Abstract
The septation initiation network (SIN) serves to coordinate cytokinesis with mitotic exit in the fission yeast Schizosaccharomyces pombe. SIN components Spg1 and Cdc7 together play a central role in regulating the onset of septation and cytokinesis. Spg1, a Ras-like GTPase, localizes to the spindle pole bodies (SPBs) throughout the cell cycle. It is converted to its GTP-bound (active) state during mitosis, only to become inactivated at one SPB during anaphase and at both SPBs as cells exit mitosis. Cdc7 functions as an effector kinase for Spg1, binding to Spg1 in its GTP-bound state, and therefore is present at both SPBs during mitosis and asymmetrically at only one during anaphase. Interestingly, the kinase activity of Cdc7 does not vary across the cell cycle, suggesting the possibility that Cdc7 kinase activity is independent of Spg1 binding. Consistent with this, we found that Cdc7 associates with Spg1 only during mitosis. To learn more about the essential role of Cdc7 kinase in the SIN and its regulation, we undertook a structure/function analysis and identified independent functional domains within Cdc7. We found that a region adjacent to the kinase domain is responsible for Spg1 association and identified an overlapping but distinct SPB localization domain. In addition Cdc7 associates with itself and exists as a dimer in vivo.
Highlights
Conserved GTPase-driven signaling pathways have emerged that serve to coordinate mitotic exit with cytokinesis [2, 3]
The kinase activity of Cdc7 has been shown to remain constant throughout the cell cycle despite the observation that Spg1 is in its GTP-bound form at the spindle pole bodies (SPBs) only during mitosis [22]
Cdc7 is a protein kinase essential for cytokinesis in fission yeast. It is an effector for the Ras superfamily GTPase Spg1, the activation of which is central to determining the timing of septation and cytokinesis in fission yeast
Summary
Yeast Strains, Media, and Genetic Methods—S. pombe strains used in this study (Table 1) were grown in yeast extract or minimal medium with appropriate supplements [24]. DNA transformations were done by electroporation [25] or lithium acetate transformation. Regulated expression of genes from various strengths of the nmt promoter in the pREP series of vectors was achieved by growth in the presence of thia-.
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