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J. Biol. Chem., Vol. 283, Issue 48, 33611-33619, November 28, 2008

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Regulation of Dynactin through the Differential Expression of p150^Glued Isoforms^*

From the Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Cytoplasmic dynein and dynactin interact to drive microtubule-based transport in the cell. The p150^Glued subunit of dynactin binds to dynein, and directly to microtubules. We have identified alternatively spliced isoforms of p150^Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150^Glued, we used RNAi to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150^Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150^Glued results in motor neuron disease; here we investigate the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell.

Received for publication, June 25, 2008 , and in revised form, September 8, 2008.

^* This work was supported, in whole or in part, by Grants GM068591 and GM48661 from the National Institutes of Health. This work was also supported by the ALS Association (to E. L. F. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Movies S1 and S2.

¹ Supported by National Institutes of Health-NIA Predoctoral Training Grant T32 AG00255.

² To whom correspondence should be addressed: University of Pennsylvania, D400 Richards Bldg., 3700 Hamilton Walk, Philadelphia, PA 19104-6085. Fax: 215-573-5851; E-mail: holzbaur{at}mail.med.upenn.edu.

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