The Tem1 small GTPase controls actomyosin and septin dynamics during cytokinesis.

Cytokinesis in budding yeast involves an actomyosin-based ring which assembles in a multistepped fashion during the cell cycle and constricts during cytokinesis. In this report, we have investigated the structural and regulatory events that occur at the onset of cytokinesis. The septins, which form an hour-glass like structure during early stages of the cell cycle, undergo dynamic rearrangements prior to cell division: the hourglass structure splits into two separate rings. The contractile ring, localized between the septin double rings, immediately undergoes contraction. Septin ring splitting is independent of actomyosin ring contraction as it still occurs in mutants where contraction fails. We hypothesize that septin ring splitting may remove a structural barrier for actomyosin ring to contract. Because the Tem1 small GTPase (Tem1p) is required for the completion of mitosis, we investigated its role in regulating septin and actomyosin ring dynamics in the background of the net1-1 mutation, which bypasses the anaphase cell cycle arrest in Tem1-deficient cells. We show that Tem1p plays a specific role in cytokinesis in addition to its function in cell cycle progression. Tem1p is not required for the assembly of the actomyosin ring but controls actomyosin and septin dynamics during cytokinesis.

A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation.

Cytokinesis in animal cells is accomplished through constriction of an actomyosin ring [1] [2] [3], which must assemble at the correct time and place in order to ensure proper division of genetic material and organelles. Budding yeast is a useful model system for determining the biochemical pathway of contractile ring assembly. The budding yeast IQGAP-like protein, Cyk1/Iqg1p, has multiple roles in the assembly and contraction of the actomyosin ring [4] [5] [6]. Previously, the IQ motifs of Cyk1/Iqg1p were shown to be required for the localization of this protein at the bud neck [6]. We have investigated the binding partner of the IQ motifs, which are predicted to interact with calmodulin-like proteins. Mlc1p was originally identified as a light chain for a type V myosin, Myo2p; however, a cytokinesis defect associated with disruption of the MLC1 gene suggested that the essential function of Mlc1p may involve interactions with other proteins [7]. We show that Mlc1p binds the IQ motifs of Cyk1/Iqg1p and present evidence that this interaction recruits Cyk1/Iqg1p to the bud neck. Immunofluorescence staining shows that Mlc1p is localized to sites of polarized cell growth as well as the bud neck before and independently of Cyk1p. These results demonstrate that Mlc1p is important for the assembly of the actomyosin ring in budding yeast and that this function is mediated through interaction with Cyk1/Iqg1p.

The multiple roles of Cyk1p in the assembly and function of the actomyosin ring in budding yeast.

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis.

Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.