Interaction Mapping of a Dynein Heavy Chain
IDENTIFICATION OF DIMERIZATION AND INTERMEDIATE-CHAIN BINDING DOMAINS*
- From the ‡Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Albany, New York 12201-0509, the§Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611-3072, and the¶Department of Biomedical Sciences, State University of New York, Albany, New York 12201-0509
Abstract
Cytoplasmic dynein is a multisubunit microtubule-based motor protein that is involved in several eukaryotic cell motilities. Two dynein heavy chains each form a motor domain that connects to a common cargo-binding tail. Although this tail domain is composed of multiple polypeptides, subunit organization within this region is poorly understood. Here we present an in vitrodissection of the tail-forming region of the dynein heavy chain fromDictyostelium. Our work identifies a sequence important for dimerization and for binding the dynein intermediate chain. The core of this motif localizes within an ∼150-amino acid region that is strongly conserved among other cytoplasmic dyneins. This level of conservation does not extend to the axonemal dynein heavy chains, suggesting functional differences between the two. Dimerization appears to occur through a different mechanism than the heavy chain-intermediate chain interaction. We corroborate the in vitro interactions with in vivo expression of heavy chain fragments in Dictyostelium. Fragments lacking the interaction domain express well, without an obvious phenotype. On the other hand, the region crucial for both interactions appears to be lethal when overexpressed.
Footnotes
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↵* This work was supported in part by a Grant GM51532 from the National Institutes of Health (to M. P. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed: Wadsworth Center, Empire State Plaza, P. O. Box 509, Albany, NY 12201-0509. Tel.: 518-486-1490; Fax: 518-474-7992; E-mail:Michael.Koonce{at}wadsworth.org.
- Abbreviations:
- DHC
-
dynein heavy chain
- DIC
-
dynein intermediate chain
- aa
-
amino acid(s)
- Pipes
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1,4-piperazinediethanesulfonic acid
- PAGE
-
polyacrylamide gel electrophoresis
- HSS
-
high speed supernatant(s)
- BSA
-
bovine serum albumin
- DSP
-
dithiobis(succinimidyl propionate)
- EDC
-
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
-
- Received December 28, 1998.
- Revision received March 8, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
