The Bub2-dependent mitotic pathway in yeast acts every cell cycle and regulates cytokinesis.
In eukaryotes an abnormal spindle activates a conserved checkpoint consisting of the MAD and BUB genes that results in mitotic arrest at metaphase. Recently, we and others identified a novel Bub2-dependent branch to this checkpoint that blocks mitotic exit. This cell-cycle arrest depends upon inhibition 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, we find that Bub2 and Bfa1 physically associate across the entire cell cycle and bind to Tem1 during mitosis and early G1. Bfa1 is multiply phosphorylated in a cell-cycle-dependent manner with the major phosphorylation occurring in mitosis. This Bfa1 phosphorylation is Bub2-dependent. Cdc5, but not Cdc15 or Dbf2, partly controls the phosphorylation of Bfa1 and also Lte1. Following spindle checkpoint activation, the cell cycle phosphorylation of Bfa1 and Lte1 is protracted and some species are accentuated. Thus, the Bub2-dependent pathway is active every cell cycle and the effect of spindle damage is simply to protract its normal function. Indeed, function of the Bub2 pathway is also prolonged during metaphase arrests imposed by means other than checkpoint activation. In metaphase cells Bub2 is crucial to restrain downstream events such as actin ring formation, emphasising the importance of the Bub2 pathway in the regulation of cytokinesis. Our data is consistent with Bub2/Bfa1 being a rate-limiting negative regulator of downstream events during metaphase.
Lee
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SE
Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK.
Jensen
S
S
Frenz
L M
LM
Johnson
A L
AL
Fesquet
D
D
Johnston
L H
LH
D064173:Anaphase-Promoting Complex-Cyclosome; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004789:Enzyme Activation / Q000187:drug effects; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016193:G1 Phase / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors; D015151:Immunoblotting; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D008677:Metaphase / Q000187:drug effects; D008938:Mitosis / Q000187:drug effects; D020559:Monomeric GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D015739:Nocodazole / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
C119117:BFA1 protein, S cerevisiae; C070351:BUB2 protein, S cerevisiae; C082279:CDC15 protein; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; C089357:LTE1 protein, S cerevisiae; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; C089965:TEM1 protein, S cerevisiae; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; C085768:DBF2 protein, S cerevisiae; C085769:DBF20 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C081927:CDC5 protein, S cerevisiae; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; D008025:Ligases; D015739:Nocodazole