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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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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.
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fig6: 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.

Mentions: Based on the high incidence of coiled-coil domains in the predicted tertiary structure of PF2, it is likely that PF2 interacts with other axonemal polypeptides, specifically other components of the DRC and outer doublet microtubules. To identify those polypeptides, isolated axonemes from the HA-tagged strain were treated with varying concentrations of the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and then analyzed on Western blots. EDC has previously proven useful in the identification of interactions between subunits of several other axonemal structures (for examples see King et al., 1991; Yang et al., 2000). After a 1-h exposure of isolated axonemes to 1 mM EDC, three new bands migrating at ∼125, 130, and 145 kD, as well as a number of higher molecular mass (>250 kD) bands, could be detected (Fig. 6)Figure 6.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172716&req=5

fig6: 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.
Mentions: Based on the high incidence of coiled-coil domains in the predicted tertiary structure of PF2, it is likely that PF2 interacts with other axonemal polypeptides, specifically other components of the DRC and outer doublet microtubules. To identify those polypeptides, isolated axonemes from the HA-tagged strain were treated with varying concentrations of the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and then analyzed on Western blots. EDC has previously proven useful in the identification of interactions between subunits of several other axonemal structures (for examples see King et al., 1991; Yang et al., 2000). After a 1-h exposure of isolated axonemes to 1 mM EDC, three new bands migrating at ∼125, 130, and 145 kD, as well as a number of higher molecular mass (>250 kD) bands, could be detected (Fig. 6)Figure 6.

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