Control of mitotic exit in budding yeast. In vitro regulation of Tem1 GTPase by Bub2 and Bfa1.
The elimination of mitotic kinase activity at the end of mitosis is essential for progression to the next stage of the eukaryotic cell cycle. In budding yeast, this process is controlled by a regulatory cascade called the mitotic exit network. Extensive genetic data indicate that mitotic exit network activity is determined by a GTP-binding protein, Tem1, and its putative regulators, Bub2, Bfa1, and Lte1. Here we describe the purification and in vitro activities of Tem1, Bub2, and Bfa1. We describe the nucleotide binding properties of Tem1 and characterize its intrinsic GTPase activity. The combination of Bfa1 and Bub2 acts as a two-component GTPase-activating protein for Tem1. In the absence of Bub2, Bfa1 inhibits the GTPase and GTP exchange activities of Tem1. This inhibition is elicited by either the N- or C-terminal regions of Bfa1, which also retain some ability to co-activate GTPase activity in the presence of Bub2. Although the C-terminal region of Bfa1 binds to Bub2, no interaction of the N-terminal half of Bfa1 with Bub2 was detected despite their combined GAP activity. Therefore, we propose that Bfa1 acts both as an adaptor to connect Bub2 and Tem1 and as an allosteric effector that facilitates this interaction.
Geymonat
Marco
M
Divisions of Yeast Genetics, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Spanos
Ad
A
Smith
Susan J M
SJ
Wheatley
Edward
E
Rittinger
Katrin
K
Johnston
Leland H
LH
Sedgwick
Steven G
SG
D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008938:Mitosis; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D004718:Saccharomycetales / Q000378:metabolism* / Q000502:physiology; D013997:Time Factors
C119117:BFA1 protein, S cerevisiae; C070351:BUB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C089965:TEM1 protein, S cerevisiae; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins