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TITLE
Centrin phosphorylation is required for flagellar excision in Chlamydomonas reinhardtii
SYNOPSIS
Centrin, a 20-kD contractile phosphoprotein with four calcium-binding EF-hands, is present in the flagellar transition region between the basal body and the flagellum in Chlamydomonas. When Chlamydomonas cells are subjected to experimental treatments (e.g., alcohol or detergent treatment, mechanical shear, and extreme pH), they excise their flagella in a process known to involve calcium ions. We have isolated a centrin mutant that fails to excise its flagella in response to stimulation, and we demonstrate that this mutant is deficient in centrin phosphorylation. Upon restoration of the excision-competent phenotype by plasmid rescue, the transformed cells regain centrin phosphorylation. We conclude that centrin phosphorylation is required for flagellar excision in Chlamydomonas.
INTRODUCTION
Centrin is localized in three distinct areas (Wright et al., 1985; Schutze et al., 1987; Salisbury et al., 1988, 1989).
Centrin is a member of the superfamily of calcium-binding proteins.
The excision process is known to involve calcium ions (Huber et al., 1986; Rosenbaum and Carlson, 1969) and is mediated by a calcium-sensitive alteration of transition-zone structure (Sanders and Salisbury, 1989).
In this study we demonstrate that centrin phosphorylation is required for flagellar excision.
MATERIALS AND METHODS
Cell Culture
Wild-type strain 2677 was obtained from Duke and maintained in Sager and Granick (1954) medium.
Flagellar Excision
Cells were harvested by centrifugation and subjected to pH shock.
Monoclonal and Polyclonal Antibodies
The polyclonal serum, 08/28, has been characterized elsewhere (Salisbury et al., 1984).
The monoclonal antibody, 17E10, was raised according to the procedure of Salisbury and coworkers (1986).
Plasmid DNA
The pMN24 plasmid, containing the cloned wild-type nitrate reductase gene (Fernandez et al., 1989), was prepared according to the procedures of Tam and Lefebvre (1993).
Transformation Protocol
Cells were transformed according to the procedure of Kindle (1990).
DNA-blot Analysis and Library Screening
Genomic DNA was isolated by the method of Weeks et al. (1986) as modified by Tam and Lefebvre (1993).
Isolation of Genomic Sequence Flanking the Integrated Plasmid
A 9.0-kb genomic fragment was cloned by plasmid rescue as described by Tam and Lefebvre (1993).
Western Immunoblot Analysis
Proteins were separatred by SDS-PAGE (Laemmli, 1970).
Protein transfer to nitrocellulose and immunostaining were carried out according to the methods of Towbin and coworkers (1979), with modifications.
Two-dimensional Electrophoresis
Two-dimensional electrophoresis was performed according to the methods of O'Farrell (1975), with modifications.
Isolation of Excision Mutant
The pMN24 plasmid was digested and introduced into nit1 mutant cells by transformation.
Each transformed cell line was isolated and scored for flagellar excision autonomy.
Verification of the plasmid insert was done by DNA-blot analysis (Figure 1).
Linkage of the Excision Mutation to the Integrated NIT1 Plasmid
The mutant was backcrossed to a strain carrying the nit1 mutation and analyzed for cosegregation of the Nit and excision-incompetent phenotypes (Table I).
RESULTS
Linkage of the Excision Mutation to the Integrated NIT1 Plasmid
The results are consistent with the conclusion that insertion of the plasmid DNA into the nuclear genome resulted in the mutant phenotype.
Two-dimensional Electrophoresis
Two-dimensional electrophoresis reveals that the mutant does not have a phosphorylated isoform of centrin (Figure 2a).
Following rescue of the excision phenotype by transformation of cells with intact DNA, the mutant cells do have a phosphorylated isoform of centrin and do shed their flagella (Figure 2b).
Western Immunoblot Analysis
Western analysis confirms the two-dimensional electrophoresis results: mobility shifts of centrin before and after rescue are consistent with centrin forms plus and minus phosphorylation (Figure 3).
DISCUSSION
We show that centrin phosphorylation is required for flagellar excision.
We conclude that, because the centrin gene was not disrupted in the mutant, some other gene was disrupted whose product is involved in centrin phosphorylation.
Further studies are required to characterize the altered gene product(s).
[Literature cited should follow here, then tables, figure legends, and figures.]
Dr. Anne B. Thistle, 334 Conradi Building
644-5131, thistle@bio.fsu.edu
Office hours: TBA and by appointment