Subcellular distribution of SUMO-1 after DMSO or MG132 treatment and immunoprecipitation of nucleolar extract using anti-SUMO-1. A, immunofluorescence analysis of HeLa cells cultured in the presence of DMSO as control (a–c) and with 10 μm MG132 overnight (d–f). Double staining of DNA (blue; Hoechst staining) and SUMO-1 (green) is shown in the right panels (c and f), the middle panels show DNA (b and e), and the left panels show SUMO-1 (a and d). In the control cells, SUMO-1 proteins are distributed throughout the nucleoplasm (a) excluding the nucleoli (c). In the treated cells, SUMO-1 proteins localize inside the nucleoli (f). B, nucleolar localization of SUMO-1 in HeLa cells treated with DMSO (a–c) and with MG132 overnight (d–f). Shown are high magnification images of SUMO-1 staining (green) (a and d) and pyronin Y staining (red) (b and e) and the merged images of pyronin Y and SUMO-1 staining (c and f). Only in HeLa cells treated with MG132 staining of SUMO-1 and pyronin Y overlaps, confirming that SUMO-1 species translocated into nucleolar structures after proteasome inhibition. C, immunoprecipitation (IP), with anti-SUMO-1 antibody, of HeLa cells nucleolar extract treated with MG132 overnight (right) and with DMSO (left). As expected SUMO-1 proteins are enriched only after proteasome inhibition. D, anti-ubiquitin Western blot (WB) of nucleolar extracts before and after MG132 treatment. The extracts were normalized with anti-nucleophosmin.

Quantitative proteomics strategy to identify nucleolar SUMO-1 targets. Arg0- or Arg0 and Lys0-labeled HeLa cells were treated with MG132 overnight, and Arg10- or Arg10 and Lys8-labeled HeLa cells were treated with DMSO as control. Labeled cells were mixed in a 1:1 ratio, and the nucleoli were purified. SUMO-1 target proteins were immunoprecipitated (IP) and separated by SDS-PAGE, and the gel lane was cut into slices. Proteins were in-gel digested with trypsin, identified by mass spectrometry, and quantified by MSQuant and Max Quant.

Biological process analysis. Analysis was performed with the on-line software PANTHER using the data set reported in Table I. The p value was set at >0.05. The Bonferroni correction for multiple testings was used. Only categories with significant differences are shown.

Novel SUMO-1 target identification. A, histones mixture in vitro reaction. A mixture of calf thymus total histones was incubated in the presence or absence (as control) of His-SUMO-1, Ubc9, Aos1/Uba2, inorganic pyrophosphatase, and ATP in sumoylation buffer. The reaction mixture was analyzed by anti-SUMO-1 Western blot (WB) and SDS-PAGE. When SUMO-1 is added to the mixture, a new band of 45 kDa appears, and the amount of free SUMO-1 decreases indicating that SUMO-1 has been conjugated to one of the histones of the mixture. The Coomassie-stained band possibly corresponding to sumoylated histone was cut, trypsin-digested, and analyzed by nLC-MS/MS. This analysis identified histone H1.3 (supplemental Table S3) as the target of the sumoylation reaction. The identification of SUMO-1 protein confirms that the histone is sumoylated because the molecular mass of the band corresponds to the mass of the histone H1 plus SUMO-1. B, histone H3 in vitro reaction. Purified histone H3 was incubated in the presence or absence of SUMO-1 in the same reaction buffer described above, and the mixture was incubated without histone H3 as a control of the reaction. Proteins were separated by SDS-PAGE. Sumoylated histone H3 was detected by specific antibody as a band migrating at about 35 kDa, which was not present in the control. Western blot with anti-SUMO-1 confirms the transfer of SUMO-1 to the substrate. The sumoylation of histone H3 was also confirmed by nLC-MS/MS analysis of the band described above. * indicates a probable histone H3 dimer; ** indicates the complex Ubc9-SUMO-1. C, endogenous sumoylation of p160. NIH 3T3 cells were infected with p160-FLAG retrovirus. Immunoprecipitation (IP) with anti-FLAG affinity resin and immunoblotting with anti-SUMO-1 indicate that p160 is indeed sumoylated. Immunoprecipitation of not infected cells was used as control.

Subcellular distribution of SUMO-1 after DMSO and MG132 treatment at different time points. Shown is immunofluorescence analysis of HeLa cells cultured in the presence of DMSO as control (a, e, and i) and with 10 μm MG132 for 1 h (b, f, and l), 6 h (c, g, and m), and 12 h (d, h, and n). Double staining of DNA (blue; Hoechst staining) and SUMO-1 (green) is shown in the lower panels (i–n), the middle panels show DNA (e–h), and the upper panels show SUMO-1 (a and d). As expected, in the control cells, SUMO-1 proteins are distributed throughout the nucleoplasm (a) excluding the nucleoli (i). In treated cells, SUMO-1 proteins localize first in more defined dots (b and l); after 6 h, more and bigger dots localized around the nucleoli are detected (c and m) and are finally found inside nucleolar structures (12 h) (d and n).

Purification of nucleolar SUMO-1 proteins. Arg0-labeled HeLa cells were treated with MG132, and Arg10-labeled HeLa cells were treated with DMSO as control. Equal amounts of HeLa cells were mixed. SUMO-1 target proteins were immunoprecipitated (IP) from nucleolar lysate using anti-SUMO-1 antibody cross-linked to protein G-Sepharose beads. Immunoprecipitated proteins were eluted in non-reducing Laemmli buffer and then in reducing buffer. A, Western blot (WB) analysis shows that most of the SUMO-1 targets are eluted in non-reducing conditions. B, SDS-PAGE of purified SUMO-1 proteins. The gel lane was excised in 24 slices, and proteins were in-gel digested with trypsin. C, subcellular localization of the identified proteins.

Purification of in vitro His-SUMO-1 target proteins. An in vitro reaction of HeLa nuclear extract was performed with His-SUMO-1, which was previously bound to Ni²⁺ beads. The reaction mixture was obtained by incubating Ubc9, inorganic pyrophosphatase, and ATP in sumoylation buffer with and in the absence of SUMO-1 as control. A, anti-SUMO-1 Western blot (WB) of purified His-SUMO-1-conjugated proteins shows an enrichment of conjugated species. B, SDS-PAGE of purified His-SUMO-1 proteins stained by silver shows a very specific signal in the reaction mixture compared with the control (ctrl). The gel was excised in 34 slices for nLC-MS/MS analysis.