The Roadblock Light Chain Binds a Novel Region of the Cytoplasmic Dynein Intermediate Chain

this study, we sought to complete the analysis of the organization of the cytoplasmic dynein complex by determining how the roadblock light chains incorporated into the complex. We report here that both mammalian roadblock light chains, roadblock-1 and roadblock-2, bind directly to the intermediate chains of cytoplasmic dynein. The hypothesis that individual roadblock light chains specifically bound to different intermediate chain isoforms was also tested. It was found that both roadblock light chains bind all IC74-1and IC74-2 intermediate chain isoforms. We have mapped the binding region to an approximately 73 amino acid domain of the intermediate chain, of which only a 53 amino acid portion is hypothesized to be capable of binding the roadblock light chain. This domain is located significantly downstream of the previously reported Tctex1 and LC8 binding sites, indicating that a single intermediate chain could bind each of the three light chain families.


Summary
Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, roadblock-1 and roadblock-2. We find that both members
The cytoplasmic dynein complex is comprised of a motor domain and a cargo-binding domain, both of which are crucial for the proper functioning of the motor complex (14,15). Each domain is comprised of different cytoplasmic dynein subunits. The heavy chains of cytoplasmic dynein comprise the motor domain. This domain consists of both a microtubule binding site and an ATP hydrolysis site that is required to generate the power necessary to move the complex along the microtubule (16). The major form of cytoplasmic dynein contains two identical heavy chains per complex, each with a molecular weight of ~530 kDa. In contrast, the cargo-binding domain is comprised of  (21,22).
Three different mammalian light chain families were recently identified with molecular weights between 10 and 14 kDa: Tctex1, LC8, and roadblock. Unlike the two intermediate chain genes, which are closely related, each of the three light chain families has a distinct primary sequence. The Tctex1 light chain family is composed of two related members, Tctex1 and rp3 (23,24). Tctex1 and rp3 family members are differentially expressed in various tissues (24). Furthermore, Tctex1, but not rp3, binds to rhodopsin (25,26). In addition to Tctex1 and rp3, there are also several more distantly related members of this family (27). The LC8 light chain family is comprised of three different members, LC8a, LC8b, and LC8c (28). Yeast two-hybrid screens have found multiple binding partners to LC8 family members including neuronal nitric oxide synthase (29) and the BCL-2 family protein, BIM (30). The roadblock light chain family is comprised of two members, roadblock-1 and roadblock-2 (31). Mutations of the roadblock gene in Drosophila exhibited defects in both axonal transport and mitosis (31

Experimental Procedures
Cloning of the human roadblock light chains. The human roadblock sequences (31) were used to design primers specific for roadblock-1 and roadblock-2. Roadblock-1 was amplified out of a human brain cDNA library by PCR using roadblock-1 specific primers and then inserted into pBluescript M13-vector (Stratagene) using the Sma1 site.
Roadblock-2 was amplified from an EST (ATCC #1994230) using roadblock-2 specific primers and similarly introduced into the pBluescript M13-vector.
Yeast two-hybrid assay. Human roadblock-1 was subcloned into the pCR vector (Clontech) after EcoRI and XmaI restriction sites were introduced by PCR mutagenesis.
Roadblock-1 was then digested out of the pCR vector and ligated into the pGADT7 activation domain vector of the Matchmaker system (Clontech). Rat intermediate chain constructs were similarly introduced into either the pGBKT7 or pGBT9 DNA-binding domain vector (Clontech). HF-7C yeast were co-transformed according to the Gietz LiAc Yeast Transformation protocol (36). Best results were obtained when the heat shock step was increased to two hours at 42° C. The yeast were resuspended in 100 µl of sterile water and 15 µl were dropped onto both minus (-) 2 and minus (-) 3 plates. The -2 plates, lacking both leucine (Leu) and tryptophan (Trp), showed co-transformation.
The -3 plates, lacking Leu, Trp and histidine (His), demonstrate protein-protein interactions. To eliminate false positives arising from "leaky" HIS3 expression, 5 mM of a competitive inhibitor of the HIS3 pathway, 3-AT (3-amino-1,2,4-triazole), was added to the -3 plates. The plates were incubated at 30° C for 5-8 days. Positive interactions were 7 confirmed with a $-galactosidase assay that screened for the independent expression of the lacZ reporter gene.
Yeast two-hybrid library screen. Mouse roadblock-1 and roadblock-2 were cloned into a "bait" pAS2 DNA-binding domain vector (Clontech) and used to screen a mouse brain "prey" library. Sequencing of the individual "prey" plasmids and using the sequences to screen appropriate databases identified candidate roadblock light chain binding partners.
The interaction of roadblock-1 and roadblock-2 and the candidate "prey" proteins was then verified by rescreening in a pair-wise yeast two-hybrid assay. Blot Overlay assay. The design and purification of full length IC74-1A, IC74-2C, and the truncations is described elsewhere (18,37). These constructs and purified bovine brain cytoplasmic dynein (38) were resolved on 4-16% or 4-20% gradient mini gels and transferred to 0.2 µm polyvinylidene difluoride (PVDF) membrane (BioRad) in a Hoeffer blotting apparatus using 10 mM CAPS buffer pH 11 (Calbiochem). The blot overlay assay was as previously described (18) except in vitro synthesized radiolabeled light chain was used to probe the blot. The blot was then placed in a Phosphoimage cassette (Molecular Dynamics) and incubated at room temperature for approximately one week.
The phosphoimage was analyzed with ImageQuant software. The location of the IC74-   (24). Similarly, the roadblock related protein, LC7, is part of the IC-LC complex in flagellar dynein (39,40). Thirdly, both the Tctex1 and LC8 light chains have been recently demonstrated to bind directly to the intermediate chains (32)(33)(34)(35). To determine whether the roadblock-1 light chain also binds to the intermediate chains of cytoplasmic dynein, a yeast two-hybrid assay was used. A roadblock-1/pGADT7 construct and three intermediate chain yeast two-hybrid constructs (either pGBT9 or pGBKT7) were made. There are two different cytoplasmic dynein intermediate chain genes (18). The intermediate chain genes are alternatively spliced to produce a number of different isoforms. We screened the binding of roadblock-1 to one IC74-1 product, IC74-1B, and two IC74-2 products, IC74-2B and IC74-2C. The results indicate that roadblock-1 interacts with each of these intermediate chains (Fig. 1). The interactions were confirmed with a $-galactosidase assay (not shown). This experiment confirmed our hypothesis that the roadblock-1 light chain interacts with the intermediate chains. Using the blot overlay assay, we determined that roadblock-2 also binds specifically to the intermediate chains of cytoplasmic dynein. As a positive control, we used the Tctex1 light chain, which has been shown to directly bind to the intermediate chains (32). Some low level non-specific binding in the 45-60 kDa region of the blot was observed for the three light chains. These blot overlay results confirm that roadblock-1 interacts with the intermediate chain and also demonstrates that the related family member, roadblock-2, interacts with cytoplasmic dynein intermediate chain.

Roadblock-1 and roadblock-2 bind multiple intermediate chains in a yeast two-
hybrid assay. To further confirm the interaction between the roadblock light chains and the intermediate chains, we used both roadblock-1 and roadblock-2 to screen a brain yeast two-hybrid library (Fig. 3). This approach also allowed us to determine if roadblock-1 and roadblock-2 interacted with any other subunits of cytoplasmic dynein. This library has previously been shown to contain the Tctex1 and LC8 light chains (27).  (18). Note that the 1-125aa fragment of IC74-2C is equivalent to a 1-159aa fragment of IC74-1A, which contains both splicing regions. 35 (26). However, when the wheat germ extract is used for the in vitro synthesis reaction we found no immunoprecipitation of either Tctex1 or the two roadblock light chains without the addition of exogenous intermediate chain (Fig. 4). This is consistent with findings that the cytoplasmic dynein intermediate chain, and other dynein subunits, cannot be identified in plant databases (42). The results from this experiment indicate that both roadblock light chains bind IC74-2C in vitro and that the roadblock binding site is C-terminal to the Tctex1 site. intermediate chain antibody (Fig. 5A). Each of the six intermediate chain constructs was identified as well as smaller fragments that are the products of premature translation termination (18). The blots were then incubated with either 35 S labeled roadblock-1 or Tctex1 and the binding of the light chain to the fragments was determined by autoradiography (Fig. 5). Roadblock-1 strongly bound to full length IC74-1A, the 1-404aa fragment, and the 1-314aa fragment. The intensity of each of these three bands appears to be equivalent. Roadblock-1 did not bind to the 1-242aa, 1-228aa, 1-150aa, or the 1-123aa fragments. This indicates that the roadblock binding domain on the intermediate chain is between amino acids 242 and 314. In contrast, Tctex1 bound to full length IC74-1A, the 1-404aa fragment, the 1-242aa fragment, and the 1-228aa fragment.
Tctex1 did not bind to either the 1-150aa or the 1-123aa fragment. The results from this blot overlay assay indicate that the Tctex1 binding site is between 123aa and 228aa on the intermediate chain. This is consistent with recent results that indicate that the Tctex1 binding site on IC74-1A is between amino acids 139-157 (32). Surprisingly, Tctex1 consistently did not interact with the 1-314aa IC741A fragment. This could be due to the

Discussion
Prior to this work, the roadblock light chain family was the only subunit whose location in the cytoplasmic dynein complex was unknown. The data in this report establish that Consistent with this hypothesis, each of these subunits has multiple isoforms and many appear to be responsible for particular functions. Both Tctex1 and the LC8 light chains have been identified as binding partners in many yeast two-hybrid assays (29,(55)(56)(57). We demonstrate that both roadblock light chains bind to all the intermediate chain isoforms. Previously, it has only been hypothesized that the Tctex1 and LC8 cytoplasmic dynein light chains bind both IC74-1 and IC74-2 intermediate chain isoforms (32,33). In this study, we confirm part of this hypothesis by determining that Tctex1 directly binds to both IC74-1A and IC74-2C. It is thought that cytoplasmic dyneins with the different intermediate chain isoforms are responsible for specific cellular functions. For example, the IC74-2C isoform is ubiquitous and in many cells is sufficient for constitutive dynein function (17,19). The distribution of other intermediate chains is more restricted, for example, IC74-1A is only found in neurons, suggesting it has a neuron specific function. This hypothesis is supported by the observation that the expression of IC74-1A and the other intermediate chain isoforms are developmentally regulated in brain, and that the changes in expression levels occur just prior to axon extension (17,19,61). Changes in the relative expression levels of the roadblock light chains have also been observed. In hepatocellular carcinoma tissue, the expression levels of roadblock-1 and roadblock-2 are altered relative to control tissue samples (62). In addition, the roadblock-1 light chain is highly expressed in the visual cortex and upon sensory stimulation, its mRNA and protein levels are rapidly down regulated (63). It is thus possible that independent regulation of the expression levels of the different interacting cytoplasmic dynein subunit isoforms creates different domains for specific cargo binding or dynein regulation.
It has also been suggested that the light chains may serve as an intramolecular glue responsible for keeping the cytoplasmic dynein complex intact. Since there are both two members of each light chain family, and two intermediate chains per complex, it is possible that light chain dimers reinforce the integrity of the cargo-binding base (27,28,34 (18,20,64,65). We report here that both    Purified bovine brain cytoplasmic dynein was resolved on a 4-16% gradient SDS-polyacrylamide gel and transferred to PVDF. The blots were incubated with 35 S labeled light chain, either Tctex1, roadblock-1 (RB-1) or roadblock-2 (RB-2), and exposed to a phosphor screen. The autoradiograph of each blot is shown. To confirm that it was the intermediate chain (IC) that was specifically recognized, a blot was screened with the  C. Schematic diagram of the -2 and -3 plates. Co-transformation with pACT2 and pAS2 [1] was used as a positive control for the -2 co-transformation plate and as a negative control for the -3 interaction plate. A positive control for both the -2 and -3 plates was the direct interaction of pVA3-1 + pTD1-1 [2]. Roadblock-1 and roadblock-2 were cloned into the pAS2 vector and used to screen a brain library. Roadblock-1 [3][4][5] and roadblock-2 [7][8][9] were found to bind to IC74-2A, IC74-2B, and an IC74-1 fragment.
The IC74-1 fragment was sequenced and found to be missing the first 187aa. This indicates that this N-terminal 187aa region does not contain the roadblock binding site.