Bub1 kinase acts as a signalling hub for the entire Cryptococcus neoformans spindle assembly checkpoint pathway

Abstract

Cryptococcus neoformans (Cn) is an important human pathogen and a model system for basidiomycetes. Here we carry out a dissection of its spindle assembly checkpoint (SAC), focusing on Bub1 and Bub3. In many eukaryotes, including humans, Saccharomyces cerevisiae and Schizosaccharomyces pombe, Bub1 underwent gene duplication, generating paralogues referred to as Bub1 and BubR1 (or Mad3). Bub1 has upstream signalling functions at kinetochores, whilst BubR1/Mad3 is a component of the downstream mitotic checkpoint complex (MCC) that delays anaphase onset until all chromosomes are correctly attached. Here we demonstrate that the single CnBub1 protein carries out all the checkpoint roles of both Bub1 kinase and Mad3/BubR1. Proteomic analysis reveals kinetochore targeting via Spc105KNL1 and interactions with all downstream SAC components and effectors (Cdc20 and the anaphase promoting complex/cyclosome). We demonstrate that CnBub1 kinase activity is required to maintain prolonged checkpoint arrest. Thus CnBub1 acts as a SAC signalling hub and is a future target for anti-mitotic drugs.

mps1andmadmutations reduceCryptococcus neoformanstitan cell viability

“…Here we have demonstrated that the checkpoint signalling roles of Mps1 kinase, Mad1 and Mad2 and their molecular interactions are very well conserved in Cryptococcus neoformans. In a second study, we demonstrated that Bub1 and Bub3 functions are also very well conserved [46]. One important difference in the SAC is that Cryptococcus lacks a BubR1/Mad3 gene as, unlike in humans, S.cerevisiae and S.pombe, the CnBub1 gene hasn't undergone duplication and CnBub1 carries out the functions of both Bub1 (as a kinetochore-bound SAC signalling scaffold) and Mad3 (as part of the diffusible APC/C inhibitor) [46].…”

“…In a second study, we demonstrated that Bub1 and Bub3 functions are also very well conserved [46]. One important difference in the SAC is that Cryptococcus lacks a BubR1/Mad3 gene as, unlike in humans, S.cerevisiae and S.pombe, the CnBub1 gene hasn't undergone duplication and CnBub1 carries out the functions of both Bub1 (as a kinetochore-bound SAC signalling scaffold) and Mad3 (as part of the diffusible APC/C inhibitor) [46]. Here we have also shown that Mps1 phospho-regulation of Mad1 is conserved in Cryptococcus.…”

“…9 demonstrates that all the deletion strains have reduced titan cell viability but, importantly, that this is not due to loss of SAC signalling. Point mutations that abolish the SAC, such as mad1-T667A and bub1-cd1 [46], did not display reduced titan cell viability. The simplest interpretation of this is that Mad1, Mad2 and Mps1 have an additional function(s) that is required for full viability of titan cells.…”

“…This indicates that the viability of titan cells is severely compromised by mutations in SAC genes. However, when we tested specific alleles of SAC genes (bub1-cd1 [46] and mad1-667A), rather than null mutants, we found that their viability was not compromised even though these alleles are checkpoint defective. Our interpretation of this experiment is that a sub-set of the checkpoint proteins (Mad1, Mad2 and particularly Mps1 kinase) have a novel function that is particularly important for the viability of titan cells and/or their daughters.…”

Shugoshin maintains mitotic arrest in response to improper kinetochore-microtubule interactions independent of the Bub1-pH2A axis

“…To study if the Bub1 kinase activity required for shugoshin's function is evolutionarily conserved in C. neoformans, we utilized the kinase-dead (bub1-kd) mutant of Bub1. In vitro assays reported earlier (Leontiou et al, 2022) revealed that Bub1 could phosphorylate the reconstituted human H2A containing nucleosomes, and kinase-dead mutants of Bub1 fail to phosphorylate. We performed a genetic interaction assay by scoring the TBZ sensitivity of sgo1Δ, bub1-kd, and sgo1Δ bub1-kd mutants (Fig 5B).…”