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Fig. 6. Addition of a sumoylation consensus sequence converts Cdc10 and Cdc12 into Siz1 substrates. (A) Lysates from strains expressing Cdc10-His₈-HA (Cdc10), Cdc12-His₈-HA (Cdc12), or versions of these with a sumoylation site consensus sequence between the C terminus of the protein and the tag (Cdc10-S and Cdc12-S) were analyzed by denaturing Ni-NTA chromatography, SDS-PAGE, and immunobloting with antibodies against HA (left panel) or Smt3 (right panel). Bands corresponding to unmodified and sumoylated (Su) proteins are indicated. The second SUMO-containing species in the Cdc10-S sample (right panel) is visible on a much darker exposure of the HA blot. (B) Whole cell lysates from strains expressing the indicated forms of Cdc10 and Cdc12 were analyzed by SDS-PAGE and immunobloting with antibodies against HA (top panel) or Smt3 (bottom panel). Strains lacked SIZ1 or had been arrested with nocodazole as indicated. Bands corresponding to unmodified and sumoylated septins are indicated. Su₂-Cdc10 indicates the second sumoylated form of Cdc10, but is not necessarily a di-sumoylated species. Positions of sumoylated Cdc11 and Cdc3 were determined based on previous results (Johnson and Blobel, 1999). (C) Whole cell lysates prepared from siz1 ${Delta}$ cells expressing Cdc10-S (top panels) or Cdc12-S (bottom panels) and Siz1 variants shown in Fig. 3A were analyzed by SDS-PAGE and immunoblotting with an antibody against HA. Lanes are numbered as in Fig. 3D. Bands corresponding to unmodified and sumoylated septins are indicated.

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