) 408.0 17.5 23.3 O HVO_0861 (SufB homolog) associated with SAMP2 HVO_0966 b 292655126 R ibose 1,5 bisphosphate isomerase K210 (3x) 788.3 <1.0 788.3 J K210 samp2ylated d HVO_1081 292655239 G lutaredoxin homolog K31 (2x) 10.7 5.5 1.9 O HVO_1289 292655444 OsmC like protein K59 (2x) 33.0 <1.0 33.0 O K59 samp2ylated, HVO_1289 protein accumulates in panA HVO_1495 292655647 F ructose PTS enzyme IIB K83 (2x) 7.0 <1.0 7.0 S HVO_1577 292655727 Transcriptional regulator ( winged HTH and CBS domains ) K52 (1x) 25.0 16.5 1.5 K SAMP2 associated HVO_1611 292655761 C onserved hypothetical protein K49 (1x) s.r. s.r. s.r. S HVO_1655 292655804 T hioredoxin like K74 ( 3 x) s.r. s.r. s.r. O HVO_1864 292655999 MoaE MobB domain protein K240 (2x) K247 (2x) 58.7 <1.0 58.7 H K240 and K247 samp1ylated HVO_1896 292656029 R ibosomal protein S24e K69 (1x) s.r. s.r. s.r. J HVO_2011 292656139 C onserved hypothetical protein K129 (1x) 60.3 <1.0 60.3 S HVO_2104 292656231 A rabitol PTS enzyme IIB K40 (3x) K42 (3x) 10 <1.0 10 G HVO_2177* 292656305 SAMP3 K18 (3x) K55 (3x) K62 (3x) 1282 1801 0.7 H HVO_2328 292656456 I sochorismatase family protein K90 (2x) 68.0 <1.0 68.0 Q K90 samp2ylated HVO_B0057 292493996 CobJ precorrin 3B C17 methyltransferase K341 (1x) 53.0 <1.0 53.0 H encoded in an apparent operon with SAMP2 associated HVO_B0053 HVO_B0324 292494257 C onserved hypothetical protein K297 (2x) 34.3 <1.0 34.3 S

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Table 5 4 a HVO _0359, homolog of eukaryotic EF isopeptidase activity 1 (Gonen et al. 1996) b HVO_0966, homolog of the ribose 1,5 bisphosphate isomerase of the proposed 2 novel pathway of AMP metabolism in the hyperthermophilic archaeon Thermococcus kodakarensis 3 (Aono et al. 2012; Sato et al. 2007) c Modification site wi th lysine residue covalently attached by an 4 isopeptide bond to SAMP3 indicated by amino acid residue number deduced from genome sequence 5 with the exception of HVO_2177* (SAMP3), the latter of which is based on Met22 as the biological start 6 codon. A ll samp3 ylated sites were detected in wild type (vs. ubaA ) strains expressing Flag SAMP3 A90K 7 See Methods for details on cell culture and conjugate isolation. The n umber of biological replicates in 8 which a site was detected in its modified form via high quality M S/ MS spectra is in parentheses 9 d Normalized spectral count average ( ) and ratio (R) of the avera ges based on LC MS/MS analysis of 10 biological replicates of Flag SAMP3 A90K fractions purified from wild type (S3) and mutant (C, 11 control). e Clusters of Orthologous Groups (COGs) based on Wolf et al. (Wolf et al. 2012) 12 f Proteins/protein homologs found associated with and covalently modified by SAMP1/2, accumulated 13 after treatment with proteasome inhibitor clastolactacystin lactone (CLBL), and enhanced by deletion 14 of the Rpt like AAA ATPase PAN A ( panA ) were as previously described (Humbard et al. 2010; Hepowit 15 et al. 2012; Kirkland et al. 2008; Kirkland et al. 2007) Proteins not found associate d with SAMP1/2 are 16 indicated by ( ). Note: s.r., detection of two or more samp3ylat ed peptides in single replicate 17 18

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List of References Aono, R., Sato, T., Yano, A., Yoshida, S., Nishitani, Y., Miki, K., Imanaka, T., and Atomi, H. (2012) Enzymatic characterization of AMP phosphorylase and ribose 1,5 bisphosphate isomerase functioning in an archaeal AMP metabolic pathway. J Bacteriol 194 : 6847 6855. Gonen, H., Dickman, D., Schwartz, A. L., and Ciechanover, A. (1996) Protein synthesis elongation factor EF 1 is an isopeptidase essential for ubiquitin dependent degradation of certain proteolytic substrates. Adv Exp Med Biol 389:209 19. : 2 09 219. Hepowit, N. L., Uthandi, S., Miranda, H. V., Toniutti, M., Prunetti, L., Olivarez, O., De Vera, I. M., Fanucci, G. E., Chen, S., and Maupin Furlow, J. A. (2012) Archaeal JAB1/MPN/MOV34 metalloenzyme (HvJAMM1) cleaves ubiquitin like small archaeal modifier proteins (SAMPs) from protein conjugates. Mol Microbiol : 10. Humbard, M. A., Miranda, H. V., Lim, J. M., Krause, D. J., Pritz, J. R., Zhou, G., Chen, S., Wells, L., and Maupin Furlow, J. A. (2010) Ubiquitin like small archaeal modifier proteins (SAMPs) in Haloferax volcanii Nature 463 : 54 60. Kirkland, P. A., Gil, M. A., Karadzic, I. M., and Maupin Furlow, J. A. (2008) Genetic and proteomic analyses of a proteasome activating nucleotidase A mutant of the haloarchaeon Haloferax volcanii J Bacte riol 190 : 193 205. Kirkland, P. A., Reuter, C. J., and Maupin Furlow, J. A. (2007) Effect of proteasome inhibitor clasto lactacystin lactone on the proteome of the haloarchaeon Haloferax volcanii Microbiology 153 : 2271 2280. Sato, T., A tomi, H., and I manaka, T. (2007 ) Archaeal type III RuBisCOs function in a pathway for AMP metabolism. Science 315 : 1003 1006. Wolf, Y. I., Makarova, K. S., Yutin, N., and Koonin, E. V. (2012) Updated clusters of orthologous genes for Archaea: a complex ancestor of the A rchaea and the byways of horizontal gene transfer. Biol Direct 7: 46 47.