Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

Abstract

a These authors contributed equally to this work. b Current address: Abcam, AbstractChromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein, Sid4, at the SPB, and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Polo-like kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1's SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

Localization of the ubiquitin ligase Dma1 to the fission yeast contractile ring is modulated by phosphorylation

“…Proteins were resolved by PAGE (see below), transferred by electroblotting to a polyvinylidene difluoride (PVDF) membrane (Immobilon FL; Millipore, Bedford, MA, USA), blocked with Odyssey Blocking Buffer (LI‐COR Biosciences, Lincoln, NE, USA), and incubated with primary antibodies at the following concentrations/dilutions: 1 : 10 000 anti‐Cdc2 (anti‐PSTAIR, Sigma‐Aldrich), 0.4 μg·mL −1 anti‐GFP (Roche, Basel, Switzerland), 1 μg·mL −1 anti‐FLAG (Sigma‐Aldrich), 1 : 2000 anti‐Sid4 serum, or 1 : 5000 anti‐Dma1 serum (VU377) [10] overnight at 4 °C. Primary antibodies were detected with secondary antibodies coupled to IRDye680 or IRDye800 (LI‐COR Biosciences, Lincoln, NE, USA) and visualized using an Odyssey Infrared Imaging System (LI‐COR Biosciences).…”

“…These 20 µL reactions were incubated with agitation at 30 °C for 120 min before adding SDS sample buffer. Ubiquitination products and Dma1 were detected by immunoblotting with a 1 : 15 000 dilution of anti‐Dma1 serum [10].…”

“…Dma1 has a very complex localization pattern during the cell cycle. It dynamically localizes to spindle pole bodies (SPB) during mitosis [5,10] and ubiquitinates the SIN scaffold protein Sid4 that is detected exclusively at SPBs [6,11]. Dma1 binding and ubiquitination of Sid4 requires casein kinase 1 (CK1)‐mediated Sid4 phosphorylation, which provides a docking site for Dma1’s FHA domain [12].…”

“…Sid4 ubiquitination then antagonizes the SPB localization of the Polo‐like kinase Plo1 [5,6], the major SIN activator [13–15] so that SIN signaling is inhibited and cytokinesis is delayed. Dma1 also localizes to the site of cell division through an unknown mechanism and for an unknown purpose, first in precursor nodes that eventually form the cytokinetic ring (CR) [16], then to the CR; Dma1 leaves the site of division transiently during late anaphase, only to return prior to cell division [10]. Dma1 overproduction or tethering Dma1 to SPBs blocks SIN activity and results in cell death [5,10,17], indicating its levels, localization, and activity must be properly regulated.…”

“…We previously determined that Dma1 auto‐ubiquitination influences its SPB localization and function in the mitotic checkpoint. Specifically, Dma1 auto‐ubiquitination prevents its binding to Sid4 and therefore Dma1 auto‐ubiquitination is implicated in checkpoint silencing [10]. Here, we investigated potential regulation of Dma1 by phosphorylation.…”