Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition

Vittoria Matafora,Alfonsina D'Amato,Francesco Blasi,Silvia Mori,Angela Bachi

doi:10.1074/mcp.m900079-mcp200

Vittoria Matafora, Alfonsina D'Amato + Show 3 more

Open Access

https://doi.org/10.1074/mcp.m900079-mcp200

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Abstract

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity.

Highlights

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates
Small Ubiquitin-like MOdifier (SUMO)-1-positive staining was seen as dots dispersed throughout the nucleus; whereas after MG132 treatment, the staining accumulated in well defined structures (Fig. 1A) in agreement with previous studies [25, 26]
In this study we demonstrated that a crosstalk exists between SUMO-1 and the ubiquitin-proteasome system

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS and 100 units/ml penicillin and streptomycin (Invitrogen). 1.3 mg of HeLa nuclear extract and 6 mg of HeLa cytosolic extract were incubated with 100 ␮g of His-SUMO-1 previously bound to Ni2ϩ beads (Qiagen), 30 ␮g of Ubc9, 0.5 units/ml inorganic pyrophosphatase, and 10 mM ATP in sumoylation buffer (10 mM MgCl2, 0.1 mM DTT, 50 mM Tris-HCl, pH 7.5) for 1 h at room temperature [39]. The His-SUMO-1-conjugated proteins were eluted from beads with 500 mM imidazole in 50 mM Tris-HCl, 150 mM NaCl. Proteins were separated by 10% SDS-PAGE, stained by silver staining [40], and excised in 34 slices for LC-MS/MS analysis. A mixture of calf thymus total histones (1 ␮g) or purified histone H3 (1 ␮g) (Sigma-Aldrich) was incubated in the presence (or absence as control) of His-SUMO-1 (1 ␮g), Ubc (10 ng), Aos1/Uba (150 ng), 0.5 units/ml inorganic pyrophosphatase, and 10 mM ATP in sumoylation buffer for 1 h at room temperature. The experiments were performed in biological triplicate (experiments I, II, and III)

RESULTS

Nucleoli Nucleoli

DISCUSSION