Abstract
In the fission yeast Schizosaccharomyces pombe, septation and constriction of the actomyosin ring for cell division are positively regulated by the Spg1 GTPase, a member of the Ras superfamily. Spg1 is negatively regulated by Byr4 and Cdc16, which together form a two-component GTPase-activating protein for the Spg1 GTPase. To better understand how Byr4 regulates septation, Byr4 mutants were tested for in vitro functions. This analysis revealed that Byr4 contained one Cdc16-binding site and four Spg1-binding sites (SBS), designated SBS1-SBS4. Although mutants with a single SBS bound Spg1 and inhibited GTP dissociation, the equilibrium binding affinity of these mutants was 28-280-fold weaker than Byr4. Because some Byr4 mutants with multiple SBSs bound Spg1 tighter than the corresponding mutants with a single SBS, multiple SBSs probably interact to cause the high affinity binding of Byr4 to Spg1. A region of Byr4 that bound Spg1, SBS4, and the region that bound Cdc16, Cdc16-binding site, was necessary and sufficient to form Cdc16-dependent Spg1GAP activity that was similar to that of wild-type Byr4 with Cdc16.
Highlights
In many eukaryotes, including Schizosaccharomyces pombe, cell division results from the constriction of an actomyosin ring that is perpendicular to the mitotic spindle
To further understand how Byr4 regulates septation, Byr4 mutants were assayed for binding to Cdc16 or Spg1 and for Spg1GAP activity with Cdc16
SBS4 and Cdc16-binding site (CBS) Are Necessary and Sufficient to Form Spg1GAP Activity with Cdc16 —Because a two-component GTPaseactivating protein (GAP) is unique to Byr4 and Cdc16, we determined what regions of Byr4 were required for this reaction
Summary
In many eukaryotes, including Schizosaccharomyces pombe, cell division results from the constriction of an actomyosin ring that is perpendicular to the mitotic spindle (reviewed in Ref. 1). To further understand how Byr4 regulates septation, Byr4 mutants were assayed for binding to Cdc16 or Spg1 and for Spg1GAP activity with Cdc16. A C-terminal fragment of Byr4, which contained SBS4 and CBS, was necessary and sufficient for Spg1GAP activity with Cdc16.
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