Identification and functional analysis of the spindle checkpoint component mad³⁺ in Schizosaccharomyces pombe

• Main Author: Millband, David Nicholas
• Published: University of Edinburgh 2001
• Subjects: 571.29
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.657776

The spindle checkpoint delays the metaphase to anaphase transition in response to spindle and kinetochore defects. Genetic screens in budding yeast originally identified the Mad and Bub proteins (and later the Mps1 kinase) as being core components of this conserved regulatory pathway. This work describes the identification and phenotypic characterisation of the fission yeast open reading frame (ORF), <i>SPCC895.02. </i>Based initially upon sequence homology and supported later by biochemical data we believe <i>SPCC895.02</i> encodes the fission yeast homologue of the Mad3 protein. This thesis comprises three key parts. In the first instance evidence is presented proving that the putative <i>mad3⁺. </i>ORF does indeed encode a component of the spindle checkpoint. Fission yeast cells devoid of <i>mad3⁺ </i>are unable to arrest their cell cycle in the presence of microtubule depolymerising agents, re-replicate their DNA and furthermore precociously separate their sister chromatids when spindle integrity is compromised. Secondly, a detailed localisation study was undertaken in which the endogenous <i>mad3⁺</i> ORF was tagged with the green fluorescence protein (GRF). Mad3-GFP is recruited to unattached kinetochores early in mitosis and accumulations there upon prolonged checkpoint activation. Furthermore, Mad3-GFP kinetochore localisation was found to be dependent upon the Bub1p kinase, Bub3p and also the Mph1p (Mps1 homologue) kinase, but not upon Mad1p or Mad2p. Finally, biochemical analysis of Mad3p interactions revealed genetic and biochemical interactions with Mad2p and the checkpoint effector S1p1/Cdc20p, suggesting an important role for Mad3p in transducing the inhibitory ‘wait anaphase’ signal to the anaphase promoting complex/cyclosome (APC/C).