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Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Bowman AB, Patel-King RS, Benashski SE, McCaffery JM, Goldstein LS, King SM - J. Cell Biol. (1999)

Bottom Line: The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here.Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein.We propose that roadblock/LC7 family members may modulate specific dynein functions.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0683, USA.

ABSTRACT
Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.

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A robl/LC7-like protein is present in cytoplasmic dynein. (A) Western blot analysis was performed on samples from the fractionation of a rat brain homogenate. Blots were probed with mAb 74-1 and the R7178 rabbit polyclonal to detect IC74 of cytoplasmic dynein and the Mr ∼12,000 robl/LC7-like protein, respectively. (B) Rat brain proteins eluted from microtubules with ATP were sedimented in a 5–20% sucrose gradient. Samples were probed with the 74-1 and R7178 antibodies. The robl/LC7-like protein precisely comigrates with IC74 of cytoplasmic dynein. (C) Rat brain homogenate immunoprecipitates of cytoplasmic dynein (antibody 74-1), kinesin (antibody H-2), dynactin (antibody 50-1), and a bead control were probed with the R7178 antibody. The robl/LC7-like protein is detectable only in the cytoplasmic dynein (IC74) sample. (D) Drosophila embryo immunoprecipitates of cytoplasmic dynein (antibody 74-1) or the bead control were probed with the 6883 anti-robl antibody and 74-1 anti-IC74 antibody. The roadblock protein was only precipitated in the cytoplasmic dynein (IC74) sample. (E) Equally loaded Drosophila robl  and wild-type larval homogenates were probed with the 6883 anti-robl antibody and with the 3A5 anti-tubulin antibody. The roadblock protein is undetectable in the robl  larvae, whereas tubulin is detected at about equal levels in  and wild-type larvae.
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Figure 8: A robl/LC7-like protein is present in cytoplasmic dynein. (A) Western blot analysis was performed on samples from the fractionation of a rat brain homogenate. Blots were probed with mAb 74-1 and the R7178 rabbit polyclonal to detect IC74 of cytoplasmic dynein and the Mr ∼12,000 robl/LC7-like protein, respectively. (B) Rat brain proteins eluted from microtubules with ATP were sedimented in a 5–20% sucrose gradient. Samples were probed with the 74-1 and R7178 antibodies. The robl/LC7-like protein precisely comigrates with IC74 of cytoplasmic dynein. (C) Rat brain homogenate immunoprecipitates of cytoplasmic dynein (antibody 74-1), kinesin (antibody H-2), dynactin (antibody 50-1), and a bead control were probed with the R7178 antibody. The robl/LC7-like protein is detectable only in the cytoplasmic dynein (IC74) sample. (D) Drosophila embryo immunoprecipitates of cytoplasmic dynein (antibody 74-1) or the bead control were probed with the 6883 anti-robl antibody and 74-1 anti-IC74 antibody. The roadblock protein was only precipitated in the cytoplasmic dynein (IC74) sample. (E) Equally loaded Drosophila robl and wild-type larval homogenates were probed with the 6883 anti-robl antibody and with the 3A5 anti-tubulin antibody. The roadblock protein is undetectable in the robl larvae, whereas tubulin is detected at about equal levels in and wild-type larvae.

Mentions: Previously, the highly conserved LC8 protein and Tctex1 were found in both cytoplasmic and flagellar dyneins. The robl mutant phenotypes and the identification of a homologous sequence in organisms lacking motile cilia/flagella (C. elegans), raised the obvious possibility that robl/LC7-like proteins may be present in cytoplasmic dynein. Accordingly, we examined samples from the stepwise ATP-dependent microtubule affinity purification of cytoplasmic dynein from rat brain homogenates for the presence of a robl/LC7-like protein (Fig. 8 A). The R7178 antibody detected a single band of Mr ∼12,000 in the initial microtubule pellet. Some robl/LC7, and a similar fraction of IC74, remained in the supernatant. Most of the robl/LC7 protein co-purified with microtubules through a buffer wash and GTP elution. Some of the protein was eluted from microtubules with ATP and nearly all of the remainder could be stripped by treatment with 1 M NaCl. In contrast, most IC74 was ATP-eluted. We previously observed that different DLCs do not show precisely the same pattern during elution from microtubules, perhaps because they mark specific subsets of cytoplasmic dynein with distinct microtubule binding characteristics (King et al. 1996a, King et al. 1998). To further address the association of the robl/LC7-like protein with cytoplasmic dynein, the ATP eluate was sedimented through a 5–20% sucrose gradient. Immunological analysis of the resulting fractions revealed that the robl/LC7-like protein sedimented at ∼18 S and precisely copurified with the IC74 component of cytoplasmic dynein (Fig. 8 B).


Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Bowman AB, Patel-King RS, Benashski SE, McCaffery JM, Goldstein LS, King SM - J. Cell Biol. (1999)

A robl/LC7-like protein is present in cytoplasmic dynein. (A) Western blot analysis was performed on samples from the fractionation of a rat brain homogenate. Blots were probed with mAb 74-1 and the R7178 rabbit polyclonal to detect IC74 of cytoplasmic dynein and the Mr ∼12,000 robl/LC7-like protein, respectively. (B) Rat brain proteins eluted from microtubules with ATP were sedimented in a 5–20% sucrose gradient. Samples were probed with the 74-1 and R7178 antibodies. The robl/LC7-like protein precisely comigrates with IC74 of cytoplasmic dynein. (C) Rat brain homogenate immunoprecipitates of cytoplasmic dynein (antibody 74-1), kinesin (antibody H-2), dynactin (antibody 50-1), and a bead control were probed with the R7178 antibody. The robl/LC7-like protein is detectable only in the cytoplasmic dynein (IC74) sample. (D) Drosophila embryo immunoprecipitates of cytoplasmic dynein (antibody 74-1) or the bead control were probed with the 6883 anti-robl antibody and 74-1 anti-IC74 antibody. The roadblock protein was only precipitated in the cytoplasmic dynein (IC74) sample. (E) Equally loaded Drosophila robl  and wild-type larval homogenates were probed with the 6883 anti-robl antibody and with the 3A5 anti-tubulin antibody. The roadblock protein is undetectable in the robl  larvae, whereas tubulin is detected at about equal levels in  and wild-type larvae.
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Related In: Results  -  Collection

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Figure 8: A robl/LC7-like protein is present in cytoplasmic dynein. (A) Western blot analysis was performed on samples from the fractionation of a rat brain homogenate. Blots were probed with mAb 74-1 and the R7178 rabbit polyclonal to detect IC74 of cytoplasmic dynein and the Mr ∼12,000 robl/LC7-like protein, respectively. (B) Rat brain proteins eluted from microtubules with ATP were sedimented in a 5–20% sucrose gradient. Samples were probed with the 74-1 and R7178 antibodies. The robl/LC7-like protein precisely comigrates with IC74 of cytoplasmic dynein. (C) Rat brain homogenate immunoprecipitates of cytoplasmic dynein (antibody 74-1), kinesin (antibody H-2), dynactin (antibody 50-1), and a bead control were probed with the R7178 antibody. The robl/LC7-like protein is detectable only in the cytoplasmic dynein (IC74) sample. (D) Drosophila embryo immunoprecipitates of cytoplasmic dynein (antibody 74-1) or the bead control were probed with the 6883 anti-robl antibody and 74-1 anti-IC74 antibody. The roadblock protein was only precipitated in the cytoplasmic dynein (IC74) sample. (E) Equally loaded Drosophila robl and wild-type larval homogenates were probed with the 6883 anti-robl antibody and with the 3A5 anti-tubulin antibody. The roadblock protein is undetectable in the robl larvae, whereas tubulin is detected at about equal levels in and wild-type larvae.
Mentions: Previously, the highly conserved LC8 protein and Tctex1 were found in both cytoplasmic and flagellar dyneins. The robl mutant phenotypes and the identification of a homologous sequence in organisms lacking motile cilia/flagella (C. elegans), raised the obvious possibility that robl/LC7-like proteins may be present in cytoplasmic dynein. Accordingly, we examined samples from the stepwise ATP-dependent microtubule affinity purification of cytoplasmic dynein from rat brain homogenates for the presence of a robl/LC7-like protein (Fig. 8 A). The R7178 antibody detected a single band of Mr ∼12,000 in the initial microtubule pellet. Some robl/LC7, and a similar fraction of IC74, remained in the supernatant. Most of the robl/LC7 protein co-purified with microtubules through a buffer wash and GTP elution. Some of the protein was eluted from microtubules with ATP and nearly all of the remainder could be stripped by treatment with 1 M NaCl. In contrast, most IC74 was ATP-eluted. We previously observed that different DLCs do not show precisely the same pattern during elution from microtubules, perhaps because they mark specific subsets of cytoplasmic dynein with distinct microtubule binding characteristics (King et al. 1996a, King et al. 1998). To further address the association of the robl/LC7-like protein with cytoplasmic dynein, the ATP eluate was sedimented through a 5–20% sucrose gradient. Immunological analysis of the resulting fractions revealed that the robl/LC7-like protein sedimented at ∼18 S and precisely copurified with the IC74 component of cytoplasmic dynein (Fig. 8 B).

Bottom Line: The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here.Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein.We propose that roadblock/LC7 family members may modulate specific dynein functions.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0683, USA.

ABSTRACT
Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.

Show MeSH
Related in: MedlinePlus