Abstract
Cytoplasmic dynein is the multisubunit protein complex responsible for many microtubule-based intracellular movements. Its cargo binding domain consists of dimers of five subunits: the intermediate chains, the light intermediate chains, and the Tctex1, Roadblock, and LC8 light chains. The intermediate chains have a key role in the dynein complex. They bind the three light chains and the heavy chains, which contain the motor domains, but little is known about how the two intermediate chains interact. There are six intermediate chain isoforms, and it has been hypothesized that different isoforms may regulate specific dynein functions. However, there are little data on the potential combinations of the intermediate chain isoforms in the dynein complexes. We used co-immunoprecipitation analyses to demonstrate that all combinations of homo- and heterodimers of the six intermediate chains are possible. Therefore the formation of dynein complexes with different combinations of isoforms is not limited by interaction between the various intermediate chains. We further sought to identify the domain necessary for the dimerization of the intermediate chains. Analysis of a series of truncation and deletion mutants showed that a 61-amino-acid region is necessary for dimerization of the intermediate chain. This region does not include the N-terminal coiled-coil, the C-terminal WD repeat domain, or the three different binding sites for the Tctex1, LC8, and Roadblock light chains. Analytical gel filtration and covalent cross-linking of purified recombinant polypeptides further demonstrated that the intermediate chains can dimerize in vitro in the absence of the light chains.
Highlights
Cytoplasmic dynein 12 is a motor complex responsible for the transport of membranous vesicles and different cargo proteins toward the
Biochemical analyses showed that cytoplasmic dynein 1 can be fractionated into two complexes, one containing the heavy chains and the light intermediate chains and the second composed of the intermediate chains and three light chain families [25,26,27], but it is not known how the intermediate chains might interact directly or whether their interaction requires one or more of the light chain families
We demonstrate that all the cytoplasmic dynein intermediate chain isoforms are competent to form hetero- and homodimers in vivo
Summary
Cytoplasmic dynein 12 is a motor complex responsible for the transport of membranous vesicles and different cargo proteins toward the. A role for the LC8 light chain as a molecular glue has been suggested by its presence in complexes unrelated to dynein [3, 28] Supporting this hypothesis are the observations that an N-terminal fragment of the intermediate chain becomes more ordered upon binding of the LC8 or Tctex light chains [29, 30]. The region of the intermediate chain necessary for dimerization is shown to be a previously uncharacterized region of 61 amino acids This region is immediately N-terminal to the Roadblock binding region, and it does not overlap with the previously identified intermediate chain subdomains: the N-terminal coiled-coil domain, the C-terminal WD repeat domain, and the three distinct light chain binding domains. In vitro analysis of purified intermediate chain fragments confirmed that none of the light chains are necessary for intermediate chain dimerization
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