The Bub2-dependent mitotic pathway in yeast acts every cell cycle and regulates cytokinesis

In eukaryotes an abnormal spindle activates a conserved checkpoint consisti ng of the MAD and BUB genes that results in mitotic arrest at metaphase, Re cently, we and others identified a novel Bub2-dependent branch to this chec kpoint that blocks mitotic exit. This cell-cycle arrest depends upon inhibi tion of the G-protein Tem1 that appears to be regulated by Bfa1/Bub2, a two -component GTPase-activating protein, and the exchange factor Lte1, Here, w e find that Bub2 and Bfa1 physically associate across the entire cell cycle and bind to Tem1 during mitosis and early G1. Bfa1 is multiply phosphoryla ted in a cell-cycle-dependent manner with the major phosphorylation occurri ng in mitosis. This Bfa1 phosphorylation is Bub2-dependent. Cdc5, but not C dcl5 or Dbf2, partly controls the phosphorylation of Bfa1 and also Lte1, Fo llowing spindle checkpoint activation, the cell cycle phosphorylation of Bf a1 and Lte1 is protracted and some species are accentuated. Thus, the Bub2- dependent pathway is active every cell cycle and the effect of spindle dama ge is simply to protract its normal function. Indeed, function of the Bub2 pathway is also prolonged during metaphase arrests imposed by means other t han checkpoint activation. In metaphase cells Bub2 is crucial to restrain d ownstream events such as actin ring formation, emphasising the importance o f the Bub2 pathway in the regulation of cytokinesis. Our data is consistent with Bub2/Bfa1 being a rate-limiting negative regulator of downstream even ts during metaphase.