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First published online 3 August 2004
doi: 10.1242/jcs.01297

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Cpc1, a Chlamydomonas central pair protein with an adenylate kinase domain

Hui Zhang^* and David R. Mitchell

Department of Cell and Developmental Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA

View larger version (21K): [in a new window]   Fig. 1. (A) Restriction map of the plasmid rescued from insertional allele cpc1-2, which contains a complete pUC119 vector (shaded) and another small fragment of pUC119 (hatched) embedded within Chlamydomonas genomic sequences (unshaded). (B) Southern blot of genomic DNA from Chlamydomonas wild-type strain 137c and from two insertional alleles of cpc1, digested with PvuII and probed with PstI-MscI fragment labeled 60A8L in (A). Hybridization of this probe to bands that differ from the wild type in both mutants indicates that 60A8L is close to the insertion site in both alleles.

View larger version (15K): [in a new window]   Fig. 2. (A) Map of overlapping bacterial artificial chromosome (BAC) clones selected with cpc1 insertion site probe 60A8L. Indicated to the right are the number of fast-swimming (rescued) transformants and the total number of arg+ colonies screened. NT, not tested. All four tested BAC clones rescued the cpc1 mutant phenotype, indicating that the CPC1 gene lies in the region of overlap between BACs 6H3 and 28H16. The orientation of this BAC contig on chromosome III is indicated by arrows pointing toward the telomere (tel) and centromere (cen). (B) Restriction map of BAC 28H16 (top line) shows the location of the insertion site in mutation cpc1-2 (arrow). Subclones ES6, ES10 and SS3, and three smaller fragments of ES10 (A, B, C) shown below the restriction map were tested for their ability to rescue the mutation. Rescue was only seen with ES10. H, HindIII; E, EcoRI; S, SalI.

View larger version (43K): [in a new window]   Fig. 3. Thin-section electron micrographs of wild-type Chlamydomonas axonemes (WT), cpc1-2 axonemes and axonemes from cpc1 cells transformed with plasmid ES10 [cpc1-2(ES10)]. Diagram to the right summarizes central pair structures as seen in cross sections (modified from Mitchell and Sale, 1999). Central pair structures missing in cpc1 are fully restored in transformants (arrows). Bar, 100 nm.

View larger version (15K): [in a new window]   Fig. 4. Diagram of transcription units in ES10 and the domain structure of the predicted translation product, Cpc1. (A) Directional arrows show one complete and one partial transcription unit in ES10. The left-pointing partial transcription unit was identified from BLAST searches of EST databases and with the ES10 sequence and includes only the 3' untranslated region of a gene as reported in the Chlamydomonas genome database v1.0 (predicted product genie 3.63). The right-pointing complete transcription unit and corresponding ORF were identified from sequence analysis of ES10, cDNA clones a-e (see text) and EST sequences 1-7 (corresponding to AV626984, BU650028, BE337526, BG857322, BG859077 and BE337525, respectively). (B) Distribution of sequences predicted to form -helical coiled coils (CC) and sequences with similarity to adenylate kinases (Ad Kin) and to calcium binding domains (EF). (C) Distribution of regions with highest similarity to a mammalian homolog, rat Kpl2 (AAD56310). Sequences of genomic clone ES10 and the CPC1 cDNA have been deposited in GenBank (Accession no. AY601881 and AY601882).

View larger version (40K): [in a new window]   Fig. 5. (A) Cpc1 sequences predicted to fold into an EF hand, with residues important for coordination of Ca2+ underlined. (B) Alignment of portions of the Cpc1 adenylate kinase domain with the complete sequence of human adenylate kinase isoform 2 (AAC52061). Identical residues are shaded black, conservative replacements in grey. Cpc1 sequences homologous to adenylate kinase are separated into four segments, with segment 1 spanning the phosphate binding loop (P-loop, Walker A motif) and segment 3 encompassing the highly conserved Walker B motif. Arrows (segments 1 and 4) indicate arginine residues that may be important for substrate interactions. The RxxH sequence in segment 4, part of the LID domain, is common to most eukaryotic adenylate kinases.

View larger version (52K): [in a new window]   Fig. 6. Western blotting of flagellar axonemes from wild-type cells and three central pair defective Chlamydomonas strains. (A) 6% SDS polyacrylamide gel of wild-type and pf18, pf16 and cpc1 mutant axonemes stained with Coomassie blue. Dots next to the wild-type sample mark two high molecular weight proteins, CP3 and CP4, missing from central pair assembly-defective strain pf18. The sizes of molecular weight standards (in kDa) are shown along the left edge. (B) Western blot of the gel in (A) probed with anti-Cpc1A. A single band is seen in the wild type and at reduced amounts in pf16 axonemes. The immunoreactive band aligns with the location of CP4 in (A).

View larger version (66K): [in a new window]   Fig. 7. Purification of Cpc1 by Chlamydomonas flagellar fractionation. (A,B) 6% SDS polyacrylamide gel (A) and corresponding western blot (B) of wild-type axonemes (Ax), the insoluble material following successive extractions with 0.5 M NaCl (NP) and 0.2 M KI (KP) and the material solubilized by KI (KS). Western blotting with anti-Cpc1A shows that Cpc1 is extracted by KI. Stoichiometric amounts were loaded in each lane. Dots in (A) mark the location of high molecular weight central pair proteins CP3 and CP4.

View larger version (69K): [in a new window]   Fig. 8. Sucrose density gradient analysis of KI extracts from Chlamydomonas axonemes. (A,B) cpc1 axonemes (A) and wild-type axonemes (B) were sedimented on 5-20% sucrose gradients and resulting fractions were analyzed by SDS-PAGE (bottom of gradient to the left). Gels were stained with Coomassie blue. The sizes of molecular weight standards (in kDa) are shown along the right edge of each gel. (C) Western blotting of gel in (B) with anti-Cpc1A shows that Cpc1 sediments in two peaks with approximate sedimentation coefficients of 16S and 9S. Five proteins [marked in (B) with arrows], including CP3, CP4 (Cpc1) and bands of 135 kDa, 79 kDa, and 56 kDa co-sediment with Cpc1 in the faster sedimenting (16S) peak. None of these bands are seen in KI extracts from cpc1 axonemes (A).

View larger version (10K): [in a new window]   Fig. 9. Double-reciprocal plot of the variation of in vitro Chlamydomonas flagellar beat frequency with ATP concentration. Intercepts provide a Frequencymax of 64.9 Hz and a Km(freq) of 0.14 mM ATP.

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