Affinity Chromatography Demonstrates a Direct Binding between Cytoplasmic Dynein and the Dynactin Complex

doi:10.1074/jbc.270.48.28806

Affinity Chromatography Demonstrates a Direct Binding between Cytoplasmic Dynein and the Dynactin Complex *

From the (1) Cell Biology Graduate Group, University of Pennsylvania School of Medicine and the
(2) Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104

^§ To whom correspondence should be addressed:
143 Rosenthal Bldg., Dept. of Animal Biology, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6048.
Tel.: 215-573-3257; Fax: 215-898-9923; holzbaur{at}pobox.upenn.edu.

Abstract

We used affinity chromatography to probe for a direct binding interaction between cytoplasmic dynein and dynactin. Purified cytoplasmic dynein was found to bind to an affinity column of p150, the largest polypeptide in the dynactin complex. To test the specificity of the interaction, we loaded rat brain cytosol onto the p150 affinity column and observed that cytoplasmic dynein from cytosol was specifically retained on the column. Preincubation of the p150 affinity matrix with excess exogenous dynein intermediate chain resulted in a significant reduction of dynein binding, suggesting that p150 may be interacting with dynein via this polypeptide. Therefore we constructed an affinity column of recombinant dynein intermediate chain and observed that dynactin was retained from rat brain cytosol. These results demonstrate that the native dynein and dynactin complexes are capable of direct in vitro interaction mediated by a direct binding of the dynein intermediate chain to the p150component of the dynactin complex. We have mapped the site of this interaction to the amino-terminal region of p150, which is predicted to form an α-helical coiled-coil. Regulation of the dynein-dynactin interaction may prove to be key in the control mechanism for cytoplasmic dynein-mediated vesicular transport.

Footnotes

↵* This work was supported by Grant GM48661 from the National Institutes of Health (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 by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Arp1

actin-related protein 1

DHC

dynein heavy chain

DIC

dynein intermediate chain

LIC

light intermediate chain

PIPES

1,4-piperazinediethanesulfonic acid

BSA

bovine serum albumin

PAGE

polyacrylamide gel electrophoresis.
↵²M. K. Tokito, D. S. Howland, V. M.-Y. Lee, and E. L. F. Holzbaur, submitted for publication.
↵³Vaughn, K. T., and Vallee, R. B. (1995) J. Cell Biol., in press.
↵⁴C. M. Waterman-Storer, S. Kuznetsov, S. Karki, G. M. Langford, D. G. Weiss, and E. L. F. Holzbaur, submitted for publication.
↵⁵P. Farshori and E. L. F. Holzbaur, manuscript in preparation.
- Received May 9, 1995.
- Revision received July 13, 1995.