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A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product.

Rupp G, Porter ME - J. Cell Biol. (2003)

Bottom Line: The pf2-4 mutant displays an altered waveform that results in slow swimming cells.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.

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.

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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.
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fig4: 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.

Mentions: Previous studies have shown that pf2 axonemes lack five axonemal polypeptides known as DRC subunits 3–7 (Table I; Huang et al., 1982; Piperno et al., 1992, 1994). Dikaryon rescue experiments tentatively identified DRC subunit 4 as the gene product of the PF2 locus (Piperno et al., 1994). To determine if PF2 encodes a structural component of the axoneme or a polypeptide extrinsic to the axoneme that is required for assembly of the DRC, an epitope-tagged construct was used to rescue the pf2 mutant phenotype. PF2 is predicted to contain numerous coiled-coil domains throughout its length (Fig. 4Figure 4.


A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product.

Rupp G, Porter ME - J. Cell Biol. (2003)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172716&req=5

fig4: 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.
Mentions: Previous studies have shown that pf2 axonemes lack five axonemal polypeptides known as DRC subunits 3–7 (Table I; Huang et al., 1982; Piperno et al., 1992, 1994). Dikaryon rescue experiments tentatively identified DRC subunit 4 as the gene product of the PF2 locus (Piperno et al., 1994). To determine if PF2 encodes a structural component of the axoneme or a polypeptide extrinsic to the axoneme that is required for assembly of the DRC, an epitope-tagged construct was used to rescue the pf2 mutant phenotype. PF2 is predicted to contain numerous coiled-coil domains throughout its length (Fig. 4Figure 4.

Bottom Line: The pf2-4 mutant displays an altered waveform that results in slow swimming cells.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.

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.

Show MeSH
Related in: MedlinePlus