Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe

The Schizosaccharomyces pombe Cdc4 protein is required for the formation an d function of the contractile ring, presumably acting as a myosin light cha in. By using NMR spectroscopy, we demonstrate that purified Cdc4p is a mono meric protein with two structurally independent domains, each exhibiting a fold reminiscent of the EF-hand class of calcium-binding proteins. Although Cdc4p has one potentially functional calcium-binding site, it does not bin d calcium in vitro. Three variants of Cdc4p containing single point mutatio ns responsible for temperature-sensitive arrest of the cell cycle at cytoki nesis (Gly-19 to Glu, Gly-82 to Asp, and Gly-107 to Ser) were also characte rized by NMR and circular dichroism spectroscopy. In each case, the amino a cid substitution only leads to small perturbations in the conformation of t he protein. Furthermore, thermal unfolding studies indicate that, like wild -type Cdc4p, the three mutant forms are all extremely stable, remaining com pletely folded at temperatures significantly above those causing failure of cytokinesis in intact cells. Therefore, the altered phenotype must arise d irectly from a disruption of the function of Cdc4p rather than indirectly t hrough a disruption of its overall structure. Several mutant alleles of Cdc 4p also show interallelic complementation in diploid cells. This phenomenon can be explained if Cdcp4 has more than one essential function or, alterna tively, if two mutant proteins assemble to form a functional complex. Based on the structure of Cdc4p, possible models for interallelic complementatio n including interactions with partner proteins and the formation of a myosi n complex with Cdc4p fulfilling the role of both an essential and regulator y light chain are proposed.