Figure 5. FIGURE 5: Sumoylation of mouse PML in yeast.

Yeast strain yRA1 was transformed with PML cloned in pRA20 (V5 tag, TRP1 marker, 93%-GAL1 promoter) and with a plasmid expressing 6xHis-Myc-Smt3 under control of the CUP promoter (HIS3 marker) or with empty vector. Expression of PML and Smt3 was induced by addition of galactose (2% [w/v]) and CuSO₄ (100 μM), respectively. Cells were lysed and equivalent amounts of protein extract were analyzed by SDS-PAGE and anti-V5 (A) or anti-Myc (B) immunoblotting. The band marked with an asterisk (*) in panel A is an example of PML modified by a single Smt3 moiety that, in cells overexpressing 6xHis-myc-Smt3, gives rise to a slower migrating species (arrow head) due to covalent attachment of the 6xhis-Myc-tagged Smt3.