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Vol. 19, Issue 3, 1152-1161, March 2008
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-Tubulin Mutation Results in Inefficient Cycling with CCT and a Deficient Interaction with TBCB
*Department of Biochemistry, New York University Medical Center, New York, NY 10016; Institut Cochin, Université René Descartes, Paris F-75014, France; and Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom
Submitted September 5, 2007;
Revised December 26, 2007;
Accepted January 4, 2008
Monitoring Editor: Tim Stearns
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin gene, TUBA1A, have recently been identified that cause these diseases. Here, we show that one such mutation, R264C, leads to a diminished capacity of de novo tubulin heterodimer formation. We identify the mechanisms that contribute to this defect. First, there is a reduced efficiency whereby quasinative -tubulin folding intermediates are generated via ATP-dependent interaction with the cytosolic chaperonin CCT. Second, there is a failure of CCT-generated folding intermediates to stably interact with TBCB, one of the five tubulin chaperones (TBCA–E) that participate in the pathway leading to the de novo assembly of the tubulin heterodimer. We describe the behavior of the R264C mutation in terms of its effect on the structural integrity of -tubulin and its interaction with TBCB. In spite of its compromised folding efficiency, R264C molecules that do productively assemble into heterodimers are capable of copolymerizing into dynamic microtubules in vivo. The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Neuronal migration disorders are a group of brain developmental diseases characterized by neuronal migration defects that result in severe cortical malformations. In its most severe form (lissencephaly), the surface of the brain has a smooth appearance, four abnormal cortical layers, and a variably hypoplastic cerebellum (Dobyns and Truwit, 1995; Dobyns et al., 1996). In a less severe form (pachygyria), the disease is characterized by a simplified pattern of folds on the surface of the brain. In either event, a consequence of impaired neuronal migration is not only aberrant lamination in the brain, but ultimately behavioral deficits: humans with lissencephaly/pachygyria are mentally retarded and frequently suffer from epilepsy (Francis et al., 2006). The disease involves mutations in a number of genes.
In classical lissencephaly, the cause is ascribable to sporadic mutations in the LIS1 gene, resulting in haploinsufficiency. Lis1 is a ubiquitously expressed 45-kDa protein; it plays multiple functional roles that include interactions with microtubules via cytoplasmic dynein. Lis1 binds to dynein via several distinct sites (Faulkner et al., 2000; Smith et al., 2000; Tai et al., 2002) and to several other proteins (e.g., mNudC, mNudE, and NudEL) originally identified in Aspergillus as being involved in nuclear distribution (Efimov and Morris, 2000; Kitagawa et al., 2000), thus linking Lis1 with nucleokinesis. Lis1 also binds to catalytic dimers of the brain cytosolic platelet activating factor acetylhydrolase, which in turn enhances N-methyl-D-aspartate receptor currents (Tabuchi et al., 1997). Importantly, suppression of Lis1 via small interfering RNA causes an accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. There is also an abolition of interkinetic nuclear oscillations of the radial progenitors in the ventricular zone and an accompanying inhibition of cell division (Hatten, 2005; Tsai et al., 2005).
In its X-linked form, lissencephaly is caused by both familial and sporadic mutations in the gene (DCX) encoding the microtubule associated protein doublecortin (Gleeson et al., 1998). It has been demonstrated that doublecortin coassembles with brain tubulin, stimulating polymerization into microtubules that contain 13 protofilaments (Francis et al., 1999). DCX associates with the dyenin complex, an interaction that might be mediated by the previously identified interaction between DCX and Lis1 (Tanaka et al., 2004). Mutations that cause lissencephaly are clustered in the two tubulin binding domains of DCX, and their presence impairs polymerization of microtubules both in vitro and in vivo (Sapir et al., 2000), thus implicating correct binding of DCX to microtubules as critical for proper neuronal migration. DCX does not bind to the tubulin heterodimer, but it does nucleate microtubule growth and acts as a potent anticatastrophe factor that stabilizes microtubules by linking adjacent protofilaments in the microtubule lattice. The enrichment of DCX at the distal ends of neuronal processes has led to the suggestion that the function of DCX in neurons is to drive the assembly and stabilization of noncentrosomal microtubules in DCX-enriched zones (Moores et al., 2004, 2006). Surprisingly, knockout of Dcx in mice does not result in defects in cortical neuronal lamination or positioning (Corbo et al., 2002). However, suppression of DCX expression reveals that the protein is required for radial migration in rat neocortex (Bai et al., 2003), and the absence of DCX does result in a severe morphological defect that implicates its function in the maintenance of bipolar shape and nuclear translocation during migration in the adult forebrain (Koizumi et al., 2006). DCX may also function in roles that are not directly related to microtubules. For example, DCX interacts with c-Jun NH2-terminal kinase (JNK) and JNK interacting protein, implicating the involvement of DCX in a signaling pathway (Gdalyahu et al., 2004).
Perturbations of the laminar architecture of the cortex also result from mutations in the Aristaless-related homeobox gene ARX (Str
mme et al., 2002), the extracellular matrix protein reelin (Hong et al., 2000), and the very-low-density lipoprotein receptor (VLDLR) (Boycott et al., 2005). ARX has been identified as the gene associated with an X-linked human brain malformation, and with abnormal genitalia (Kitamura et al., 2002). Reelin was first implicated in neuronal migration by studying the naturally occurring mouse mutant reeler, which displays an inversion of the normal inside-out order of cortical neurons (D'Arcangelo et al., 1995; Trommsdorff et al., 1999). Together, mutations in DCX, LIS1, ARX, reelin, and VLDLR account for <50% of the cases of the lissencephaly/pachygyria spectrum of phenotypes (Francis et al., 2006). Very recently, mutations in the -tubulin gene TUBA1A have also been shown to cause neuronal migration disorders (Keays et al., 2007; Poirier et al., 2007). These observations reinforce the notion that a common feature of many forms of lissencephaly/pachygyria is the disruption of key elements of microtubule behavior. In mutations involving -tubulin, this might occur either via defects in the tubulin heterodimer assembly pathway or via a mechanism in which proper interactions between -tubulin and critical effectors of microtubule function are compromised. Here, we describe the elucidation of the mechanisms whereby a recurrent pachygyria-causing mutation, R264C, in TUBA1A results in a compromised efficiency of de novo /β-tubulin heterodimer formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin sequence. These products were also cloned into the pcDNA3.1+ vector. All constructs were checked by DNA sequencing. Transcription/translation reactions were done at 30°C for 60 min in 25 µl of rabbit reticulocyte lysate (TNT; Promega, Madison, WI) containing [35S]methionine (specific activity, 1000 Ci/µmol; 10 µCi/µl). In kinetic experiments, aliquots (1.5 µl) were withdrawn from the reaction at various times, diluted into 5 µl of gel loading buffer (gel running buffer supplemented with 10% glycerol and 0.1% bromphenol blue), and stored on ice before resolution on a nondenaturing gel (Tian et al., 1997). For the generation of -tubulin–labeled heterodimers, transcription/translation reactions were chased for a further 15 min after the addition of native bovine brain tubulin to 0.2 mg/ml. In some experiments, the labeled product was isolated on microcolumns of DEAE Sephacel as described previously (Tian et al., 1997). Labeled reaction products were detected by autoradiography after resolution on either SDS-polyacrylamide gel electrophoresis (PAGE) or on native polyacrylamide gels as described previously (Tian et al., 1997).
Sensitivity to Proteolytic Digestion
Tubulin heterodimers containing [35S]methionine-labeled wild-type or R264C mutant TUBA1A sequences were generated by in vitro transcription/translation and purified as described above. The reaction products were subjected to digestion with proteinase K, and the reaction products were analyzed by SDS-PAGE as described previously (Tian et al., 2006).
In Vitro Folding Reactions
In vitro folding assays were done in folding buffer containing CCT, ATP, guanosine triphosphate (GTP), tubulin chaperones (TBCB, TBCC, TBCD, and TBCE), and purified native tubulin heterodimer as described previously (Tian et al., 1995b; Tian et al., 1996). Target proteins (i.e., wild type or R264C TUBA1A -tubulin) were expressed as either unlabeled or 35S-labeled proteins in Escherichia coli, and the inclusion bodies were purified and unfolded in 8 M urea as described previously (Gao et al., 1992). Reaction products were analyzed either by electrophoresis on native polyacrylamide gels as described previously (Zabala and Cowan, 1992), or on a 2.2-ml gel filtration column of Superose 6 equilibrated and run on a Smart system (GE Healthcare, Piscataway, NJ) in 0.15 M NaCl, 10 mM Tris-HCl, pH 7.4, and 1 mM EGTA. Back reactions (see text) were done by incubating purified wild-type or R264C tubulin heterodimers [35S]methionine-labeled by in vitro transcription/translation in their -subunit with a twofold molar excess of TBCD and a fourfold stoichiometric excess of TBCB for 30 min at 30°C as described previously (Tian et al., 1997). In some experiments, folding reactions were done with unlabeled target proteins in reactions containing CCT, ATP, and 20 µM [-32P]GTP (specific activity, 100 Ci/mmol) (Tian et al., 1995a). In kinetic experiments done with [-32P]GTP, reactions were terminated at various time intervals by withdrawing an aliquot from the reaction mixture and quenching further formation of labeled IQ intermediates by the addition of unlabeled GTP to a final concentration of 2 mM. Samples were stored on ice before analysis on nondenaturing gels. In experiments to determine the stability of IQ intermediates, folding reactions done for 1 h in the presence of [-32P]GTP were quenched with unlabeled GTP and the incubation continued at 30°C for various time intervals before analysis by native PAGE.
Microtubule Copolymerization Experiments
Products of in vitro translation reactions or CCT-mediated in vitro folding reactions were mixed with depolymerized bovine brain microtubules, and they were taken through successive cycles of polymerization and depolymerization as described previously (Tian et al., 1997). At the end of each of these cycles, aliquots containing equal amounts of depolymerized material were removed and analyzed by either native gel electrophoresis or by scintillation counting.
Cell Culture, Transfections, and Immunofluorescence
Constructs were transfected into HeLa cells grown on glass coverslips in DMEM containing 10% fetal calf serum by using the FuGENE6 transfection reagent (Roche Diagnostics, Indianapolis, IN). Thirty-six hours posttransfection, cells were fixed with paraformaldehyde. In regrowth experiments to determine the dynamic behavior of microtubules, cells were incubated for 2 h with 10 µM nocodazole 36 h posttransfection, restored to drug-free medium, and fixed at various brief intervals thereafter. Cells were stained with a polyclonal anti-FLAG antibody and a monoclonal anti-β-tubulin antibody (Sigma-Aldrich, St. Louis, MO).
Computational Methods
A model of human -tubulin was built by homology modeling using available structures (Research Collaboratory for Structural Bioinformatics PDB code 1TUB). The image in Figure 5A was rendered using PyMOL (http://www.pymol.org).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Tubulin Mutation Results in Inefficient Native Heterodimer Production on Translation In Vitro/β-tubulin heterodimer, the subunit from which microtubules polymerize, cannot occur spontaneously. Rather, the process involves interaction with a series of molecular chaperones (Tian et al., 1997; Cowan and Lewis, 2002; Szymanski, 2002). Newly synthesized - and β-tubulin polypeptides are first captured in a partly unfolded conformation by the chaperone prefoldin, which delivers them to the ATP-dependent chaperonin CCT (Vainberg et al., 1998). Successive cycles of iterative annealing within the chaperonin cavity result in the generation of folding intermediates that are quasi-native in the sense that they contain a folded GTP-binding pocket (Tian et al., 1995a), but these molecules are still not competent to assemble so as to produce functional heterodimers. For this to occur, a further series of interactions is required with a series of downstream tubulin chaperones termed TBCA–E (Figure 1A).
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) to become incorporated into tubulin heterodimers by expressing the mutant protein in rabbit reticulocyte lysate in the presence of [35S]methionine. The reticulocyte-derived cell-free cocktail contains all the components required for de novo tubulin heterodimer assembly (Cleveland et al., 1978). The reaction products were analyzed by electrophoresis under both denaturing (SDS-PAGE) and nondenaturing gel conditions. We found that the R264C mutant was expressed as efficiently as its wild type counterpart as judged by the yield of labeled protein analyzed under denaturing conditions (Figure 1B). However, upon analysis under native conditions, the yield of product that migrated as native heterodimer was reduced by approximately fivefold in the case of the mutant protein (Figure 1C). The reduced level of tubulin heterodimer formation in the case of the R264C mutant was reflected at least in part by an enhanced retention of total label in the CCT/-tubulin binary complex (
11% in the wild-type protein vs. 18% in the R264C mutant). Consistent with this result, kinetic analysis of these reactions showed a slower rate of accumulation of native heterodimer in the case of the R264C mutant (Supplemental Figure 1), suggesting one or more defects in the heterodimer assembly pathway. To confirm that the material produced in reduced yield in these reactions was indeed native, we subjected the wild type and mutant cell-free translation products to two successive cycles of polymerization and depolymerization with added purified bovine brain microtubules. In this experiment, we found that wild-type and mutant heterodimers produced by transcription/translation in rabbit reticulocyte lysate cocycled with brain tubulin with indistinguishable efficiency (Figure 1D). We conclude that although the R264C mutant -tubulin protein is compromised in terms of its ability to assemble into native tubulin heterodimers in good yield, once assembled, mutant-containing heterodimers can efficiently copolymerize into microtubules in vitro.
R264C Mutant -Tubulin Acquires Bound GTP at a Reduced Rate and Does Not Stably Interact with TBCB
We first sought to determine whether the R264C mutation resulted in a compromised ability of the -tubulin target protein to acquire a quasinative state (termed IQ, defined as containing GTP bound at the N-site; Tian et al., 1995a) as a result of ATP-dependent cycling with CCT. To do this, equal amounts of unlabeled, urea-unfolded wild-type or mutant polypeptides made by expression in E. coli were presented by sudden dilution into buffer containing CCT, ATP, and [-32P]GTP. After incubation at 30°C, the reaction products were resolved on a nondenaturing gel. The result of this experiment showed that the mutant polypeptide acquired labeled GTP as a result of ATP-dependent cycling with chaperonin much more slowly than its wild-type counterpart (Figure 2, A and C). To see whether the reduced rate of formation of IQ intermediates of the R264C mutant reflected a relative instability of this species, we measured the half-life of IQ intermediates in pulse-chase experiments. This experiment showed that wild-type and mutant IQ intermediates had essentially the same stability (Figure 2, B and D). We conclude that the R264C mutation results in a compromised capacity of CCT to generate quasinative folding intermediates as a result of the chaperonin's ability to efficiently cycle its target protein.
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-tubulin IQ intermediates to interact with the downstream chaperones that participate in the de novo formation of /β tubulin heterodimers. To do this, [35S]methionine labeled -tubulins were expressed in E. coli as insoluble inclusion bodies, purified, unfolded in 8 M urea, and diluted into in vitro folding reactions containing CCT, ATP, GTP, and one or more of the chaperones (TBCB–E) required for de novo incorporation of -tubulin into the heterodimer. After incubation at 30°C, the reaction products were resolved by native PAGE. In these experiments, we noted a conspicuous absence of the TBCB/-tubulin intermediate in reactions done with the mutant target protein and TBCB alone. In addition, in fully reconstituted reactions containing TBCC, TBCD, and TBCE (with or without TBCB), the yield of native heterodimers produced in reactions done with the mutant target protein was reduced by a factor of about five compared with parallel control reactions done with the wild-type protein (Figure 3A). We also analyzed the products of in vitro folding reactions done with CCT and TBCB by gel filtration on Superose 6. In the case of wild type -tubulin, in addition to the 800-kDa binary complex formed between CCT and the target protein, a labeled product migrating with an apparent molecular mass of 90 kDa was observed. This material has the expected migration characteristics of a complex (TBCB/-tubulin) formed between -tubulin and a single molecule of TBCB. However, in the case of the R264C mutant, no product migrating at 90 kDa was formed (Figure 3B). Together, these data suggested that quasinative mutant -tubulin polypeptides produced by interaction with CCT are unable to form a stable cocomplex with TBCB.
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-tubulin subunit by coupled transcription/translation in rabbit reticulocyte lysate. The labeled heterodimers were purified by ion exchange chromatography and incubated with a molar excess of TBCD in the presence of TBCB. Under these conditions, the heterodimer is disrupted by interaction with TBCD such that the β-subunit forms a TBCD/β-tubulin complex, whereas the uncoupled 35S-labeled -subunit is free to form a cocomplex (TBCB/-tubulin) with TBCB (Tian et al., 1997; Bhamidipati et al., 2000). Analysis by native PAGE of the products of these reactions showed that, in contrast to wild-type -tubulin, the labeled heterodimer-derived R264C mutant-containing -tubulin polypeptide completely failed to yield detectable quantities of the TBCB/-tubulin intermediate (Figure 3C). The apparent disappearance of the input label in back reactions done with the R264C -tubulin mutant presumably reflects either its irreversible denaturation in the absence of a functional interaction with a stabilizing component, or its existence in an unstable complex with TBCB that does not survive native gel electrophoresis. In either event, most of the radioactivity is presumably lost to the reaction vessel walls (Tian et al., 1997), although we note the presence of a slightly enhanced signal at the gel origin that probably represents aggregated material. We conclude that R264C mutant -tubulin polypeptides generated either as a result of ATP-dependent interaction with CCT or via disruption of native heterodimers are unable to stably interact with TBCB.
Enhanced R264C Mutant Heterodimer Production in the Presence of an Excess of TBCB
We reasoned that the failure of the R264C mutant -tubulin to yield the characteristic TBCB/-tubulin intermediate might reflect a reduced affinity of the mutant polypeptide for the TBCB chaperone, and that this reduced affinity might be compensated in tubulin heterodimer assembly reactions containing higher concentrations of TBCB. To test this idea, we repeated our CCT-driven forward folding reactions by using increasing relative concentrations of TBCB. In reactions done with wild-type target protein, the inclusion of increasing amounts of TBCB did not enhance the yield of native heterodimers; instead, with a rising stoichiometric excess of TBCB over TBCC, TBCD, and TBCE, the reaction products contained an enhanced abundance of the TBCB/-tubulin intermediate (Figure 4A). In contrast, parallel reactions done with the R264C target protein did result in the production of a slightly increased yield of heterodimers as a function of a greater abundance of TBCB, with no evidence of a TBCB/-tubulin intermediate (Figure 4B). The material migrating as heterodimer in these reactions was competent to assemble into microtubules as shown by its ability to cocycle with native brain microtubules through successive cycles of polymerization and depolymerization in vitro (data not shown). The ability of a superabundance of TBCB to contribute to an enhanced yield of native R264C mutant-containing heterodimers is consistent with the notion that a consequence of the mutation is a lowered affinity for interaction with TBCB, with a resulting compromise in the efficiency of productive heterodimer assembly.
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-Tubulin Structure-tubulin. The location of R264 places it at a loop between helix 8 and strand 7 of -tubulin such that the arginine side chain forms a hydrogen bond with the carbonyl oxygen of residue D424 in the C-terminal helix (helix 12) (Figure 5A). Mutation of R264 to a cysteine residue would result in a disruption of this interaction, destabilizing the tertiary structure formed between the C-terminal -helix and the R264 loop and possibly also the local structure of the loop itself. We reasoned that such a destabilization might lead to a decrease in overall structural stability, and that this might be reflected in an enhanced susceptibility of the mutant protein to proteolytic digestion. To test this prediction, we prepared [35S]methionine-labeled wild-type and R264C mutant protein by translation in reticulocyte lysate, and we subjected the reaction products to limited digestion with proteinase K. The result of this experiment showed that mutant-containing heterodimers were indeed more sensitive to proteolytic attack, with the more rapid appearance of a degradation product a few kilodaltons smaller than the intact protein (Figure 5B). This could reflect cleavage of a more exposed C-terminal domain in the R264C polypeptide.
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-tubulin in vivo. TUBA1A differs from other -tubulin isotypes by as little as a single conservative amino acid substitution; therefore, it is not possible to generate antisera that can distinguish TUBA1A from other highly homologous -tubulin isotypes upon transfection into cultured cells. Moreover, because TUBA1A is universally expressed, we required a means to distinguish the transgene from its endogenously expressed counterpart. We therefore engineered a tag such that the transgene could be uniquely identified. Initially, we placed a FLAG tag at the C terminus of the molecule in such a way that the introduced epitope substituted for the naturally encoded C-terminal tyrosine residue. The design of this construct was based on the observation that an identically placed FLAG tag yielded heterodimers that were polymerization competent in vitro and in vivo both in the case of the wild-type mouse homologue of TUBA1A (tuba1) and in the case of a mutation (S140G) in this gene (Keays et al., 2007). As expected, we found that a C-terminal tag placed in this manner had no effect on the ability of wild-type TUBA1A tubulin to incorporate into microtubules upon transfection into HeLa cells. However, we were surprised to discover that the corresponding tagged R264C mutant protein completely failed to incorporate into microtubules (data not shown). This result was particularly puzzling because heterodimers containing the untagged mutant -tubulin were clearly polymerization competent in vitro (Figure 1C). To resolve this apparent paradox, we engineered FLAG-tagged constructs in which we deleted sequences encoding 11 amino acids from the C-terminus of the -tubulin polypeptide. The rationale behind this approach was based on the fact that up to 12 C-terminal amino acids of -tubulin can be removed without compromising either assembly of the heterodimer or its incorporation into microtubules in vivo (Gu and Cowan, 1989), whereas removal of at least some of this highly acidic C-terminal domain might be expected to compensate for the acidic charges conferred by the FLAG epitope, which itself contributes a net charge of –3.
To ensure that these C-terminally truncated FLAG-tagged molecules behaved in the same manner as their unmodified counterparts, we compared their properties in in vitro translation and in CCT-mediated folding reactions. Consistent with the behavior of the corresponding untagged proteins, we found that the yield of mutant product migrating as tubulin heterodimer was reduced relative to the wild-type counterpart by a factor of 5 upon expression in rabbit reticulocyte lysate (Figure 6A). The wild-type and mutant C-terminally truncated cell-free translation products both cycled efficiently through successive cycles of polymerization and depolymerization with native brain tubulin (Figure 6B). Moreover, in CCT-mediated in vitro folding reactions, the wild-type (but not the R264C mutant) protein yielded the characteristic TBCB/-tubulin intermediate in reactions done in the presence of TBCB (Figure 6C). Thus, removal of 11 residues from the C terminus and substitution with a FLAG epitope had no significant influence on the in vitro behavior of wild-type or mutant -tubulins compared with intact, untagged molecules. We therefore expressed the FLAG-tagged C-terminally deleted sequences by transfection in HeLa cells, and we examined their microtubules by double label immunofluorescence by using an anti-FLAG antibody (to detect the transgene) and an anti-β-tubulin antibody (to detect the overall microtubule network). We found that tagged C-terminally truncated wild-type and R264C mutant -tubulin both incorporated into interphase and mitotic HeLa cell microtubules in vivo (Figure 6D). We conclude that the R264C mutation does not preclude assembly of the -tubulin polypeptide into heterodimers, and that these heterodimers are competent to copolymerize into microtubules. We further infer that, in the R264C mutant, addition of a FLAG tag at the C terminus has a disruptive effect on heterodimer formation as a result of the addition of excessive negative charge, but that this effect can be compensated by removal of negatively charged residues that are not essential for heterodimer assembly or incorporation into microtubules in vitro or in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Tubulin-tubulin as a cause of lissencephaly/pachygyria expands the list of genes in which mutations can cause these diseases. Some of these genes (e.g., TUBA1A, LIS1, and DCX) are involved either directly or indirectly in contributing to microtubule behavior. Here, we show that one of the pachygyria-causing -tubulin mutations, R264C, results in several defects in the de novo tubulin heterodimer assembly pathway. This pathway involves several chaperone proteins. Prefoldin, a multisubunit complex assembled from six subunits in the range 14–23 kDa, functions by binding to nascent - and β-tubulin polypeptides and delivering them to the cytosolic chaperonin CCT (Hansen et al., 1999; Spiess et al., 2004). The chaperonin provides a locally sequestered folding environment in which tubulin polypeptides can avoid becoming kinetically trapped in off-pathway configurations, and yields quasinative folding intermediates (termed IQ) defined by the formation of a native (N-site) GTP-binding pocket as a result of one or more cycles of iterative annealing within the chaperonin cavity (Tian et al., 1995a). Notwithstanding their acquisition of bound GTP, these IQ intermediates are incapable of assembly into polymerization-competent heterodimers. For this to occur, they must first interact with a series of downstream chaperones (termed cofactors, or TBCA-E). TBCA and TBCD interact with β-tubulin folding intermediates, whereas TBCB and TBCE interact with -tubulin folding intermediates. The TBCD/β-tubulin and TBCE/-tubulin complexes associate to form a supercomplex that constitutes a tubulin heterodimer assembly machine (Tian et al., 1997); interaction of this supercomplex with TBCC results in the triggering of GTP hydrolysis by β-tubulin. This latter reaction results in the release of the newly formed heterodimer (Cowan and Lewis, 2002) (Figure 1A).
We found no significant difference in overall yield between wild-type and mutant -tubulin polypeptides upon translation in vitro and analysis of the products under denaturing conditions (Figure 1B). Conversely, the yield of native assembly competent mutant-containing heterodimers was lower by a factor of 5 compared with a control reaction done with wild-type -tubulin (Figure 1C). It follows that there must be one or more compromised interactions in the complex chaperone-dependent pathway leading to de novo tubulin heterodimer formation.
We identified two steps in the folding pathway in which the R264C mutation behaved differently from its wild-type counterpart. First, in the essential ATP-dependent interaction between unfolded polypeptides and CCT, the rate at which GTP-containing IQ intermediates are generated is much lower in the case of the mutant protein compared with the wild-type protein (Figure 2, A and C). This is unlikely to be a structural effect of the mutation on the GTP binding pocket itself, because R264 is located far from the N-site. Consistent with this conclusion, pulse chase experiments showed wild-type and mutant IQ intermediates to have indistinguishable half-lives (Figure 2, B and D). An effect of TBCB on the rate of generation of productive IQ intermediates can also be ruled out, because IQ production can be uncoupled from the action of cofactors (including TBCB) without affecting the yield of native tubulin heterodimers (Tian et al., 1995a). The reduced yield of mutant IQ molecules generated via interaction with CCT could, therefore, reflect either a lower affinity of the mutant target protein for the chaperonin, or a reduced efficiency with which mutation-containing intermediates undergoing cycles of ATP-dependent binding to and release from the chaperonin acquire a native GTP binding pocket. In either event, the R264C mutation results in a reduction of the rate at which potentially productive quasinative -tubulin folding intermediates can form via iterative cycles of annealing of the target protein with the chaperonin.
Second, in reactions done in vitro using purified components, we found that the R264C mutant -tubulin protein failed to yield the characteristic TBCB/-tubulin folding intermediate in either ATP-driven folding reactions containing CCT and TBCB (Figure 3, A and B) or in back-reactions supplemented with TBCB in which native tubulin heterodimers were disrupted by interaction with TBCD (Figure 3C). The efficient de novo production of tubulin heterodimers in vitro is absolutely dependent on the presence of TBCB (Tian et al., 1997). The failure of R264C mutant -tubulin to interact stably with TBCB, together with the reduced rate (and hence efficiency) with which CCT produces R264C quasinative GTP-containing intermediates, explains the relatively poor translational yield of polymerization competent mutant-containing heterodimers. Nonetheless, we found that we could generate some assembly competent mutant -tubulin-containing heterodimers in vitro in the presence of TBCB-E, and that the yield of mutant-containing heterodimers generated in these reactions could be enhanced by supplying a large excess of TBCB (Figure 4). Thus, the mutant protein may still be capable of a functional interaction with TBCB, although with a much lower affinity compared with its wild-type counterpart.
Role of TBCB
Among the various chaperone proteins that participate in de novo tubulin heterodimer formation, genetic experiments with model organisms have shown that TBCB, TBCC, TBCD, and TBCE are all essential to life in higher eukaryotes (Grishchuk and McIntosh, 1999; Cowan and Lewis, 2002; Radcliffe and Toda, 2000; Steinborn et al., 2002). It makes sense, therefore, that mutations resulting in compromised functioning of these proteins would result in microtubule phenotypes. In TBCE, for example, deletion mutations have been described in mouse (Martin et al., 2002) and in humans (Parvari et al., 2002) that lead to microtubule defects. No examples of inherited mutations in TBCB are known. However, there is persuasive evidence that TBCB plays an important role in neuronal development: induced perturbations in the level of TBCB in neurons results in either longer axons (upon TBCB depletion) or abnormalities in growth cone microtubules (upon TBCB overexpression) (Lopez-Fanarraga et al., 2007) that are reminiscent of neuronal changes in giant axonal neuropathy, an autosomal recessive disorder caused by mutation in the gene encoding gigaxonin (Bomont et al., 2000). The molecular mechanisms underlying this disease are not well understood, but it has been shown that gigaxonin interacts with TBCB and controls its degradation through the ubiquitin-proteasome pathway (Wang et al., 2005). There is also evidence that microtubule dynamics are influenced via the p21-activated kinase 1-induced phosphorylation of TBCB (Vadlamudi et al., 2005). Together with our demonstration that the pachygyria-causing R264C mutation results in a reduced affinity of the mutant polypeptide for TBCB, these data underscore the importance of TBCB in contributing to tubulin biosynthesis and in influencing microtubule behavior.
Structural Implications
TBCB possesses two distinct domains: an N-terminal ubiquitin-like (Ubl) domain and a C-terminal domain containing a conserved glycine-rich cytoskeletal-associated protein (CAP-Gly) domain (Tian et al., 1997; Lytle et al., 2004). The Ubl domain of TBCB is considered to contain an -tubulin interacting surface because in Schizosaccharomyces pombe, the Ubl domain of the TBCB orthologue Alp11B is essential for viability (Radcliffe and Toda, 2000; Lytle et al., 2004). Moreover, the Ubl domain of TBCE, which (like TBCB) is an -tubulin binding component in the tubulin heterodimer assembly pathway (Figure 1A), is required for this chaperone's activity (Tian et al., 2006; unpublished observations). The C-terminal domain of TBCB (which is not essential for viability in S. pombe) may also contribute to -tubulin binding, because a CAP-GLY domain is present in many proteins, including several involved in modulating plus-end microtubule dynamics (reviewed in Carvalho et al., 2003), and this domain contains a conserved motif (GKNDG) that is responsible for targeting to the extreme C-terminal EEY/F motif present in -tubulin (Weisbrich et al., 2007). However, the 10 carboxy-terminal residues of -tubulin are structurally disordered (Nogales et al., 1999), and they are identical in the wild-type and R264C polypeptides. Moreover, the CAP-Gly domain of TBCB lacks the highly basic groove present in proteins that bind to the C terminus of -tubulin (Li et al., 2002; Mishima et al., 2007). Molecular modeling analyses (data not shown) of the predicted interaction between -tubulin and the N-terminal Ubl domain of TBCB suggest that docking occurs at the region around the loop in -tubulin that contains R264, consistent with the observation that the mutant protein fails to stably interact with TBCB. However, it is possible that there is an additional (and perhaps cooperative) contribution to -tubulin binding via the C-terminal CAP-Gly domain.
Expression of R264C Tubulin In Vivo
We found that the ability of a FLAG-tagged form of the R264C mutant protein to assemble into polymerization competent heterodimers was dependent on the positioning of the tag. Thus, addition of the tag close to the authentic C terminus resulted in a protein that failed to yield productively folded heterodimers in vitro or in vivo, even though the corresponding wild-type protein tagged in an identical manner folded to the native state. Conversely, addition of the identical tag to a carboxy-terminally truncated form of either the wild-type or mutant protein yielded assembly competent heterodimers. The highly acidic 12 C-terminal amino acids of -tubulin are not required for functional heterodimer formation (Gu and Cowan, 1989), and they are structurally disordered (Nogales et al., 1999). It therefore seems likely that the R264C mutation renders the protein more structurally sensitive to overall charge changes in the flexible carboxy-terminal region.
On expression by transfection in HeLa cells, a C-terminally truncated FLAG-tagged form of the R264C mutant -tubulin became incorporated into interphase and mitotic spindle microtubules in a manner that was indistinguishable from parallel controls done with the wild-type protein (Figure 6). This is despite the failure of the R264C mutant to stably interact with TBCB in vitro (Figure 3). We also found no significant difference in the rate of growth of centrosomally anchored microtubules in HeLa cells that had been transfected with sequences encoding either wild-type or R264C mutant TUBA1A, although there was a relatively high diffuse signal in cells expressing the mutant sequence during early times of recovery from drug-induced depolymerization (Figure 7). These data suggest that the R264C mutation might affect the efficiency with which heterodimers can be assembled into microtubules in vivo, although we note that these experiments were done under transient transfection conditions using a powerful (CMV-derived) promotor such that the level of expressed protein would be much higher than that encountered in the developing brains of afflicted individuals. A reduction in polymerization efficiency could reflect structural changes in -tubulin resulting from the mutation, for example via destabilization incurred as a result of loss of the hydrogen bond interaction between residues R264 and D424. However, we cannot discount any effects conferred by addition of the C-terminal FLAG tag.
TUBA1A and its homologues in other vertebrate species are dominantly expressed in differentiating neurological cells, especially during post-natal development (Lewis et al., 1985; http://www.ncbi.nlm.nih.gov/UniGene/, follow "Expression Profile" link). Although other -tubulin isotypes (e.g., TUBA2, TUBA6) are also expressed in brain tissue, the TUBA1A -tubulin isotype is likely to be a major contributor of -tubulin to the overall tubulin heterodimer pool in migrating cells, especially during the critical period of neuronal migration upon which proper cortical lamination depends. A paucity of assembly competent heterodimers resulting from compromised interactions among components of the heterodimer assembly pathway such as those described here could result in a limiting abundance of neuronal microtubules, and this in turn could affect neuronal migration events. We predict that other neuronal migration disease-causing mutations in - or β-tubulins will also be characterized either by defects in the heterodimer assembly pathway such as those described here, or by a failure to interact with any of a variety of effectors that are required for the proper modulation of microtubule behavior during neuronal migration.
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Footnotes |
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Address correspondence to: Nicholas J. Cowan (cowann01{at}med.nyu.edu)
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REFERENCES |
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