Dataset Information

Ubiquitin S65 proteomics: Association of ubiquitin mutants with ubiqiutin binding domains

ABSTRACT: Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin itself can be post-translationally modified by phosphorylation, with nearly every serine, threonine, and tyrosine residue having the potential to be phosphorylated. However, the effect of this modification on ubiquitin function is largely unknown. Here, we performed in vivo and in vitro characterization of the effects of phosphorylation of yeast ubiquitin at position serine 65. We find ubiquitin S65 phosphorylation to be regulated under oxidative stress, occurring in tandem with the restructuring of the ubiquitin landscape into a highly polymeric state. Phosphomimetic mutation of S65 recapitulates the oxidative stress phenotype, causing a dramatic accumulation of ubiquitylated proteins and a proteome-wide reduction of protein turnover rates. Importantly, this mutation impacts ubiquitin chain disassembly, chain linkage distribution, and substrate targeting. These results demonstrate that phosphorylation represents an additional mode of ubiquitin regulation with broad implications in cellular physiology.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Danielle Swaney

LAB HEAD: Judit Villen

PROVIDER: PXD002202 | Pride | 2015-07-07

REPOSITORIES: Pride

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Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover.

Swaney Danielle L DL Rodríguez-Mias Ricard A RA Villén Judit J

EMBO reports 20150703 9

Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin can itself be phosphorylated at nearly every serine, threonine, and tyrosine residue. However, the effect of this modification on ubiquitin function is largely unknown. Here, we characterized the effects of phosphorylation of yeast ubiquitin at serine 65 in vivo and in vitro. We find this post-translational modification to be regulated under oxida ...[more]

PMID: 26142280

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Project description:Post translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Since little is known about crosstalk between different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk between different PTMs. In addition various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin upon inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, while the total protein level decreased immediately afterwards. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.

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