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. 2003 Jul 7;162(1):47-57.
doi: 10.1083/jcb.200303019.

A Subunit of the Dynein Regulatory Complex in Chlamydomonas Is a Homologue of a Growth Arrest-Specific Gene Product

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A Subunit of the Dynein Regulatory Complex in Chlamydomonas Is a Homologue of a Growth Arrest-Specific Gene Product

Gerald Rupp et al. J Cell Biol. .
Free PMC article

Abstract

The dynein regulatory complex (DRC) is an important intermediate in the pathway that regulates flagellar motility. To identify subunits of the DRC, we characterized a Chlamydomonas motility mutant obtained by insertional mutagenesis. The pf2-4 mutant displays an altered waveform that results in slow swimming cells. EM analysis reveals defects in DRC structure that can be rescued by reintroduction of the wild-type PF2 gene. Immunolocalization studies show that the PF2 protein is distributed along the length of the axoneme, where it is part of a discrete complex of polypeptides. PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest-specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin. PF2 and its homologues appear to be universal components of motile axonemes that are required for DRC assembly and the regulation of flagellar motility. The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures.

Figures

Figure 1.
Figure 1.
Cloning the PF2 gene. (A) Partial restriction maps of the region containing the PF2 gene from wild-type and pf2-4 (9B11). Also indicated is the location of the NIT1 plasmid insertion in 9B11, now known as pf2-4, and the position of the flanking clone FC-1 (black box) representing the NotI/BamHI restriction fragment recovered from the 9B11 minilibrary. S, SacI sites; N, NotI sites. (B) Southern blot of wild-type (wt) and pf2-4 genomic DNA digested with the indicated restriction enzymes and hybridized with FC-1. (C) Alignment of three overlapping phage clones recovered with FC-1 (black boxes) and two plasmid clones, derived from λG1, that contain the PF2 gene. Plasmid p9-3HA is the epitope-tagged PF2 construct. Also shown are the number of rescued strains obtained by cotransformation of pf2-4 and pf2-1 with the selected clones. nd, not determined.
Figure 1.
Figure 1.
Cloning the PF2 gene. (A) Partial restriction maps of the region containing the PF2 gene from wild-type and pf2-4 (9B11). Also indicated is the location of the NIT1 plasmid insertion in 9B11, now known as pf2-4, and the position of the flanking clone FC-1 (black box) representing the NotI/BamHI restriction fragment recovered from the 9B11 minilibrary. S, SacI sites; N, NotI sites. (B) Southern blot of wild-type (wt) and pf2-4 genomic DNA digested with the indicated restriction enzymes and hybridized with FC-1. (C) Alignment of three overlapping phage clones recovered with FC-1 (black boxes) and two plasmid clones, derived from λG1, that contain the PF2 gene. Plasmid p9-3HA is the epitope-tagged PF2 construct. Also shown are the number of rescued strains obtained by cotransformation of pf2-4 and pf2-1 with the selected clones. nd, not determined.
Figure 1.
Figure 1.
Cloning the PF2 gene. (A) Partial restriction maps of the region containing the PF2 gene from wild-type and pf2-4 (9B11). Also indicated is the location of the NIT1 plasmid insertion in 9B11, now known as pf2-4, and the position of the flanking clone FC-1 (black box) representing the NotI/BamHI restriction fragment recovered from the 9B11 minilibrary. S, SacI sites; N, NotI sites. (B) Southern blot of wild-type (wt) and pf2-4 genomic DNA digested with the indicated restriction enzymes and hybridized with FC-1. (C) Alignment of three overlapping phage clones recovered with FC-1 (black boxes) and two plasmid clones, derived from λG1, that contain the PF2 gene. Plasmid p9-3HA is the epitope-tagged PF2 construct. Also shown are the number of rescued strains obtained by cotransformation of pf2-4 and pf2-1 with the selected clones. nd, not determined.
Figure 2.
Figure 2.
Analysis of the PF2 transcription unit. (A) Partial restriction map of wild-type genomic DNA in the region of the PF2 transcription unit. Also shown are selected SacI fragments (A, C, and D) and a NotI fragment (B) that were used to map the boundaries of the plasmid insertion and determine the size of the transcription unit. (B) Northern blot loaded with total RNA isolated from wild-type (wt) and pf2 mutant cells before (0) and 45 min after deflagellation. This blot was hybridized with probe C, which recognized an ∼2.5-kb transcript that is up-regulated in wild-type cells and missing in pf2 mutants. (B, bottom) Loading control. The blot was rehybridized with a CRY1 probe, which encodes the ribosomal S14 protein subunit (Nelson et al., 1994). (C) Diagram of the intron–exon structure of the PF2 transcription unit. Open rectangles indicate exons and solid lines indicate introns. The predicted translation start (ATG) site and the putative polyadenylation signal (Poly A) are indicated. Also shown are the single nucleotide alterations that resulted in the pf2-1 and pf2-2 mutations, and the location of the epitope tag in exon 12.
Figure 2.
Figure 2.
Analysis of the PF2 transcription unit. (A) Partial restriction map of wild-type genomic DNA in the region of the PF2 transcription unit. Also shown are selected SacI fragments (A, C, and D) and a NotI fragment (B) that were used to map the boundaries of the plasmid insertion and determine the size of the transcription unit. (B) Northern blot loaded with total RNA isolated from wild-type (wt) and pf2 mutant cells before (0) and 45 min after deflagellation. This blot was hybridized with probe C, which recognized an ∼2.5-kb transcript that is up-regulated in wild-type cells and missing in pf2 mutants. (B, bottom) Loading control. The blot was rehybridized with a CRY1 probe, which encodes the ribosomal S14 protein subunit (Nelson et al., 1994). (C) Diagram of the intron–exon structure of the PF2 transcription unit. Open rectangles indicate exons and solid lines indicate introns. The predicted translation start (ATG) site and the putative polyadenylation signal (Poly A) are indicated. Also shown are the single nucleotide alterations that resulted in the pf2-1 and pf2-2 mutations, and the location of the epitope tag in exon 12.
Figure 2.
Figure 2.
Analysis of the PF2 transcription unit. (A) Partial restriction map of wild-type genomic DNA in the region of the PF2 transcription unit. Also shown are selected SacI fragments (A, C, and D) and a NotI fragment (B) that were used to map the boundaries of the plasmid insertion and determine the size of the transcription unit. (B) Northern blot loaded with total RNA isolated from wild-type (wt) and pf2 mutant cells before (0) and 45 min after deflagellation. This blot was hybridized with probe C, which recognized an ∼2.5-kb transcript that is up-regulated in wild-type cells and missing in pf2 mutants. (B, bottom) Loading control. The blot was rehybridized with a CRY1 probe, which encodes the ribosomal S14 protein subunit (Nelson et al., 1994). (C) Diagram of the intron–exon structure of the PF2 transcription unit. Open rectangles indicate exons and solid lines indicate introns. The predicted translation start (ATG) site and the putative polyadenylation signal (Poly A) are indicated. Also shown are the single nucleotide alterations that resulted in the pf2-1 and pf2-2 mutations, and the location of the epitope tag in exon 12.
Figure 3.
Figure 3.
Structural defects in pf2 axonemes. Longitudinal images of wild-type and mutant axonemes were aligned and computer image averaged. Shown here are grand averages of axonemes isolated from (A) wild type, (B) pf2-4, (C), pf2-1, and (D) a rescued pf2 strain (pf2-4r). The arrows in A–D indicate the region of the 96-nm repeat that is occupied by the DRC structure. (E) Model of axoneme structures within the 96-nm axoneme repeat. The proximal and distal RSs are labeled S1 and S2, respectively; the outer dynein arms (OA) are shown on top, and the DRC is indicated as a crescent-shaped structure above the distal RS (S2). Difference plots between wild type (wt) and pf2-4 (F), and wt and pf2-1 (G) demonstrate that the DRC is missing in both mutants. Note that the DRC is restored in the rescued strain, pf2-4r (D).
Figure 4.
Figure 4.
PF2 protein structure and epitope tagging. (A) Diagrammatic representation of the domain structure of the PF2 polypeptide. Indicated are predicted coiled-coil domains (gray, shaded boxes) and the location of the epitope tag. (B) Western blot analysis of whole axonemes isolated from wild type, pf2-4, and a pf2-4 strain rescued with the epitope-tagged PF2 gene (pf2-4r:3HA). Blots were visualized with a reversible total protein stain (left) before immunolabeling with an antibody directed against the HA epitope (right). The HA antibody recognized a single band in pf2-4r:3HA migrating at ∼60 kD.
Figure 4.
Figure 4.
PF2 protein structure and epitope tagging. (A) Diagrammatic representation of the domain structure of the PF2 polypeptide. Indicated are predicted coiled-coil domains (gray, shaded boxes) and the location of the epitope tag. (B) Western blot analysis of whole axonemes isolated from wild type, pf2-4, and a pf2-4 strain rescued with the epitope-tagged PF2 gene (pf2-4r:3HA). Blots were visualized with a reversible total protein stain (left) before immunolabeling with an antibody directed against the HA epitope (right). The HA antibody recognized a single band in pf2-4r:3HA migrating at ∼60 kD.
Figure 5.
Figure 5.
Localization of the epitope-tagged PF2 protein in Chlamydomonas flagella. The epitope-tagged, rescued strain, pf2-4r:3HA, was stained with an antibody against the HA tag and then imaged by differential interference contrast (A and C) or indirect immunofluorescence light microscopy (B and D). The HA-tagged PF2 is present along the entire length of the flagella in pf2-4r:3HA cells and can also been seen in two spots in the basal body region (B and D). pf2-4 mutant (E and F) or wild-type cells (G and H) labeled with the HA antibody or secondary antibody alone (not depicted) show only background cell body autofluorescence.
Figure 6.
Figure 6.
Identification of polypeptides that interact with PF2. Axonemes from the HA-tagged pf2-4 rescued strain were treated with 0, 1, 5, or 10 mM EDC and then used to prepare four identical Western blots. The first blot was stained for total protein. The remaining blots were labeled with antibodies directed against either the HA epitope (HA), α-tubulin (YOL 1/34), or β-tubulin (Tu27B). Asterisks mark three cross-linked products formed as a result of treating the HA-tagged PF2 axonemes with 1 mM EDC.
Figure 7.
Figure 7.
PF2 homologues. (A) Clustal W alignment of the deduced amino acid sequence of the Chlamydomonas reinhardtii (Cr) PF2 gene (AY309087–AY309089) with homologues identified by BLAST. The shaded areas represent identical or conservatively substituted amino acids in >50% of the sequences. The underlined region represents a putative microtubule-binding domain identified in the trypanosome and human orthologues (Hill et al., 2000). The abbreviation and GenBank/EMBL/DDBJ accession nos. for the sequences are as follows: Danio rerio (Dr), AI584969; Drosophila melanogaster (Dm), AE003427 and AA440118; Homo sapiens (Hs), AF050079; Mus musculus (Mm), U19859; Trypanosoma bruzi (Tb), AAB81499; and Trypanosoma cruzi (Tc), AQ44061. Gas11, growth arrest–specific 11; Gas8, growth arrest–specific 8. (B) Phylogenetic analysis of the Cr PF2 sequence and its homologues.

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References

    1. Afzelius, B.A. 1995. Role of cilia in human health. Cell Motil. Cytoskeleton. 32:95–97. - PubMed
    1. Brenner, D.G., S. Lin-Chao, and S.N. Cohen. 1989. Analysis of mammalian cell genetic regulation in situ by using retrovirus-derived “portable exons” carrying the Escherichia coli lacZ gene. Proc. Natl. Acad. Sci. USA. 86:5517–5521. - PMC - PubMed
    1. Brokaw, C.J., and R. Kamiya. 1987. Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil. Cytoskeleton. 8:68–75. - PubMed
    1. Burgess, S.A., D.A. Carter, S.D. Dover, and D.M. Woolley. 1991. The inner dynein arm complex: compatible images from freeze-etch and thin section methods of microscopy. J. Cell Sci. 100:319–328. - PubMed
    1. Debuchy, R., S. Purton, and J.-D. Rochaix. 1989. The arginosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. EMBO J. 8:2803–2809. - PMC - PubMed

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