Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2‐7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2‐7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2‐7 when cells entered S‐phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45–MCM–GINS helicase at DNA replication origins.

Fission yeast ste9/srw1 is a WD‐repeat protein highly homologous to budding yeast Hct1/Cdh1 and Drosophila Fizzy‐related that are involved in activating APC/C (anaphase‐promoting complex/cyclosome). We show that APCste9/srw1 specifically promotes the degradation of mitotic cyclins cdc13 and cig1 but not the S‐phase cyclin cig2. APCste9/srw1 is not necessary for the proteolysis of cdc13 and cig1 that occurs at the metaphase–anaphase transition but it is absolutely required for their degradation in G1. Therefore, we propose that the main role of APCste9/srw1 is to promote degradation of mitotic cyclins when cells need to delay or arrest the cell cycle in G1. We also show that ste9/srw1 is negatively regulated by cdc2‐dependent protein phosphorylation. In G1, when cdc2–cyclin kinase activity is low, unphosphorylated ste9/srw1 interacts with APC/C. In the rest of the cell cycle, phosphorylation of ste9/srw1 by cdc2–cyclin complexes both triggers proteolysis of ste9/srw1 and causes its dissociation from the APC/C. This mechanism provides a molecular switch to prevent inactivation of cdc2 in G2 and early mitosis and to allow its inactivation in G1.

The chitin synthase that makes the primary septum during cell division in budding yeasts is an important therapeutic target with an unknown activation mechanism. We previously found that the C2-domain of the Saccharomyces cerevisiae Inn1 protein plays an essential but uncharacterised role at the cleavage site during cytokinesis. By combining a novel degron allele of INN1 with a point mutation in the C2-domain, we screened for mutations in other genes that suppress the resulting defect in cell division. In this way we identified 22 dominant mutations of CHS2 (Chitin Synthase II) that map to two neighbouring sites in the catalytic domain. Whereas Chs2 in isolated cell membranes is normally almost inactive, unless protease treatment is used to bypass inhibition, the dominant suppressor allele Chs2-V377I has enhanced activity in vitro. We show that Inn1 associates with Chs2 in yeast cell extracts, and interacts in the yeast two-hybrid assay with the amino-terminal 65% of Chs2 that contains the catalytic domain. In addition to compensating for mutations in the Inn1 C2-domain, the dominant CHS2 alleles also suppress cytokinesis defects produced by lack of the Cyk3 protein, and our data support a model whereby the C2-domain of Inn1 acts in conjunction with Cyk3 to regulate the catalytic domain of Chs2 during cytokinesis. These findings suggest novel approaches for developing future drugs against important fungal pathogens.

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role. Cytokinesis is the process by which a cell divides in two and occurs once cells have replicated and segregated their chromosomes. Eukaryotic cells assemble a molecular machine called the actomyosin ring that drives cytokinesis. Contraction of the actomyosin ring is coupled to ingression of the plasma membrane and extracellular matrix remodelling. In eukaryotes, glycosyltransferases that synthesise polysaccharides of the extracellular matrix are emerging as essential factors during cytokinesis. Defects associated with the function of those glycosyltransferases induce the failure of cell division, which promotes the formation of genetically unstable tetraploid cells. Budding yeast cells contain a glycosyltransferase called Chs2 that makes a special layer of extracellular matrix and is essential during cell division. Our findings provide new insights into the molecular mechanism by which the cytokinesis regulators Inn1 and Cyk3 finely regulate the activity of glycosyltransferase Chs2 at the end of mitosis. In addition we isolated a group of actomyosin ring components that form complexes together with Chs2 and Inn1 at the cleavage site, which we have named ‘ingression progression complexes’. These complexes coordinate the contraction of the actomyosin ring, ingression of the plasma membrane and extracellular matrix remodelling in a precise manner. Chs2 is indeed a key factor for coordinating these events. It appears that similar principles could apply to other eukaryotic species, such as fission yeast even if the identity of the relevant glycosyltransferase has changed over the evolution. Taking into account the conservation of the basic cytokinetic mechanisms future studies should try to determine whether a glycosyltransferase similar to Chs2 plays a key role during cytokinesis in human cells.