Fig. 3. Different domains in Siz1 participate in sumoylation of different substrates. (A) Diagram of the Siz variants used in experiments in B-D. Portions corresponding to Siz2 sequence are shaded grey. All variants also include a C-terminal HA epitope tag. (B) Lysates from siz1 siz2 cells expressing the indicated HA-tagged Siz variants were analyzed by SDS-PAGE and immunoblotting with an antibody against the HA epitope. The band corresponding to Siz1-HA, as well as constructs 3 and 5 is indicated with an arrowhead. Open arrows indicate two bands corresponding to construct 4 [the upper band is sumoylated (data not shown)]. Asterisks designate bands that cross-react with the Ab. (C) (Left) Lysates from siz1 siz2 cells that lacked the major sumoylation sites in all three septins (EJY411) and contained the indicated constructs were analyzed by SDS-PAGE and immunoblotting with an Ab against Smt3. (Right) Lysates from siz1 or siz2 strains that lacked the major septin sumoylation sites were analyzed by immunoblotting with an Ab against Smt3. Dots between panels indicate position ofSIZ2-dependent SUMO conjugates. (D) siz1 siz2 cells contained Siz variants as indicated over the lanes and tagged substrates as indicated below each pair of panels. Cdc3 and Pol30 were tagged with only HA and were analyzed by subjecting whole cell lysates to SDS-PAGE and immunoblotting with an Ab against HA. The Cdc3-HA cultures were arrested with nocodazole, and the Pol30-HA cultures were treated for 2 hours with 0.2 % MMS. The sumoylated form of Pol30 indicated is the mono-sumoylated Lys164 conjugate. Other substrates bore HA and His₈ tags and were analyzed by denaturing Ni-NTA affinity chromatography followed by SDS-PAGE and immunoblotting with antibodies against HA (lower panels) or Smt3 (top panels). Designations are as in Fig. 1. The white asterisk in lane 4 of the Cdc3 panel indicates the band corresponding to the construct 4 fusion protein, which is visible in this experiment because a whole cell lysate was analyzed.