FIGURE 1.

A, TAP-Rsp5p was purified from yeast and resolved on a 7.5% SDS-PAGE, and proteins were visualized by silver staining. Arrowhead indicates Rsp5p, bar indicates region of gel excised for MS/MS analysis. B, schematic diagram of Rsp5p shows conserved domains and position of SUMOylation residues. C, the LC-MS/MS peptide sequences in which SUMOylation was observed are listed (modified residues indicated with asterisk). D, His-FLAG-Smt3p (+) or an empty vector (−) was transformed into a Tap-Rsp5p strain, and total cell lysates were subjected to pulldown assays in the presence of 6 m guanidine hydrochloride with His-Select resin. Input and pulldown samples were immunoblotted with anti-FLAG antibodies for Smt3p, anti-Rsp5p, and anti-GAPDHp antibodies. Asterisks indicate two major species of Rsp5p that pull down with His-FLAGSmt3p.