Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe.
The Schizosaccharomyces pombe Cdc4 protein is required for the formation and function of the contractile ring, presumably acting as a myosin light chain. By using NMR spectroscopy, we demonstrate that purified Cdc4p is a monomeric 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 bind calcium in vitro. Three variants of Cdc4p containing single point mutations responsible for temperature-sensitive arrest of the cell cycle at cytokinesis (Gly-19 to Glu, Gly-82 to Asp, and Gly-107 to Ser) were also characterized by NMR and circular dichroism spectroscopy. In each case, the amino acid substitution only leads to small perturbations in the conformation of the protein. Furthermore, thermal unfolding studies indicate that, like wild-type Cdc4p, the three mutant forms are all extremely stable, remaining completely folded at temperatures significantly above those causing failure of cytokinesis in intact cells. Therefore, the altered phenotype must arise directly from a disruption of the function of Cdc4p rather than indirectly through a disruption of its overall structure. Several mutant alleles of Cdc4p also show interallelic complementation in diploid cells. This phenomenon can be explained if Cdcp4 has more than one essential function or, alternatively, if two mutant proteins assemble to form a functional complex. Based on the structure of Cdc4p, possible models for interallelic complementation including interactions with partner proteins and the formation of a myosin complex with Cdc4p fulfilling the role of both an essential and regulatory light chain are proposed.
Slupsky
C M
CM
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Desautels
M
M
Huebert
T
T
Zhao
R
R
Hemmingsen
S M
SM
McIntosh
L P
LP
D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology*; D003598:Cytoskeletal Proteins; D020832:EF Hand Motifs; D044783:F-Box Proteins; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D018583:Pliability; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000166:cytology*; D029702:Schizosaccharomyces pombe Proteins; D013696:Temperature; D044767:Ubiquitin-Protein Ligases
D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D044783:F-Box Proteins; D029702:Schizosaccharomyces pombe Proteins; C513274:cdc4 protein, S pombe; D044767:Ubiquitin-Protein Ligases