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• W2014030046 abstract "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, ROBL-1 andROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from theIC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an ∼72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain. 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, ROBL-1 andROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from theIC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an ∼72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain. intermediate chain light chain amino acids 3-(cyclohexylamino)propanesulfonic acid Cytoplasmic dynein is responsible for transporting a number of different cargoes to the minus-ends of microtubules (1Paschal B.M. Vallee R.B. Nature. 1987; 330: 181-183Crossref PubMed Scopus (414) Google Scholar), including kinetochores, endosomes, lysosomes, and viruses (2Pfarr C.M. Coue M. Grissom P.M. Hays T.S. Porter M.E. McIntosh J.R. Nature. 1990; 345: 263-265Crossref PubMed Scopus (365) Google Scholar, 3Steuer E.R. Wordeman L. Schroer T.A. Sheetz M.P. Nature. 1990; 345: 266-268Crossref PubMed Scopus (401) Google Scholar, 4Harada A. Takei Y. Kanai Y. Tanaka Y. Nonaka S. Hirokawa N. J. Cell Biol. 1998; 141: 51-59Crossref PubMed Scopus (286) Google Scholar, 5Lin S.X. Collins C.A. J. Cell Sci. 1992; 101: 125-137Crossref PubMed Google Scholar, 6Leopold P.L. Kreitzer G. Miyazawa N. Rempel S. Pfister K.K. Rodriguez-Boulan E. Crystal R.G. Hum. Gene Ther. 2000; 11: 151-165Crossref PubMed Scopus (216) Google Scholar, 7Ye G.J. Vaughan K.T. Vallee R.B. Roizman B. J. Virol. 2000; 74: 1355-1363Crossref PubMed Scopus (144) Google Scholar). In addition, cytoplasmic dynein has been implicated in the positioning of the Golgi apparatus, the assembly and orientation of the mitotic spindle, and the transport of microtubules (8Corthesy-Theulaz I. Pauloin A. Rfeffer S.R. J. Cell Biol. 1992; 118: 1333-1345Crossref PubMed Scopus (239) Google Scholar, 9Fath K.R. Trimbur G.M. Burgess D.R. J. Cell Biol. 1994; 126: 661-675Crossref PubMed Scopus (176) Google Scholar, 10Heald R. Tournebize R. Blank T. Sandaltzopoulos R. Becker P. Hyman A. Karsenti E. Nature. 1996; 382: 420-425Crossref PubMed Scopus (789) Google Scholar, 11Gaglio T. Dionne M.A. Compton D.A. J. Cell Biol. 1997; 138: 1055-1066Crossref PubMed Scopus (181) Google Scholar, 12Dillman J.F. Dabney L.P. Pfister K.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 141-144Crossref PubMed Scopus (101) Google Scholar, 13Ahmad F.J. Echeverri C.J. Vallee R.B. Baas P.W. J. Cell Biol. 1998; 140: 391-401Crossref PubMed Scopus (199) Google Scholar). 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 (14Vallee R.B. Shpetner H.S. Paschal B.M. Trends Neurosci. 1989; 12: 66-70Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 15Susalka S.J. Hancock W.O. Pfister K.K. Biochim. Biophys. Acta. 2000; 1496: 76-88Crossref PubMed Scopus (46) Google Scholar). 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 (16Gee M.A. Heuser J.E. Vallee R.B. Nature. 1997; 390: 636-639Crossref PubMed Scopus (266) Google Scholar). The major form of cytoplasmic dynein contains two identical heavy chains per complex, each with a molecular weight of ∼530,000. In contrast, the cargo-binding domain is comprised of multiple isoforms of the intermediate chains, light intermediate chains, and light chains. There are two intermediate chains per complex and these subunits have a molecular weight of ∼74,000. In mammals, there are two intermediate chain genes,IC74-11 andIC74-2. Alternative splicing and phosphorylation produce multiple intermediate chain isoforms (17Pfister K.K. Salata M.W. Dillman J.F. Vaughan K.T. Vallee R.B. Torre E. Lye R.J. J. Biol. Chem. 1996; 271: 1687-1694Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 18Vaughan K.T. Vallee R.B. J. Cell Biol. 1995; 131: 1507-1516Crossref PubMed Scopus (398) Google Scholar, 19Pfister K.K. Salata M.W. Dillman J.F. Torre E. Lye R.J. Mol. Biol. Cell. 1996; 7: 331-343Crossref PubMed Scopus (55) Google Scholar). The intermediate chains bind to the p150Glued subunit of the accessory complex, dynactin (18Vaughan K.T. Vallee R.B. J. Cell Biol. 1995; 131: 1507-1516Crossref PubMed Scopus (398) Google Scholar, 20Karki S. Holzbaur E.L. J. Biol. Chem. 1995; 270: 28806-28811Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). There are also multiple isoforms of the light intermediate chains with molecular weights of 50,000–60,000, which are the products of two light intermediate chain genes, LIC1 andLIC2 (21Gill S.R. Cleveland D.W. Schroer T.A. Mol. Biol. Cell. 1994; 5: 645-654Crossref PubMed Scopus (78) Google Scholar, 22Hughes S.M. Vaughan K.T. Herskovits J.S. Vallee R.B. J. Cell Sci. 1995; 108: 17-24PubMed Google Scholar). Three different mammalian light chain families were recently identified with molecular weights between 10,000 and 14,000: 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 (23King S.M. Dillman J.F. Benashski S.E. Lye R.J. Patel-King R.S. Pfister K.K. J. Biol. Chem. 1996; 271: 32281-32287Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24King S.M. Barbarese E. Dillman J.F. Benashski S.E., Do, K.T. Patel-King R.S. Pfister K.K. Biochemistry. 1998; 37: 15033-15041Crossref PubMed Scopus (99) Google Scholar). Tctex1 and rp3 family members are differentially expressed in various tissues (24King S.M. Barbarese E. Dillman J.F. Benashski S.E., Do, K.T. Patel-King R.S. Pfister K.K. Biochemistry. 1998; 37: 15033-15041Crossref PubMed Scopus (99) Google Scholar). Furthermore, Tctex1, but not rp3, binds to rhodopsin (25Tai A.W. Chuang J.Z. Bode C. Wolfrum U. Sung C.H. Cell. 1999; 97: 877-887Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 26Tai A.W. Chuang J.Z. Sung C.H. J. Cell Biol. 2001; 153: 1499-1509Crossref PubMed Scopus (106) Google Scholar). In addition to Tctex1 and rp3, there are also several more distantly related members of this family (27DiBella L.M. Benashski S.E. Tedford H.W. Harrison A. Patel-King R.S. King S.M. J. Biol. Chem. 2001; 276: 14366-14373Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The LC8 light chain family is comprised of three different members, LC8a, LC8b, and LC8c (28Wilson M.J. Salata M.W. Susalka S.J. Pfister K.K. Cell Motil. Cytoskeleton. 2001; 49: 229-240Crossref PubMed Scopus (41) Google Scholar). Yeast two-hybrid screens have found multiple binding partners to LC8 family members including neuronal nitric-oxide synthase (29Jaffrey S.R. Snyder S.H. Science. 1996; 274: 774-777Crossref PubMed Scopus (427) Google Scholar) and the BCL-2 family protein, BIM (30Puthalakath H. Huang D.C. O'Reilly L.A. King S.M. Strasser A. Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar). The roadblock light chain family is comprised of two members, roadblock-1 and roadblock-2 (31Bowman A.B. Patel-King R.S. Benashski S.E. McCaffery J.M. Goldstein L.S. King S.M. J. Cell Biol. 1999; 146: 165-180Crossref PubMed Scopus (148) Google Scholar). Mutations of the roadblock gene inDrosophila exhibited defects in both axonal transport and mitosis (31Bowman A.B. Patel-King R.S. Benashski S.E. McCaffery J.M. Goldstein L.S. King S.M. J. Cell Biol. 1999; 146: 165-180Crossref PubMed Scopus (148) Google Scholar). Both Tctex1 and LC8 family members were shown to bind to the intermediate chains of cytoplasmic dynein (32Mok Y.K., Lo, K.W. Zhang M. J. Biol. Chem. 2001; 276: 14067-14074Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 33Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 34Makokha M. Hare M., Li, M. Hays T. Barbar E. Biochemistry. 2002; 41: 4302-4311Crossref PubMed Scopus (92) Google Scholar, 35King S.J. Bonilla M. Rodgers M.E. Schroer T.A. Protein Sci. 2002; 11: 1239-1250Crossref PubMed Scopus (69) Google Scholar). The binding sites for these two light chains were found to be just C-terminal of the second alternative splice region in the intermediate chain gene, indicating that Tctex1 and LC8 could bind any of the intermediate chain isoforms. In addition, the binding sites for both light chains on the intermediate chain were found to be distinct, implying that both Tctex1 and LC8 could bind to the same intermediate chain. In this study, we sought to complete the analysis of the organization of the cytoplasmic dynein complex by determining how the roadblock light chains were 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 IC74-1 and IC74-2 intermediate chain isoforms. We have mapped the binding region to an ∼72 amino acid domain of the intermediate chain, of which only a 40-amino acid portion was 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. The human roadblock sequences (31Bowman A.B. Patel-King R.S. Benashski S.E. McCaffery J.M. Goldstein L.S. King S.M. J. Cell Biol. 1999; 146: 165-180Crossref PubMed Scopus (148) Google Scholar) 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 theSmaI site. Roadblock-2 was amplified from an EST (ATCC number 1994230) using roadblock-2-specific primers and similarly introduced into the pBluescript M13− vector. 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 vectors (CLONTECH). HF-7C yeast were co-transformed according to the Gietz LiAc Yeast Transformation protocol (36Gietz R.D. Woods R.A. BioTechniques. 2001; 30 (, 822–826, 828): 816-820Crossref PubMed Scopus (146) Google Scholar). Best results were obtained when the heat shock step was increased to 2 h at 42 °C. The yeast were resuspended in 100 μl of sterile water and 15 μl were dropped onto both −2 and −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 HIS3pathway, 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 confirmed with a β-galactosidase assay that screened for the independent expression of the lacZ reporter gene. 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 pairwise yeast two-hybrid assay. A T7 promoter and Kozak sequence were introduced upstream of human roadblock-1, roadblock-2, and Tctex1 cDNA by PCR. The resulting cDNA was purified and used as the template in the TnT coupled wheat germ extract system (Promega) according to the manufacturer’s directions. The amino acid mixture used was lacking in methionine and 40 μCi of translational grade [35S]methionine (Amersham Biosciences) was added per 50-μl reaction. Best results were obtained when the reaction was incubated for 2 h at 30 °C. The design and purification of full-length IC74-1A, IC74-2C, and the truncations is described elsewhere (18Vaughan K.T. Vallee R.B. J. Cell Biol. 1995; 131: 1507-1516Crossref PubMed Scopus (398) Google Scholar, 37Vaughan P.S. Leszyk J.D. Vaughan K.T. J. Biol. Chem. 2001; 276: 26171-26179Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). These constructs and purified bovine brain cytoplasmic dynein (38Dillman III, J.F. Pfister K.K. J. Cell Biol. 1994; 127: 1671-1681Crossref PubMed Scopus (161) Google Scholar) were resolved on 4–16 or 4–20% gradient mini-gels and transferred to a 0.2-μm polyvinylidene difluoride membrane (Bio-Rad) in a Hoeffer blotting apparatus using 10 mm CAPS buffer, pH 11 (Calbiochem). The blot overlay assay was as previously described (18Vaughan K.T. Vallee R.B. J. Cell Biol. 1995; 131: 1507-1516Crossref PubMed Scopus (398) Google Scholar) except in vitro synthesized radiolabeled light chain was used to probe the blot. The blot was then placed in a PhosphoImager cassette (Amersham Biosciences) and incubated at room temperature for approximately 1 week. The phosphorimage was analyzed with ImageQuant software. The location of the IC74-1A fragments was confirmed by screening the blot with the 74.1 intermediate chain antibody using enhanced chemical luminescence (ECL) (Pierce). 22.5 μl of in vitro synthesized radiolabeled light chain was incubated with 60 μg of either full-length IC74-2C, a 1–125-aa fragment of IC74-2C, or no intermediate chain. The mixture was next incubated on a shaker at 4 °C for 2 h. The mixture was then added to Protein A beads (Zymed Laboratories Inc.) coupled with a pan intermediate chain antibody, 74.1, and incubated for 3 h on a shaker at 4 °C (38Dillman III, J.F. Pfister K.K. J. Cell Biol. 1994; 127: 1671-1681Crossref PubMed Scopus (161) Google Scholar). The beads were washed twice with wash buffer (150 mm NaCl, 1 m Tris, pH 8.1, 5 mm EDTA) and then once with deionized water. The beads were resuspended in 50 μl of ×2 Laemmli sample buffer and 30 μl was resolved on a 4–20% gradient mini-gel (ISC Bioexpress). The gel was then dried for 3 h at 70 °C (Bio-Rad model 583). The dried gel was then placed in a phosphorimage cassette for approximately 1 week. We recently identified roadblock-1 as a component of purified bovine brain cytoplasmic dynein (28Wilson M.J. Salata M.W. Susalka S.J. Pfister K.K. Cell Motil. Cytoskeleton. 2001; 49: 229-240Crossref PubMed Scopus (41) Google Scholar). However, how the roadblock-1 light chain was incorporated into the cytoplasmic dynein complex was unknown. Three pieces of evidence led us to hypothesize that roadblock-1 binds to the intermediate chains of cytoplasmic dynein. First, previous experiments demonstrated that the intermediate chain base of the cytoplasmic dynein complex could be separated biochemically from the heavy chain motor domain (21Gill S.R. Cleveland D.W. Schroer T.A. Mol. Biol. Cell. 1994; 5: 645-654Crossref PubMed Scopus (78) Google Scholar). Analysis of the fractionated cytoplasmic dynein subunits revealed that Tctex1 and LC8 light chains copurify with the intermediate chains on a sucrose gradient (24King S.M. Barbarese E. Dillman J.F. Benashski S.E., Do, K.T. Patel-King R.S. Pfister K.K. Biochemistry. 1998; 37: 15033-15041Crossref PubMed Scopus (99) Google Scholar). Similarly, the roadblock-related protein, LC7, is part of the IC-LC complex in the flagellar dynein (39Pfister K.K. Witman G.B. J. Biol. Chem. 1984; 259: 12072-12080Abstract Full Text PDF PubMed Google Scholar, 40Mitchell D.R. Rosenbaum J.L. Cell Motil. Cytoskeleton. 1986; 6: 510-520Crossref PubMed Scopus (59) Google Scholar). Third, both the Tctex1 and LC8 light chains have been recently demonstrated to bind directly to intermediate chains (32Mok Y.K., Lo, K.W. Zhang M. J. Biol. Chem. 2001; 276: 14067-14074Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 33Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 34Makokha M. Hare M., Li, M. Hays T. Barbar E. Biochemistry. 2002; 41: 4302-4311Crossref PubMed Scopus (92) Google Scholar, 35King S.J. Bonilla M. Rodgers M.E. Schroer T.A. Protein Sci. 2002; 11: 1239-1250Crossref PubMed Scopus (69) Google Scholar). 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 (18Vaughan K.T. Vallee R.B. J. Cell Biol. 1995; 131: 1507-1516Crossref PubMed Scopus (398) Google Scholar). The intermediate chain genes were 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 β-galactosidase assay (not shown). This experiment confirmed our hypothesis that the roadblock-1 light chain interacts with the intermediate chains. To confirm the in vivo results that roadblock-1 binds to the intermediate chains of cytoplasmic dynein, we utilized an in vitro solid phase blot overlay assay. Purified bovine brain cytoplasmic dynein, which contains all IC74-1 and IC74-2 intermediate chain isoforms, was resolved with SDS-PAGE and blotted to polyvinylidene difluoride. The blot was incubated with 35S-labeled roadblock-1 synthesized in vitro (Fig. 2A). Roadblock-1 specifically interacted with a band of the approximate weight of the intermediate chains of cytoplasmic dynein (Fig.2B). This radiolabeled band was identified as the cytoplasmic dynein intermediate chain by immunochemistry. The other roadblock family member, roadblock-2, had not previously been shown to be a component of cytoplasmic dynein. 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 (32Mok Y.K., Lo, K.W. Zhang M. J. Biol. Chem. 2001; 276: 14067-14074Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Some low level nonspecific 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. 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 mouse brain yeast two-hybrid library (Fig. 3). This approach also allowed us to determine whether 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 (27DiBella L.M. Benashski S.E. Tedford H.W. Harrison A. Patel-King R.S. King S.M. J. Biol. Chem. 2001; 276: 14366-14373Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Interactions between both roadblock light chains and intermediate chains IC74-2A, IC74-2B, and IC74-1 fragment were identified in the screen. The IC74-1 fragment was missing the first N-terminal 187 amino acids, a segment that includes the regions that were alternatively spliced to produce either IC74-1A or IC74-1B. This indicates that both roadblock light chains were capable of binding either IC74-1A or IC74-1B. A second construct was also identified that was only missing the first 43 amino acids and was determined to be IC74-1A (not shown). This indicated that the region of roadblock binding was found between amino acids 187 and 643 (the end) of the IC74-1 intermediate chain. In this screen, no colonies encoding cytoplasmic dynein heavy chain, light intermediate chains, or members of the Tctex1 and LC8 family were identified. However, roadblock light chain was identified as a binding partner, demonstrating that these light chains self-associate. 2K. Nikulina and S. M. King, manuscript in preparation. The fact that both roadblock light chains bound a C-terminal IC-1 fragment in the yeast two-hybrid assay suggested that the binding domain for roadblock-1 and roadblock-2 was downstream of both the Tctex1- and LC8-binding sites. To further define the interaction of the roadblock light chains with the intermediate chain, an in vitro solution-binding assay was used. For these experiments, we used the ubiquitously expressed IC74-2C intermediate chain (41Susalka S.J. Pfister K.K. J. Neurocytol. 2000; 29: 819-829Crossref PubMed Scopus (61) Google Scholar). The IC74-2C isoform has the two alternative splicing regions removed. IC74-2C is found in all mammalian cell types, and in many cultured cells it is the only intermediate chain detected. Bacterially expressed IC74-2C intermediate chain and a 1–125-aa fragment of IC74-2C were purified from bacterial lysates (18Vaughan K.T. Vallee R.B. J. Cell Biol. 1995; 131: 1507-1516Crossref PubMed Scopus (398) Google Scholar). Note that the 1–125-aa fragment of IC74-2C is equivalent to a 1–159-aa fragment of IC74-1A, which contains both splicing regions.35S-Labeled light chains, roadblock-1, roadblock-2, and Tctex1, were synthesized in vitro and separately incubated with the intermediate chain or intermediate chain fragment. The pan intermediate chain antibody, 74.1, was used to immunoprecipitate the intermediate chain and any bound light chain. The epitope for the 74.1 antibody is located in the first 60 amino acids of the intermediate chain (not shown). The immunoprecipitates were resolved by SDS-PAGE, and the dried gel was analyzed by autoradiography. Note that antibody binding does not block the binding of either the roadblock light chains or Tctex1 to the intermediate chain. Both roadblock-1 and roadblock-2 co-immunoprecipitated only with the full-length intermediate chain. This demonstrates that they directly bind to IC74-2C and it confirms the yeast two-hybrid assay results (Fig.4). Because neither roadblock-1 nor roadblock-2 bound to the 1–125-aa fragment of IC74-2C, the roadblock-binding domain was located between amino acid 125 of IC74-2C and the C terminus (amino acid 612) of the intermediate chain. In contrast, Tctex1 co-immunoprecipitated with full-length IC74-2C and the 1–125 fragment of IC74-2C. This is consistent with published results that indicate that the Tctex1-binding site on IC74-2C is found between amino acids 106 and 123 (32Mok Y.K., Lo, K.W. Zhang M. J. Biol. Chem. 2001; 276: 14067-14074Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). It had previously been reported thatin vitro synthesized Tctex1 was immunoprecipitated with endogenous intermediate chain present in an in vitrotranslation mixture (26Tai A.W. Chuang J.Z. Sung C.H. J. Cell Biol. 2001; 153: 1499-1509Crossref PubMed Scopus (106) Google Scholar). However, when the wheat germ extract was 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 (42Lawrence C.J. Morris N.R. Meagher R.B. Dawe R.K. Traffic. 2001; 2: 362-363Crossref PubMed Scopus (87) Google Scholar). 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. To further define the roadblock-binding domain on the cytoplasmic dynein intermediate chain, we utilized an IC74-1A truncation series in the blot overlay assay. All constructs contained the N-terminal, which is recognized by the 74.1 intermediate chain antibody (18Vaughan K.T. Vallee R.B. J. Cell Biol. 1995; 131: 1507-1516Crossref PubMed Scopus (398) Google Scholar). The IC74-1A constructs were resolved by SDS-PAGE and transferred to polyvinylidene difluoride. To confirm the location of the intermediate chain fragments, the blot was probed with the 74.1 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 (18Vaughan K.T. Vallee R.B. J. Cell Biol. 1995; 131: 1507-1516Crossref PubMed Scopus (398) Google Scholar). The blots were then incubated with either 35S-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 1–404-aa fragment, and the 1–314-aa fragment. The intensity of each of these three bands appears to be equivalent. Roadblock-1 did not bind to the 1–242-, 1–228-, 1–150-, or the 1–123-aa fragments. This indicates that the roadblock-binding domain on the intermediate chain is between amino acids 243 and 314. In contrast, Tctex1 bound to full-length IC74-1A, 1–404-aa fragment, 1–242-aa fragment, and the 1–228-aa fragment. Tctex1 did not bind to either the 1–150- or 1–123-aa fragment. The results from this blot overlay assay indicate that the Tctex1-binding site is between 123 and 228 aa on the intermediate chain. This is consistent with recent results that indicate that the Tctex1-binding site on IC74-1A is between amino acids 140 and 157 (32Mok Y.K., Lo, K.W. Zhang M. J. Biol. Chem. 2001; 276: 14067-14074Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Surprisingly, Tctex1 consistently did not interact with the 1–314-aa IC741A fragment. This could be because of the 1–314-aa fragment not renaturing properly for Tctex1 binding, because Tctex1 bound both the smaller 1–242-aa fragment and the 1–228-aa fragment. The results of this experiment identify a roadblock-binding domain on the intermediate chain that is significantly downstream of both the Tctex1- and LC8-binding sites. 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 both roadblock-1 and roadblock-2 interact with all five known intermediate chain subunits, IC74-1A, IC74-1B, IC74-2A, IC74-2B, and IC74-2C. Three complementary methods were utilized to demonstrate that the two mammalian roadblock light chains bind the cytoplasmic dynein intermediate chain. Both roadblock-1 and roadblock-2 interact with the intermediate chain isoforms from theIC74-1" @default.
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