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• W1993764616 abstract "We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995)J. Cell Biol. 131, 1275–1290). To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an ∼2.8-fold higher affinity than to Tyr microtubules. Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of ∼120 kDa and a light chain of ∼64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules. We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995)J. Cell Biol. 131, 1275–1290). To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an ∼2.8-fold higher affinity than to Tyr microtubules. Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of ∼120 kDa and a light chain of ∼64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules. Microtubules (MTs) 1The abbreviations used are: MTs, microtubules; Glu MTs, detyrosinated microtubules; Tyr MTs, tyrosinated microtubules; IF, intermediate filament; mAb, monoclonal antibody; Pipes, 1,4-piperazinediethanesulfonic acid; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; PAGE, polyacrylamide gel electrophoresis; MAP, microtubule-associated protein. are highly dynamic structures that are present in nearly every eukaryotic cell. During cellular polarization and differentiation, MTs become stabilized, and the tubulin subunits composing these MTs become post-translationally modified. One of these modifications is detyrosination, which involves the removal of the C-terminal tyrosine residue from α-tubulin (2Argarana C.E. Barra H.S. Caputto R. Mol. Cell. Biochem. 1978; 19: 17-21Crossref PubMed Scopus (107) Google Scholar). This generates α-tubulin with a glutamate residue at the C terminus. The two forms of α-tubulin are called Tyr and Glu tubulin after their respective C termini (3Gundersen G.G. Kalnoski M.H. Bulinski J.C. Cell. 1984; 38: 779-789Abstract Full Text PDF PubMed Scopus (381) Google Scholar). Even though tubulin detyrosination accompanies the stabilization of MTs (4Schulze E. Asai D.J. Bulinski J.C. Kirschner M. J. Cell Biol. 1987; 105: 2167-2177Crossref PubMed Scopus (288) Google Scholar, 5Khawaja S. Gundersen G.G. Bulinski J.C. J. Cell Biol. 1988; 106: 141-149Crossref PubMed Scopus (209) Google Scholar, 6Webster D.R. Gundersen G.G. Bulinski J.C. Borisy G.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9040-9044Crossref PubMed Scopus (189) Google Scholar, 7Gundersen G.G. Khawaja S. Bulinski J.C. J. Cell Biol. 1989; 109: 2275-2288Crossref PubMed Scopus (117) Google Scholar, 8Gundersen G.G. Bulinski J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 90: 8827-8831Google Scholar, 9Gundersen G.G. Khawaja S. Bulinski J.C. J. Cell Biol. 1987; 105: 251-264Crossref PubMed Scopus (220) Google Scholar), several studies have demonstrated that the elevated Glu tubulin level is not responsible for the increased stability of Glu MTs (5Khawaja S. Gundersen G.G. Bulinski J.C. J. Cell Biol. 1988; 106: 141-149Crossref PubMed Scopus (209) Google Scholar, 6Webster D.R. Gundersen G.G. Bulinski J.C. Borisy G.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9040-9044Crossref PubMed Scopus (189) Google Scholar,10Kumar N. Flavin M. Eur. J. Biochem. 1982; 128: 215-222Crossref PubMed Scopus (37) Google Scholar). Nonetheless, tubulin detyrosination can serve as a marker for stable MTs in vivo. The role of enhanced tubulin modification in MT function had remained elusive until recently, when studies from this laboratory showed that stable Glu MTs function to localize vimentin intermediate filaments (IFs) in polarized fibroblasts (1Gurland G. Gundersen G.G. J. Cell Biol. 1995; 131: 1275-1290Crossref PubMed Scopus (132) Google Scholar). In this study, IFs were found to coalign with Glu MTs, and this coalignment could be disrupted by microinjection of affinity-purified antibodies to Glu tubulin, but not to Tyr tubulin. In a more recent study, microinjection of nonpolymerizable Glu tubulin, but not Tyr tubulin, into the cytoplasm disrupted the distribution of IFs without affecting the level of stable MTs. 2G. Kreitzer and G. G. Gundersen, submitted for publication. These results conclusively demonstrate that tubulin detyrosination, rather than increased MT stability, is responsible for the Glu MT-IF interaction. We hypothesize that Glu tubulin may function as a signal for the recruitment of IFs, and perhaps other cellular components, onto stable MTs in polarized cells.2 One critical element to understand the preferential interaction of IFs with Glu MTs is to determine the factors involved in mediating the interaction. Previous studies have provided evidence for cross-bridging structures or molecules that might mediate the interaction between IFs and MTs (11Hirokawa N. J. Cell Biol. 1982; 94: 129-142Crossref PubMed Scopus (492) Google Scholar, 12Svitkina T.M. Verkhovsky A.B. Borisy G.G. J. Cell Biol. 1996; 135: 991-1007Crossref PubMed Scopus (335) Google Scholar); however, none of these candidates has been shown to interact selectively with Glu versus Tyr tubulin. In this study, we have pursued the identity of factors that might be mediating the preferential interaction between IFs and Glu MTs by using antibodies to Glu tubulin as competitors for the binding of the putative cross-bridging molecule to Glu MTs in vitro. We found that antibodies to Glu tubulin reduced the binding of a kinesin to MTs more effectively than antibodies to Tyr tubulin. By directly measuring the binding affinity of kinesin for pure Glu and Tyr MTs, we confirm that kinesin binds to Glu MTs with a 2.8-fold higher affinity than to Tyr MTs. In further experiments, we found that there is a kinesin that specifically cosediments with vimentin IFs, but not with neurofilaments or actin filaments. This kinesin is composed of kinesin heavy chain and a light chain that might be novel. Taken together, our results show that kinesin can mediate the preferential interaction of vimentin IFs with Glu MTs. Taxol was a gift of Dr. Ven L. Narayanan (NCI, Bethesda, MD). Monoclonal antibody (mAb) to kinesin heavy chain, H2 (13Pfister K.K. Wagner M.C. Stenoien D.L. Brady S.T. Bloom G.S. J. Cell Biol. 1989; 108: 1453-1463Crossref PubMed Scopus (192) Google Scholar), was generously provided by Dr. G. S. Bloom (University of Texas, Southwestern, Dallas, TX). Polyclonal antibody to kinesin heavy chain, HD (14Gyoeva F.K. Gelfand V.I. Nature. 1991; 353: 445-448Crossref PubMed Scopus (179) Google Scholar), was provided by Dr. V. I. Gelfand (University of Illinois, Urbana, IL). Monoclonal antibodies to kinesin light chain, L1 and L2 (13Pfister K.K. Wagner M.C. Stenoien D.L. Brady S.T. Bloom G.S. J. Cell Biol. 1989; 108: 1453-1463Crossref PubMed Scopus (192) Google Scholar) and 63–90 (15Stenoien D.L. Brady S.T. Mol. Biol. Cell. 1997; 8: 675-689Crossref PubMed Scopus (119) Google Scholar), were kindly provided by Dr. S. T. Brady (University of Texas, Southwestern). Purified recombinant squid kinesin head, K394 (16Song Y.H. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1671-1675Crossref PubMed Scopus (126) Google Scholar), was generously provided by Dr. E. Mandelkow (Max Planck Institute, Hamburg, Germany). mAb to kinesin, SUK4, was from the Developmental Studies Hybridoma Bank maintained by Johns Hopkins University School of Medicine (Baltimore, MD) and University of Iowa (Iowa City, IA). Affinity-purified polyclonal antibodies to Glu tubulin (SG) and to Tyr tubulin (W2) were prepared as described (1Gurland G. Gundersen G.G. J. Cell Biol. 1995; 131: 1275-1290Crossref PubMed Scopus (132) Google Scholar). Purified spinal cord neurofilament proteins containing neurofilament L, M, and H and α-internexin (17Liem R.K. Hutchison S.B. Biochemistry. 1982; 21: 3221-3226Crossref PubMed Scopus (152) Google Scholar) and a construct encoding full-length vimentin, pET3a-V62-1, were generously provided by Dr. R. K. Liem (Columbia University, New York, NY). Actin purified from bakers' yeast (18Lazzarino D.A. Boldogh I. Smith M.G. Rosand J. Pon L.A. Mol. Biol. Cell. 1994; 5: 907-918Crossref PubMed Scopus (97) Google Scholar) was a gift from Dr. L. A. Pon (Columbia University). All chemicals were from Sigma unless stated otherwise. Crude preparations of MT motor proteins were obtained from 3T3 cell extracts and brain extracts according to Rickard and Kreis (19Rickard J.E. Kreis T.E. J. Cell Biol. 1990; 110: 1623-1633Crossref PubMed Scopus (85) Google Scholar). 3T3 cells were grown on 20 dishes (150 mm) to confluency and detached from the dishes by incubation in 10 mm Hepes, pH 7.0, 150 mmNaCl, and 1 mm EDTA at 37 °C. Cells were collected by centrifugation, washed in the same buffer, and lysed by sonication in 5 ml of PEMG (100 mm Pipes, pH 6.9, 1 mm EGTA, 1 mm MgCl2, and 1 mm GTP). The brain extracts were obtained by homogenizing the gray matter of calf brains in 1.3 volumes of PEMG in a blender. Cell lysate and brain homogenate were depleted of cell or tissue debris (40,000 × g, 30 min, 4 °C) and then centrifuged at 100,000 × g for 1 h at 4 °C to obtain the high-speed supernatant. The endogenous tubulin in the high-speed brain supernatant was polymerized by incubation at 37 °C for 30 min and then sedimented (40,000 × g, 30 min, 37 °C). The supernatant (H1S) was then concentrated in dialysis tubing against Aquacide (Calbiochem). Concentrated H1S and the high-speed supernatant of the 3T3 cell extract were then incubated with 10 units/ml hexokinase, 10 mmglucose, 50 μm AMP-PNP, 20 μm Taxol, and DEAE-purified bovine brain tubulin (9Gundersen G.G. Khawaja S. Bulinski J.C. J. Cell Biol. 1987; 105: 251-264Crossref PubMed Scopus (220) Google Scholar) at a final concentration of 0.2 mg/ml for 30 min at 37 °C to induce association of motor proteins with MTs. MTs and associated proteins were then sedimented and washed as described (19Rickard J.E. Kreis T.E. J. Cell Biol. 1990; 110: 1623-1633Crossref PubMed Scopus (85) Google Scholar). Proteins were eluted from MTs with PEM (PEMG without GTP), pH 6.9, containing 10 mm Mg2+ATP. Isolation of kinesin at >95% purity from bovine brain was as described (20Wagner M.C. Pfister K.K. Brady S.T. Bloom G.S. Methods Enzymol. 1991; 196: 157-175Crossref PubMed Scopus (20) Google Scholar), except that concentrated H1S (see above) was used as the starting material in our preparation. Tubulin from calf brain and HeLa cell extracts was purified by two cycles of assembly-disassembly followed by DEAE-Sephadex A-50 chromatography (9Gundersen G.G. Khawaja S. Bulinski J.C. J. Cell Biol. 1987; 105: 251-264Crossref PubMed Scopus (220) Google Scholar, 21Chapin S.J. Bulinski J.C. Methods Enzymol. 1991; 196: 254-264Crossref PubMed Scopus (18) Google Scholar). Pure Glu tubulin was prepared by incubation of calf or HeLa tubulin with pancreatic carboxypeptidase A as described (9Gundersen G.G. Khawaja S. Bulinski J.C. J. Cell Biol. 1987; 105: 251-264Crossref PubMed Scopus (220) Google Scholar). Increasing amounts of K394 in 25 mm Pipes, pH 6.9, 1 mmMgCl2, 1 mm EGTA, and 150 mm KCl was added to the same amounts of Glu or Tyr MTs (0.8 μmfinal concentration) assembled from pure HeLa cell Glu or Tyr tubulin in the presence of 20 μm Taxol. The final concentration of K394 in the incubation mixtures was between 0.16 and 0.48 μm. MTs and MT-bound K394 were separated from soluble proteins by sedimentation (100,000 × g, 7 min, 4 °C), and the levels of K394 in the MT pellets were measured by quantitative Western blotting. Glu and Tyr tubulin assembled into MTs with a similar efficiency as detected by Ponceau staining of the nitrocellulose membrane before proceeding for Western blotting (data not shown). A full-length vimentin clone (pET3a-V62-1) was used to transform Escherichia coli(DE3), and vimentin expression was induced by isopropyl-β-d-thiogalactoside (1 mm for 4 h). Cells were lysed by sonication in buffer A (6 murea, 20 mm Tris, pH 7.2, 200 μmphenylmethylsulfonyl fluoride, and 0.1 μg/ml each chymostatin, leupeptin, antipain, and pepstatin). After clarifying the lysate by centrifugation (30,000 × g, 10 min), vimentin IFs were induced to assemble by dialysis against phosphate-buffered saline, pH 7.2, with 0.1% β-mercaptoethanol overnight at 4 °C and then collected by centrifugation (60,000 × g, 1 h, 4 °C). The vimentin pellet was dissolved in buffer A, and IFs were reassembled as described above. Assembled vimentin was collected and disassembled again as described above, and the vimentin solution was passed though a DEAE-Sephadex A-50 column equilibrated with buffer A and eluted with a linear gradient of 0.1–0.5 mNaCl in buffer A. Vimentin was recovered at a single peak at 0.4–0.45m NaCl and was shown to be >90% pure by SDS-PAGE. Samples were analyzed by SDS-PAGE as described (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). Western blotting was performed as described (23Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44923) Google Scholar). Antibodies used to probe Western blots included anti-kinesin heavy chain antibodies H2 (13Pfister K.K. Wagner M.C. Stenoien D.L. Brady S.T. Bloom G.S. J. Cell Biol. 1989; 108: 1453-1463Crossref PubMed Scopus (192) Google Scholar), SUK4 (24Ingold A.L. Cohn S.A. Scholey J.M. J. Cell Biol. 1988; 107: 2657-2667Crossref PubMed Scopus (139) Google Scholar), and HD (14Gyoeva F.K. Gelfand V.I. Nature. 1991; 353: 445-448Crossref PubMed Scopus (179) Google Scholar); anti-kinesin light chain mAbs L1, L2, and 63–90 (15Stenoien D.L. Brady S.T. Mol. Biol. Cell. 1997; 8: 675-689Crossref PubMed Scopus (119) Google Scholar); anti-β-tubulin mAb 3F3 (25Moyer S.A. Baker S.C. Lessard J.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5405-5409Crossref PubMed Scopus (143) Google Scholar); and anti-Glu and anti-Tyr tubulin polyclonal antibodies SG and W2, respectively (3Gundersen G.G. Kalnoski M.H. Bulinski J.C. Cell. 1984; 38: 779-789Abstract Full Text PDF PubMed Scopus (381) Google Scholar). Alkaline phosphatase-conjugated anti-mouse or anti-rabbit antibodies were used as secondary antibodies. Blots were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. The protein concentrations of samples were determined with the BCA assay using bovine serum albumin as standard (Pierce) or the Bradford assay (40Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216412) Google Scholar) using bovine γ-globulins as standard. In a search for the cross-bridging molecule that mediates the Glu MT-IF interaction, we assembled Taxol-stabilized MTs from purified calf brain tubulin (an ∼50:50 mixture of Glu and Tyr tubulin (9Gundersen G.G. Khawaja S. Bulinski J.C. J. Cell Biol. 1987; 105: 251-264Crossref PubMed Scopus (220) Google Scholar)) and incubated them with saturating levels of affinity-purified antibodies to either Glu (SG) or Tyr (W2) tubulin and subsaturating levels of preparations of MT-interacting proteins. If there is a Glu tubulin-specific cross-bridging molecule, antibodies to Glu tubulin, but not to Tyr tubulin, should sterically interfere with the binding of the cross-bridging molecule to MTs. When we incubated MTs with SG or W2 and crude preparations of brain MT-associated proteins (MAPs), we did not see significant inhibition of individual MAP binding with either antibody (data not shown). These results are consistent with previous observations (10Kumar N. Flavin M. Eur. J. Biochem. 1982; 128: 215-222Crossref PubMed Scopus (37) Google Scholar,21Chapin S.J. Bulinski J.C. Methods Enzymol. 1991; 196: 254-264Crossref PubMed Scopus (18) Google Scholar, 26Wehland J. Willingham M.C. Sandoval I.V. J. Cell Biol. 1983; 97: 1467-1475Crossref PubMed Scopus (128) Google Scholar). With preparations of MT motor proteins from a 3T3 cell extract, SG (but not W2) blocked the sedimentation of an ∼110-kDa polypeptide with MTs (Fig. 1 a). At a 1:1 molar ratio of antibody to tubulin, SG significantly reduced the binding of the 110-kDa protein to MTs, whereas the level of the 110-kDa protein in the W2-treated sample was comparable to that in the control sample, where no antibody was added. At a 4:1 ratio of antibody to tubulin, the binding of both SG and W2 to MTs was saturated (data not shown), and the 110-kDa protein was not detectable in the SG-treated sample, but was still present in the W2-treated sample (Fig. 1 a). Other polypeptide bands were not significantly affected by either antibody (e.g. bands at ∼70 and ∼80 kDa). The 110-kDa protein ran as a doublet in some of the samples (see control (C) lane in Fig. 1 a). A mAb to Glu tubulin, 4B8 (1Gurland G. Gundersen G.G. J. Cell Biol. 1995; 131: 1275-1290Crossref PubMed Scopus (132) Google Scholar) (from J. C. Bulinski, Columbia University), also inhibited the binding of the 110-kDa protein to MTs more effectively than a mAb to Tyr tubulin, YL1/2 (27Kilmartin J.V. Wright B. Milstein C. J. Cell Biol. 1982; 93: 576-582Crossref PubMed Scopus (636) Google Scholar) (data not shown). We identified the 110-kDa polypeptide as a member of the kinesin family based on a number of criteria. The 110-kDa polypeptide bound to MTs in the absence of ATP and was eluted from MTs with 10 mm ATP (data not shown), consistent with characteristics of kinesin motor proteins (28Vale R.D. Reese T.S. Sheetz M.P. Cell. 1985; 42: 39-50Abstract Full Text PDF PubMed Scopus (1413) Google Scholar). Moreover, a mAb to kinesin heavy chain, H2, recognized the 110-kDa protein in the MT motor protein pellet (Fig. 1 b). Another mAb to kinesin (SUK4) and a polyclonal antibody to kinesin (HD) also reacted with the 110-kDa protein (data not shown). Experiments with preparations of MT motor proteins from brain extracts showed similar results, except that the polypeptide that was inhibited from binding to MTs by SG migrated at ∼120 kDa (see below, Fig. 1 c). These results demonstrate that Glu antibody is capable of inhibiting the binding of two different kinesins to MTs. We next analyzed the level of kinesin binding to MTs in the presence of SG or W2 using Western blotting with H2 antibody. As shown in Fig. 1 c, SG prevented more kinesin from binding to MTs than W2. We also analyzed the MT pellets to determine the level of tubulin antibody bound to MTs. No significant difference was observed in the levels of IgG that cosedimented with MTs (Fig. 1 d). These results rule out the possibility that the differential capability of SG and W2 to inhibit the binding of kinesin to MTs was due to differential binding of SG and W2 to MTs. To quantify the capability of SG and W2 antibodies to inhibit the binding of kinesin to MTs, we determined the percentage of inhibition of kinesin binding by SG or W2 antibodies and normalized this by the amount of SG or W2 bound to the MTs. Levels of kinesin and SG or W2 antibody bound to MTs were measured by quantitative Western blotting with H2 antibody and a commercial antibody to rabbit IgG, respectively. Table I shows the individual determinations from three identical experiments (only two determinations of the amount of bound antibody were made). On average, SG inhibited the binding of kinesin to MTs by ∼64%, whereas W2 inhibited binding only by 36%. When these levels are normalized against the amount of antibody bound to MTs, we found that at equivalent levels of antibody bound to MTs, SG inhibited the binding of kinesin to MTs 1.7-fold more than W2 did (Table I). Since we used low, subsaturating levels of kinesin in these experiments, these data suggest that kinesin was preferentially bound to Glu rather than Tyr tubulin, so that when Glu tubulin sites were blocked, more kinesin was displaced.Table IQuantification of inhibition of kinesin binding to microtubules by antibodies to Glu and Tyr tubulinKinesin bindingSGW2% of controlExp. 14475Exp. 23355Exp. 33062Average36 ± 674 ± 8Amount of MT-bound antibodiesSGW2μgExp. 14.64.4Exp. 24.03.8Average4.34.1 Open table in a new tab To directly compare the binding affinity of kinesin for Glu and Tyr MTs, we prepared tubulin from HeLa cells to generate MTs composed of pure Glu and Tyr tubulin. Tubulin purified from HeLa cells is reported to be >90% Tyr tubulin (21Chapin S.J. Bulinski J.C. Methods Enzymol. 1991; 196: 254-264Crossref PubMed Scopus (18) Google Scholar) and can be converted to Glu tubulin by carboxypeptidase A treatment (9Gundersen G.G. Khawaja S. Bulinski J.C. J. Cell Biol. 1987; 105: 251-264Crossref PubMed Scopus (220) Google Scholar). Moreover, tubulin prepared from HeLa cells is not as complex as brain tubulin since it does not contain several post-translational modifications abundant in brain tubulin (e.g. polyglutamylation (29Edde B. Rossier J. Le Caer J.-P. Desbruyeres E. Gros F. Denoulet P. Science. 1990; 247: 83-85Crossref PubMed Scopus (425) Google Scholar) and nontyrosinatable tubulin (30Paturle-Lafanechere L. Edde B. Denoulet P. Van Dorsselaer A. Mazarguil H. Le Caer J.-P. Wehland J. Job D. Biochemistry. 1991; 30: 10523-10528Crossref PubMed Scopus (162) Google Scholar)). We have confirmed that our preparations of HeLa cell tubulin and carboxypeptidase A-treated HeLa cell tubulin were predominantly Tyr (90%) and Glu (100%) tubulin, respectively. To measure the binding affinity of kinesin for Glu and Tyr MTs, we used a recombinant head domain of squid kinesin heavy chain, K394 (16Song Y.H. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1671-1675Crossref PubMed Scopus (126) Google Scholar). We incubated increasing amounts of K394 with Taxol-stabilized Glu or Tyr MTs and separated the bound kinesin from unbound by sedimenting the MTs. The amount of kinesin bound was analyzed by quantitative Western blotting. As shown in Fig. 2 a, more K394 bound to Glu MTs than to Tyr MTs at all concentrations. Scatchard plot analysis of binding assays showed that K394 bound to Glu and Tyr MTs with Kd values of 29 and 81 nm, respectively (Fig. 2 b). These results directly demonstrate that kinesin preferentially binds to Glu MTs compared with Tyr MTs. We showed above that antibodies to Glu tubulin reduced the binding of a 110-kDa (3T3 cell) and a 120-kDa (brain) kinesin to MTs more effectively than antibodies to Tyr tubulin and that recombinant kinesin heads bound to Glu MTs with a higher affinity than to Tyr MTs. These results strongly suggest that a kinesin may mediate the preferential interaction of vimentin IFs with Glu MTs. To test this possibility directly, we assembled vimentin IFs from purified recombinant vimentin and tested whether a kinesin could associate with vimentin IFs. We found a 120-kDa kinesin in the MT motor protein preparation from bovine brain cosedimented with purified vimentin IFs, as assessed by both protein staining and Western blotting (Fig. 3 a, lanes 1and 4). Experiments with preparations of MT motor proteins from 3T3 cell extracts showed a 110-kDa kinesin cosedimenting with vimentin (data not shown). The kinesin in the preparation did not sediment alone (Fig. 3 a, lanes 3 and 6). The sedimentation of this kinesin with vimentin IFs was not due to trapping by filamentous proteins since significantly less kinesin was found in the pellet from an equivalent amount of actin filaments (Fig. 3 b, compare lanes 2 and 4 with lanes 1 and 3). To distinguish whether the 120-kDa kinesin from preparations of brain MT motor proteins associated with vimentin IFs directly or through some accessory molecules, we purified kinesin from brain (see “Experimental Procedures”). Analysis of the preparation through the sequential steps of the purification (MT motor proteins (lane M), gel filtration (lane G), ion exchange (lane I), and sucrose density gradient ultracentrifugation (lane S)) showed the enrichment of the kinesin heavy (120 kDa) and light (64 kDa) chains (Fig. 4 a). For unknown reasons, our purified kinesin preparation contained relatively more light chain than that detected in kinesin preparations obtained by other laboratories (20Wagner M.C. Pfister K.K. Brady S.T. Bloom G.S. Methods Enzymol. 1991; 196: 157-175Crossref PubMed Scopus (20) Google Scholar). After the final sucrose gradient density centrifugation step, the preparation contained only the 120-kDa heavy and 64-kDa light chains (Fig. 4 a, lane S). As shown in Fig. 4 b, with the 120-kDa kinesin obtained from different stages of purification, comparable levels of kinesin (even with the purest fraction) sedimented with vimentin IFs, indicating that the 120-kDa kinesin associated with vimentin without the need for an accessory protein (however, see below for an analysis of light chain binding). More important, the 120-kDa kinesin from the purest fraction did not cosediment significantly with a comparable amount of neurofilament proteins (Fig. 4 c), showing that the interaction of kinesin is specific for vimentin IFs. Conventional kinesin holoenzyme is a tetramer composed of two ∼120-kDa heavy chains and two 60–70-kDa light chains (31Bloom G.S. Wagner M.C. Pfister K.K. Bradt S.T. Biochemistry. 1988; 27: 3409-3416Crossref PubMed Scopus (216) Google Scholar). The head domain of kinesin heavy chain is conserved among different isoforms of kinesin and kinesin-like proteins and contains both MT- and ATP-binding sites (for review, see Ref. 32Goldstein L.S.B. Annu. Rev. Genet. 1993; 27: 319-351Crossref PubMed Scopus (160) Google Scholar). We determined if kinesin head was involved in the association of kinesin with vimentin by testing whether recombinant squid kinesin head, K394, cosedimented with vimentin. As shown in Fig. 5 a, no K394 was found in the pellet after centrifugation of a vimentin/K394 incubation mixture, showing that the vimentin-binding site of kinesin is not located in the head domain. While the binding of kinesin to MTs is normally inhibited by millimolar levels of ATP, cosedimentation of the 120-kDa kinesin with vimentin IFs was not affected by the presence of 2 mm ATP (Fig. 6 b). These results show that the binding of kinesin to vimentin occurs by a mechanism distinct from that involved in kinesin-MT binding.Figure 6Cosedimentation of kinesin light chains with vimentin IFs. a, vimentin IFs were incubated with sucrose density gradient-purified kinesin, pelleted by centrifugation, and analyzed by SDS-PAGE and Coomassie Blue staining. An equivalent amount of vimentin alone was also pelleted and loaded on the same gel to serve as a control. Lane 1, pellet of vimentin + kinesin;lane 2, pellet of vimentin alone; lane 3, supernatant of vimentin + kinesin. Bands marked KHC,KLC, and V are kinesin heavy chain, kinesin light chain, and vimentin, respectively. b, cosedimentation of sucrose density gradient-purified kinesin with vimentin IFs as analyzed by Western blotting, probed first with H2 antibody (for heavy chain) and then with L1 antibody (for light chain). Lane 1, pellet of vimentin + kinesin; lane 2, kinesin alone. Note that the levels of kinesin heavy chain in lanes 1 and 2are comparable, yet the kinesin light chain is clearly detected by L1 in lane 2, but is not detectable in lane 1. c, Western blot analysis of kinesin that cosedimented with vimentin IFs using monoclonal antibodies to kinesin light chain (L1, L2, and 63–90). P and S, pellet and supernatant of vimentin + kinesin, respectively.View Large Image Figure ViewerDownload (PPT) Kinesin can be divided into three regions: a globular head domain formed by the two heavy chains, a stalk domain formed primarily by the heavy chains, and a fan-like tail domain formed by the light chains and the heavy chain carboxyl-terminal domain (for reviews, see Refs. 32Goldstein L.S.B. Annu. Rev. Genet. 1993; 27: 319-351Crossr" @default.
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• W1993764616 date "1998-04-01" @default.
• W1993764616 modified "2023-10-15" @default.
• W1993764616 title "Kinesin Is a Candidate for Cross-bridging Microtubules and Intermediate Filaments" @default.
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