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J. Biol. Chem., Vol. 276, Issue 8, 5943-5951, February 23, 2001
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From the 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.
The atomic coordinates and the structure factors (code 1GGW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Structure of Cdc4p, a Contractile Ring Protein Essential
for Cytokinesis in Schizosaccharomyces pombe*
§,
,, and§§
Department of Biochemistry and Molecular
Biology, the Department of Chemistry, and the Biotechnology
Laboratory, University of British Columbia, Vancouver, British
Columbia V6T 1Z3, the ¶ Plant Biotechnology Institute,
National Research Council of Canada, Saskatoon,
Saskatchewan S7N 0W9, and the Department of Physiology and
the ** Department of Microbiology and Immunology, University of
Saskatchewan College of Medicine, Saskatoon,
Saskatchewan S7N 5E5, Canada
*
This work was supported by grants from the Leukemia Research
Fund (to C. M. S.), the Medical Research Council of Canada (to S. M. H.), the National Cancer Institute of Canada, and the Protein Engineering Network Centers of Excellence (to L. P. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence may be addressed. Tel.: 306-975-5242;
Fax: 306-975-4839; E-mail: hemmings@cbrpbi.pbi.nrc.ca.
§§
An Alexander von Humbolt Fellow and a Canadian Institutes
of Health Research Scientist. To whom correspondence may be addressed. Tel.: 604-822-3341; Fax: 604-822-5227; E-mail:
mcintosh@otter.biochem.ubc.ca.
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