Identification of a novel region of the cytoplasmic Dynein intermediate chain important for dimerization in the absence of the light chains.
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.
Lo
Kevin W-H
KW
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.
Kan
Ho-Man
HM
Pfister
K Kevin
KK
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry; D019281:Dimerization; D004398:Dyneins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D047468:Immunoprecipitation; D010446:Peptide Fragments; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary
D002352:Carrier Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D004398:Dyneins