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First published online 31 October 2006
doi: 10.1242/jcs.03243

Journal of Cell Science 119, 4749-4757 (2006)
Published by The Company of Biologists 2006
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Multiple domains in Siz SUMO ligases contribute to substrate selectivity

Alison Reindle, Irina Belichenko, Gwendolyn R. Bylebyl, Xiaole L. Chen, Nishant Gandhi and Erica S. Johnson*

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA


Figure 1
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  		Fig. 1. Both Siz1 and Siz2 stimulate sumoylation of many proteins. Lysates from cells in which the indicated proteins had been tagged with the HA epitope and a His8 tag were subjected to denaturing Ni-NTA affinity chromatography. Eluates were analyzed by SDS-PAGE and immunoblotting with antibodies against HA (lower panels) or Smt3 (top panels). SIZ genotypes are indicated over the lanes. Bands corresponding to unmodified proteins are indicated with arrowheads. Open brackets indicate sumoylated species. For Spt7, Abf1, Rsc2 and Gcn5, sumoylated species were detected with the HA Ab on a short exposure and are shown. Other substrates required long exposures of the HA blot to detect sumoylated species (not shown). A cross-reacting band is designated with an asterisk.

 

Figure 2
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  		Fig. 2. Siz proteins and Mms21 have overlapping functions. (A) Strains of the indicated genotypes were incubated on a YPD plate for 2 days at 30°C. Doubling times of mutants are indicated in parentheses. (B) Yeast strains of the indicated genotypes containing Net-His8 HA were analyzed as in Fig. 1, with immunoblotting with anti-Smt3 (top panel) or anti-HA (bottom panel). Designations are as in Fig. 1.

 

Figure 3
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  		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.

 

Figure 4
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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.

 

Figure 5
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  		Fig. 5. The C-terminal domain of Siz1 is sufficient for cell-cycle-dependent binding to the bud neck. (A) Diagram of GFP fusions containing parts of the C-terminal domain of Siz1. Ability to localize to the bud neck is indicated. (B) Fluorescence microscopy of nocodazole-arrested siz1 cells containing GFP fusions as illustrated in A. Bar, 5 µm. (C) Lysates of siz1 cells containing GFP fusions were analyzed by SDS-PAGE and immunoblotting with an antibody against HA. Note that fusions that fail to localize to the bud neck are present in quantities comparable to those that do. Lane g was from the same exposure of the same blot, but was spliced to remove extraneous lanes. (D) siz1 Cdc3-HA cells containing the indicated Siz variants were analyzed as in Fig. 4C. Unmodified Cdc3-HA is indicated, and SUMO-Cdc3 species are indicated by open square brackets. An asterisk indicates a cross-reacting band.

 

Figure 6
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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, 1999Go). (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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© The Company of Biologists Ltd 2006