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. 2021 Apr 30;220(7):e202005130. doi: 10.1083/jcb.202005130

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© 2021 Su et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 2. — Sgo1 is SUMOylated, depending on its association with pericentromeres. (A) Sgo1 is SUMOylated in a Siz1/Siz2-dependent manner. Extracts from untagged (AMy7651), SGO1-6HA (AMy7655 and AMy18470), siz1Δ siz2Δ SGO1-6HA (AMy8121), siz1Δ SGO1-6HA (AMy29477), and siz2Δ SGO1-6HA (AMy29478) strains carrying empty vector (pRS426) or with 7×HIS-SMT3 (AMp773) were purified over Ni-NTA resin, and anti-HA immunoblot (IB) was performed on both input and eluate. Arrows and asterisks indicate SUMO-Sgo1-6HA and unmodified Sgo1-6HA, respectively. Note that unmodified Sgo1-6HA occasionally copurified because it was present in insoluble materials and bound nonspecifically to the resin. Kar2 was used as a loading control. (B) Sgo1 is SUMOylated by Siz1 and Siz2 in vitro. Purified Sgo1 was incubated with 1 µM of each SUMOylation component (E1, E2, E3, and SUMO) and ATP or missing one component as indicated. The reaction was incubated at 30°C for 3 h. (C) Sgo2 in S. pombe is SUMOylated. In vivo SUMOylation was assessed for no tag (S. pombe AM29) and two independent transformants of Sgo2-6HA (S. pombe AM1861) + empty vector (AMp1960) or + His-Myc-PMT3 (AMp1961). HA-tagged Sgo2 was probed by anti-HA Western blotting. (D) Sgo1 SUMOylation occurs in metaphase. Cells carrying SGO1-6HA and 7xHIS-SMT3 (AMy7655) were released from G1 and harvested at the indicated intervals, and SUMOylation was analyzed as described in A. Cell cycle stage was monitored by scoring spindle morphology after anti-tubulin immunofluorescence. (E) Sgo1 SUMOylation peaks in prometaphase. A pMET-CDC20 SGO1-6HA strain transformed with 7HIS-SMT3 (AMy9641) was released from G1 arrest into methionine-containing medium to repress CDC20 and arrest cells in metaphase. Cells were harvested every 15 min, and SUMO pull-down was performed as described in A. Metaphase cells were identified by scoring spindle morphology after anti-tubulin immunofluorescence. (F) Sgo1 SUMOylation is reduced upon the establishment of tension between sister kinetochores. Cells carrying pMET-CDC20 and either 7xHIS-SMT3 (AM9641) or empty vector (AMy26342) were arrested in metaphase by depletion of Cdc20 in the presence of either benomyl and nocodazole (no tension) or DMSO (tension). (G) Chromatin association promotes Sgo1 SUMOylation. Sgo1 SUMOylation was determined as described in A in wild-type (AMy7654 or 7655), bub1Δ (AMy10098), bub1-KD (catalytically inactive Bub1 kinase, AMy10102), sgo1-100 (AMy26334), and sgo1-700 (AMy26336) strains.