Figure 10.

Bir1 SUMOylation cooperates with Sgo1 SUMOylation to promote anaphase onset. (A) Bir1 is SUMOylated in a Siz1/Siz2-dependent manner. Strains were BIR1-6HA (vector control, AMy23696; HIS-SMT3, AMy 23697), BIR1-6HA siz1Δ siz2Δ (AMy23698), and bir1-K707R K732R K785R-6HA (AMy29757). SUMO pull-down was performed as described in Fig. 2 A. IB, immunoblot; M.W, molecular weight. (B) Siz1/Siz2 changes posttranslational modifications on Bir1. BIR1-6HA (AMy3533) and siz1Δ siz2Δ BIR1-6HA (AMy7826) were released from α-factor arrest. Spindle morphology was analyzed as described in Fig. 1 D. Bir1 levels were analyzed by anti-HA(HA11) Western blot. Kar2 was a loading control. (C) bir1-3R caused a modest delay in metaphase. Strains carried CDC14-GFP and YFP-TUB1 and were wild type (AMy24174), bir1-3R (AMy29736), and sgo1-4R bir1-3R (AMy29734). Cells were imaged and quantified as described in Fig. 1, E and F. Shown are the average values of three to four independent experiments, with error bars representing standard error. Statistics: two-tailed Student’s t test comparing to the wild-type strain (*, P < 0.05). (D) bir1-3R sgo1-4R-Δdb delayed metaphase to a similar extent to siz1Δ siz2Δ. Strains carried CDC14-GFP and YFP-TUB1 and were wild type (AMy24174), siz1Δ siz2Δ(AMy24313), sgo1-Δdb bir1-3R (AMy30108), and sgo1-4R-Δdb bir1-3R (AMy30064). Shown are the average values of three to four independent experiments, with error bars representing standard error. Statistics: two-tailed Student’s t test comparing to the wild-type strain (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (E) Schematic model of how Siz1/Siz2-dependent SUMOylation might promote the metaphase–anaphase transition. In prometaphase, before kinetochore–microtubule attachment, a pool of Sgo1 and CPC is SUMOylated, promoting the release of PP2A-Rts1 and dampening CPC activity. The SUMO-dependent turnover of PP2A-Rts1/CPC at kinetochores prevents microtubule release at newly attached kinetochores, thereby stabilizing the attachment and allowing tension to be generated. In response to tension, Sgo1 is released from chromatin and is subsequently degraded by APC/C to fully turn off metaphase signaling pathways. In the absence of Siz1/Siz2, disassembly and turnover of pericentromeric complexes is inefficient, so PP2A-Rts1 and CPC remain on the kinetochore as microtubule capture occurs. Persistent CPC results in microtubule detachment and unattached kinetochores, which cause spurious SAC activity and delay anaphase entry.