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Fig. 4. Siz1 specificity for Pol30 and Prp45 involves part of the conserved PINIT domain. (A) Diagram of Siz variants used in B and C. Portions corresponding to Siz2 sequence are in grey. (B) Lysates prepared from siz1 cells containing the indicated Siz variants and either Pol30-HA or Prp45-HA-His₈ were either analyzed directly (Pol30) or subjected to Ni-NTA affinity chromatography (Prp45), followed by SDS-PAGE and immunoblotting with an Ab against HA. Pol30-HA cultures were treated with 0.2% MMS. Designations are as in Fig. 1. A bracket indicates the three SUMO-Pol30 bands (the middle one is monosumoylated at Lys127, which is not Siz1 dependent). Open arrows indicate the bands corresponding to the HA-tagged Siz1-Siz2 fusions in lanes 3 and 4 of the Pol30 blot. Full-length Siz1 is not visible here. All lanes of the Pol30 panel were from the same exposure of the same blot, but lanes 3 and 4 were spliced to remove irrelevant lanes. (C) siz1 Pol30-HA cells containing the indicated Siz variants were arrested with alpha factor and released into fresh medium. Whole cell lysates made from samples taken at the indicated timepoints were analyzed as in B. Bands corresponding to unmodified Pol30 and the Lys164-linked SUMO conjugate are indicated.

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