Temporal coupling of spindle disassembly and cytokinesis is disrupted by deletion of LTE1 in budding yeast.

The mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis in budding yeast by triggering the nucleolar release and hence activation of the Cdc14 phosphatase. Activation of the MEN is tightly coordinated with spindle position in such a way that Cdc14 is only fully released upon spindle pole body (SPB) migration into the daughter cell. This temporal regulation of the MEN has been proposed to rely in part on the spatial separation of the G-protein Tem1 at the SPB and its nucleotide exchange factor Lte1 confined to the daughter cell cortex. However, the dispensability of LTE1 for survival has raised questions regarding this model. Here using real-time microscopy we show that lte1Delta mutants not only delay exit from mitosis but also uncouple the normal coordination between spindle disassembly and contraction of the actomyosin ring at cell division. These mitotic defects can be suppressed by a bub2Delta mutation or by Cdc14 over-expression suggesting that they are caused by compromised MEN activity. Thus Lte1 function is important to fine-tune the timing of mitotic exit and to couple this event with cytokinesis in budding yeast.

In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5.

The Cdc5 protein of budding yeast is a polo-like kinase that has multiple roles in mitosis including control of the mitotic exit network (MEN). MEN activity brings about loss of mitotic kinase activity so that the mitotic spindle is disassembled and cytokinesis can proceed. Activity of the MEN is regulated by a small GTPase, Tem1, which in turn is controlled by a two-component GTPase-activating protein (GAP) formed by Bfa1 and Bub2. Bfa1 has been identified as a regulatory target of Cdc5 but there are conflicting deductions from indirect in vivo assays as to whether phosphorylation inhibits or stimulates Bfa1 activity. To resolve this question, we have used direct in vitro assays to observe the effects of phosphorylation on Bfa1 activity. We show that when Bfa1 is phosphorylated by Cdc5, its GAP activity with Bub2 is inhibited although its ability to interact with Tem1 is unaffected. Thus, in vivo inactivation of Bfa1-Bub2 by Cdc5 would have a positive regulatory effect by increasing levels of Tem1-GTP so stimulating exit from mitosis.

Complexity of mitotic exit.

Completion of mitosis in budding yeast is triggered by activation of the protein phosphatase Cdc14, which is the ultimate effector of a signalling cascade, known as the mitotic exit network. Cdc14 activation leads to eradication of mitotic kinase activity, which is pivotal for mitotic exit and cytokinesis in all eukaryotes. The complexity in mitotic exit regulation is underscored by the recent discovery of a novel network, the so-called FEAR pathway that regulates early Cdc14 activation. Surprisingly, this has revealed an unexpected role for Spo12, a protein involved in meiosis, in Cdc14 activation. In this review, we will discuss these findings together with recent advances in deciphering the function of the FEAR circuit, which has unravelled an exciting new side of Cdc14.

Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccharomyces cerevisiae.

In budding yeast, activation of the small Ras-like GTPase Tem1 triggers exit from mitosis and cytokinesis. Tem1 is regulated by Bub2/Bfa1, a two-component GTPase-activating protein (GAP), and by Lte1, a putative guanine nucleotide exchange factor. Lte1 is confined to the bud cortex, and its spatial separation from Tem1 at the spindle pole body (SPB) is important to prevent untimely exit from mitosis. The pathways contributing to Lte1 asymmetry have not been elucidated. Here we show that establishment of Lte1 at the cortex occurs by an actin-independent mechanism, which requires activation of Cdc28/Cln kinase at START and Cdc42, a key regulator of cell polarity and cytoskeletal organisation. This defines a novel role for Cdc42 in late mitotic events. In turn, dissociation of Lte1 from the cortex in telophase depends on activation of the Cdc14 phosphatase. Ectopic expression of Cdc14 at metaphase results in premature dephosphorylation of Lte1 coincident with its release from the cortex. In vitro phosphatase assays confirm that Lte1 is a direct substrate for Cdc14. Our results suggest that the asymmetry in Lte1 localisation is imposed by Cdc28-dependent phosphorylation. Finally, we report a mutational analysis undertaken to investigate intrinsic Lte1 determinants for localisation. Our data suggest that an intrameric interaction between the N-and C-terminal regions of Lte1 is important for cortex association.

Mitotic exit: the Cdc14 double cross.

The release of Cdc14 from the nucleolus occurs in two waves in early and late anaphase, controlled by the FEAR and MEN pathways, respectively. Two new papers report the localisation at the spindle pole body of the Cdc14 released in early anaphase and, surprisingly, show that the two pulses of released Cdc14 have opposite effects on MEN activation.

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.

Mitotic exit: delaying the end without FEAR.

Activation of Cdc14 phosphatase, controlled by a signalling cascade known as the mitotic exit network, is the final switch that drives cells from mitosis into the next cell cycle. The recent discovery of a novel network that regulates early Cdc14 activation has revealed the unexpected existence of a two-step control of mitotic exit.