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The cytoplasmic dynein 1 cargo binding domain is formed by five subunits including the intermediate chain and the DYNLT, DYNLL, and DYNLRB light chain families. Six isoforms of the intermediate chain and two isoforms of each of the light chain families have been identified in mammals. There is evidence that different subunit isoforms are involved in regulating dynein function, in particular linking dynein to different cargoes. However, it is unclear how the subunit isoforms are assembled or if there is any specificity to their interactions. Co-immunoprecipitation using DYNLT-specific antibodies reveals that dynein complexes with DYNLT light chains also contain the DYNLL and DYNLRB light chains. The DYNLT light chains, but not DYNLL light chains, associate exclusively with the dynein complex. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that both members of the DYNLT family are capable of forming homodimers and heterodimers. In addition, both homodimers of the DYNLT family bind all six intermediate chain isoforms. However, DYNLT heterodimers do not bind to the intermediate chain. Thus, whereas all combinations of DYNLT light chain dimers can be made, not all of the possible combinations of the isoforms are utilized during the assembly of the dynein complex. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein is the motor protein responsible for the intracellular transport of various organelles and other cargoes toward microtubule minus ends. However, it remains to be determined how dynein is regulated to accomplish its varied roles. The dynein complex contains six subunits, including three classes of light chains. The two isoforms of the DYNLT (Tctex1) family of light chains, DYNLT1 and DYNLT3, have been proposed to link dynein to specific cargoes. However, no specific binding partner had been found for the DYNLT3 light chain. We find that DYNLT3 binds to Bub3, a spindle checkpoint protein. Bub3 binds exclusively to DYNLT3 and not to the other dynein light chains. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that Bub3 interacts with the cytoplasmic dynein complex. DYNLT3 is present on kinetochores at prometaphase, but not later mitotic stages, demonstrating that this dynein light chain, like Bub3 and other checkpoint proteins, is depleted from the kinetochore during chromosome alignment. Knockdown of DYNLT3 with small interference RNA increases the mitotic index, in particular, the number of cells in prophase/prometaphase. These results demonstrate that dynein binds directly to a component of the spindle checkpoint complex through the DYNLT3 light chain. Thus, DYNLT3 contributes to dynein cargo binding specificity. These data also suggest that the subpopulation of dynein, containing the DYNLT3 light chain, may be important for chromosome congression, in addition to having a role in the transport of checkpoint proteins from the kinetochore to the spindle pole. [ABSTRACT FROM AUTHOR]

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, LCS, 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. [ABSTRACT FROM AUTHOR]

The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile accepter to link a wide spectrum of molecular cargoes to the dynein motor. [ABSTRACT FROM AUTHOR]

The 8-kDa light chain of dynein (DLC8) is ubiquitously expressed in various cell types. Other than serving as a light chain of the dynein complexes, this highly conserved protein has been shown to bind a larger number of proteins with diverse biological functions. DLC8 forms a homodimer via three-dimensional domain swapping of an internal β-strand (the β2-strand) at neutral pH. The protein undergoes non-reversible dimer-tomonomer dissociation when the pH value of the protein solution decreases. The three-dimensional structure of the DLC8 monomer determined by NMR spectroscopy at pH 3.0 showed that the protein is well folded. The major conformational change accompanied by dimer dissociation is in the β2-strand of the protein, which undergoes transition from a β-strand to a nascent α-helix. The monomer form of DLC8 is not capable of binding to target proteins. Insertion of two flexible amino acid residues in the tight β1/β2-1oop dramatically stabilized the monoruer conformation of the protein. NMR studies showed that the mutation altered the conformation as well as the three-dimensional domain swapping-mediated assembly of the DLC8 dimer. The mutant DLC8 was unable to bind to its targets even at physiological pH. The threedimensional structure of the mutant protein in its monomeric form provides the structural basis of the mutation-induced stabilization of the monomer conformation. Based on the experimental data, we conclude that the formation of the β2-strand swapping-mediated dimer is mandatory for the structure and function of DLC8. We further note that the DLC8 dimer represents a novel mode of three-dimensional domain swapping. [ABSTRACT FROM AUTHOR]