Abstract
p150Glued is the largest polypeptide in the dynactin complex, a protein heteromultimer that binds to and may mediate the microtubule-based motor cytoplasmic dynein. Cloning of a cDNA encoding p150Glued from rat revealed 31% amino acid sequence identity with the product of the Drosophila gene, Glued. A dominant Glued mutation results in neuronal disruption; null mutations are lethal. However, the Glued gene product has not been characterized. To determine whether the Glued polypeptide is functionally similar to vertebrate p150Glued, we characterized the Glued protein in the Drosophila S-2 cell line. Antibodies raised against Glued were used to demonstrate that this protein sediments exclusively at 20 S, and associates with microtubules in a salt- and ATP-dependent manner. Immunoprecipitations from S-2 cytosol with the anti-Glued antibody resulted in the co-precipitation of subunits of both cytoplasmic dynein and the dynactin complex. An affinity column with covalently bound Glued protein retained cytoplasmic dynein from S-2 cytosol. Based on these observations, we conclude that Glued is a component of a dynactin complex in Drosophila and binds to cytoplasmic dynein, and therefore the mutant Glued phenotypes can be interpreted as resulting from a disruption in the function of the dynactin complex.
Highlights
P150Glued is the largest polypeptide in the dynactin complex, a protein heteromultimer that binds to and may mediate the microtubule-based motor cytoplasmic dynein
While these polypeptide were originally identified as substoichiometric components of cytoplasmic dynein preparations [7], it was subsequently found that dynein and the dynactin complex form distinct 20 S complexes in cytosol that were separable by ion exchange chromatography [2] or immunoprecipitation [3]
The recent demonstration that affinity matrices containing either covalently bound p150Glued, the largest polypeptide in the dynactin complex [2, 3], or covalently bound dynein intermediate chain are capable of retaining intact cytoplasmic dynein or dynactin complex, respectively [18], provides a clear demonstration that the two complexes are capable of a direct biochemical interaction
Summary
(Received for publication, March 3, 1995, and in revised form, September 5, 1995). From the Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104-6046. P150Glued is the largest polypeptide in the dynactin complex, a protein heteromultimer that binds to and may mediate the microtubule-based motor cytoplasmic dynein. An affinity column with covalently bound Glued protein retained cytoplasmic dynein from S-2 cytosol Based on these observations, we conclude that Glued is a component of a dynactin complex in Drosophila and binds to cytoplasmic dynein, and the mutant Glued phenotypes can be interpreted as resulting from a disruption in the function of the dynactin complex. Characterization of rat cDNA clones encoding the 150-kDa polypeptide indicated that this protein shared 31% amino acid sequence identity with the product of the Glued gene of Drosophila melanogaster [21, 22], and the 150-kDa rat polypeptide was subsequently named p150Glued [3].
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