A Proteomic Screen for Nucleolar SUMO Targets Shows SUMOylation Modulates the Function of Nop5/Nop58

Summary Posttranslational SUMO modification is an important mechanism of regulating protein function, especially in the cell nucleus. The nucleolus is the subnuclear organelle responsible for rRNA synthesis, processing, and assembly of the large and small ribosome subunits. Here, we have used SILAC-based quantitative proteomics to identify nucleolar SUMOylated proteins. This reveals a role for SUMOylation in the biogenesis and/or function of small nucleolar ribonucleoprotein complexes (snoRNPs) via the targeting of Nhp2 and Nop58. Using combined in vitro and in vivo approaches, both Nhp2 and Nop58 (also known as Nop5) are shown to be substrates for SUMOylation. Mutational analyses revealed the sites of modification on Nhp2 as K5, and on Nop58 as K467 and K497. Unlike Nop58 and Nhp2, the closely related Nop56 and 15.5K proteins appear not to be SUMO targets. SUMOylation is essential for high-affinity Nop58 binding to snoRNAs. This study provides direct evidence linking SUMO modification with snoRNP function.

. Example of the use of connected threshold connectivity to accurately define nuclear and nucleolar regions in Image J to allow the amount of nucleolar vs nuclear SUMO to be estimated. Related to Fig. 1.
Nuclear and nucleolar regions were defined based on the intensities of the staining patterns of either Hoechst (A) or anti-fibrillarin antibodies (B), respectively, as detailed in the Exp. Proc. A selection of IF images across the z-stack for a single cell is shown, along with the corresponding nuclear or nucleolar regions (black) as defined using our semiautomated Image J plug-in. Intensities resulting from staining with anti-SUMO antibodies were then summed for pixels that belonged to either or both regions and used to calculate the average percentage of nucleolar SUMO1 or 2/3. (A, B) Blots shown in Figure 2B and C (lanes 1-12) were stripped and reprobed with antibodies against SUMO2 to confirm that the Ni 2+ -NTA pulldowns had successfully enriched SUMO2-modified proteins.
(D) The majority of SUMOylated Nop58 localises to the nucleolus. Cytoplasmic, nucleoplasmic and nucleolar fractions from HeLa 6HisSUMO1 cells were subjected to denaturing Ni 2+ -NTA pulldowns, and input and eluate samples analysed by Western blotting using an anti-Nop58 antibody. The percentage of each sample that was loaded onto the gel is indicated.

Supplemental Experimental Procedures
Plasmids made for this study All plasmid sequences were verified by sequencing. belonging to these regions if they displayed intensities greater than a "high" threshold, or if they displayed intensities greater than a "low" threshold and were adjacent to another pixel that had also been defined as either nuclear or nucleolar. The same thresholds were applied across the entire z-stack for each cell. This plug-in also enabled the mapping of pixel intensities resulting from the anti-SUMO staining to the pixel location, i.e., nuclear or nucleolar, across the entire z-stack.

Immunoprecipitation of fibrillarin-containing complexes
Endogenous fibrillarin was IP'd from HeLa cells lysed in sonication buffer. Antifibrillarin 72B9 (5 μg; Cytoskeleton) was incubated with Protein-G Sepharose (~100 μg; GE) for 1 h in SB. Unbound antibody was removed by washing with SB and antibodycoated beads were added to pre-cleared lysates for IPs (3 h, 4 °C). Bound proteins were washed with SB.
In both cases, bound proteins were eluted with SDS-PAGE loading buffer, and analysed by Western blotting using the following primary antibodies: mouse anti-GFP (Roche; 1 in 1000), rabbit anti-fibrillarin serum 42 (gift from F. Fuller-Pace) or anti-Nop58 (main text). For Fig. S5C, the secondary antibodies used were anti-mouse AF680 (Invitrogen) and anti-rabbit IRDye800 (Rockland) and proteins were visualised and quantitated on an Odyssey Imager (Li-Cor Biosciences).

Cell cycle analysis and flow cytometry
To detect the percentage of GFP-positive cells in U2OS WTNop58-GFP and U2OS 2mutNop58-GFP