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Rbl2p does not bind to denatured  -tubulin. Extracts from wild-type cells (W.C.E.) were incubated either with control buffer ( Ϫ ) or with 6 M gua- nidine hydrochloride ( ϩ ) for 5 min, diluted 100-fold, and then incubated with His 6 -Rbl2p plus Ni-NTA beads. The specifically bound proteins were eluted from the beads and assayed for the presence of both  -tubulin and ␣ -tubulin by immunoblotting. The binding of  -tubulin to Rbl2p was essentially abolished by the preincubation with denaturing agent. No bound ␣ -tubulin was detected under either condition.
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The yeast protein Rbl2p suppresses the deleterious effects of excess β-tubulin as efficiently as does α-tubulin. Both in vivo
and in vitro, Rbl2p forms a complex with β-tubulin that does not contain α-tubulin, thus defining a second pool of β-tubulin
in the cell. Formation of the complex depends upon the conformation of β-tubulin. Newly synthesized...
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Context 1
... cells grown in raffinose was exposed to galactose for 10 min and then to glucose for an additional 10 min. We fractionated extracts of these cells with anti- ␣ -tubulin antibodies to isolate the ␣ /  -tubulin heterodimers and with Ni-NTA beads to bind the His 6 -Rbl2p–  -tubulin complexes. As a steady-state control, an identical culture of raffinose- grown FSY820 cells was shifted to glucose for 20 min. The distributions of the two  -tubulin proteins in whole-cell ex- tracts and in the two fractions were assayed by SDS-polyacryl- amide gel electrophoresis followed by immunoblotting. Results from a representative experiment are shown in Fig. 2A. The different fractions are represented by very different exposures because they are so different in abundance. Some Tub2-590p, the induced  -tubulin protein, is detectable in the steady-state cell extracts because the GAL promoter is weakly expressed in raffinose medium. To monitor the newly synthesized  -tubulin, we recovered the fractions associated with ␣ -tubulin and with Rbl2p and normalized the recoveries using the constitutively expressed Tub2p. Figure 2B presents an analysis of the results shown in Fig. 2A. The short exposure to galactose increased the level of Tub2-590p approximately fourfold, while the levels of wild- type  -tubulin were unaffected. The ratio of Tub2-590p to Tub2p associated with Rbl2p increased about 5.4-fold after the induction. In contrast, the ratio for the two  -tubulin proteins present as ␣ /  -tubulin heterodimer increased only about 1.6- fold. This difference in partitioning of newly synthesized  -tubulin is not the consequence of a subtle difference in the proper- ties of the two proteins. We repeated this experiment using FSY821 cells, in which the constitutive and inducible  -tubulin genes are switched. In this experiment, the levels of the induc- ible Tub2p increased sixfold after the same induction protocol. After the induction, the relative proportion of this newly syn- thesized  -tubulin protein to the constitutive Tub2-590p was 10-fold higher in the Rbl2p pool but only about 1.5-fold higher in the ␣ /  -tubulin heterodimer (data not shown). These data demonstrate that newly synthesized  -tubulin can bind to Rbl2p before it incorporates into ␣ /  -tubulin. If the opposite were true, i.e., if  -tubulin could bind to Rbl2p only after it had been in heterodimer, we would not detect any enrichment for the induced protein in the Rbl2p pool, since the heterodimer pool is at least 50-fold larger than the Rbl2p pool. It is important to note, however, that this result does not demonstrate that this order is obligatory (see Discussion). Formation of Rbl2p–  -tubulin in vitro. To determine if Rbl2p could bind only to newly synthesized  -tubulin or if instead it could bind to  -tubulin that had previously been in ␣ /  -tubulin heterodimers, we used an in vitro assay. We ex- pressed His 6 -Rbl2p in E. coli and incubated it with extracts of wild-type yeast cells and then assayed for bound proteins by using Ni-NTA beads. We found that Rbl2p bound  -tubulin in a time-dependent fashion (Fig. 3). Like the complex formed in vivo, this in vitro complex contained only a trace amount of ␣ -tubulin, which amount did not increase with time. Therefore, it is likely that the ␣ -tubulin detected represents adventitious binding. The time course demonstrates a linear rate of association between Rbl2p and  -tubulin for at least 4 h. Extrapolated back to zero time, the kinetics give evidence for a small but reproducible initial burst of complex formation. One interpre- tation of this biphasic time course is that it represents reaction with two distinct in vitro pools of  -tubulin. The low rate at which the majority of the complex forms may represent a rate-determining release of free  -tubulin from the hetero- dimer, by far the predominant population of tubulin in the extract. The initial burst could represent the diffusion-con- trolled reaction of a small equilibrium population of undimer- ized  -tubulin in the yeast extracts, which should bind to Rbl2p at the diffusion-controlled limit. The level of  -tubulin that reacts at this high rate fits well with our estimate of the level of  -tubulin not associated with ␣ -tubulin in extracts (see above). These results suggest that Rbl2p can interact with  -tubulin molecules that have previously been dimerized and hence com- pletely folded. Conversely, the ability of  -tubulin to bind to Rbl2p in vitro is abolished by denaturation. We treated wild- type yeast protein extracts with 6 M guanidine hydrochloride and then diluted the protein into solutions containing His 6 - Rbl2p. Conventional chaperone binding and folding assays often make use of substrates that are denatured by treatment with 6 M guanidine hydrochloride. Relative to the control reaction mixtures that were diluted but not exposed to denaturing agent, the amount of  -tubulin bound to Rbl2p was only barely detectable (Fig. 4). In contrast, virtually no bound ␣ -tubulin was detected in either the denatured or untreated samples. Immunoblots of these samples with anti-Rbl2p demonstrated that the amounts of Rbl2p bound to beads were the same in the treated and untreated samples. This result is consistent with the failure of  -tubulin denatured in this fashion to bind the murine Rbl2p homolog, cofactor A, in the in vitro system (7, 8). Stability of the Rbl2p–  -tubulin complex. The in vitro ex- periment described above suggests that  -tubulin can transfer from ␣ -tubulin to Rbl2p. The in vivo experiment suggests that  -tubulin can interact with Rbl2p before it interacts with ␣ -tu- bulin. A crucial issue for understanding Rbl2p function is how these two complexes of  -tubulin compare to one another. Accordingly, we measured their stabilities in vitro. We isolated the His 6 -Rbl2p–  -tubulin complex from ex- tracts of yeast cells that overproduce both proteins and then measured the dissociation of the complex by monitoring the loss of  -tubulin from the Ni-NTA beads (see Materials and Methods). Under the conditions of this experiment, the His 6 - Rbl2p protein does not dissociate from the beads. As shown in Fig. 5, the Rbl2p–  -tubulin complex dissociates exponentially through about two half-lives. These results are consistent with a simple dissociation reaction with a half-life of about 2.5 h, corresponding to a dissociation rate constant, k off , of 8 ϫ 10 Ϫ 5 s Ϫ 1 . The stability of this complex should be compared to that of the ␣ /  -tubulin heterodimer with which it can interact. The equilibrium dissociation constant for that heterodimer is re- ported to be ϳ 8 ϫ 10 Ϫ 7 M (4). We cannot measure the com- parable constant for the Rbl2p–  -tubulin complex, either di- rectly or indirectly (by measuring its association rate constant), since we do not have a source of native monomeric  -tubulin. However, the bimolecular association constants for molecules of similar sizes are on the order of 10 7 to 10 8 M Ϫ 1 s Ϫ 1 (9). If we choose the conservative value of 10 6 M Ϫ 1 s Ϫ 1 , the Rbl2p–  -tubulin complex would have a K d of ϳ 10 Ϫ 10 . That value would make the Rbl2p–  -tubulin complex significantly more stable than the ␣ /  -tubulin heterodimer. To compare the sta- bilities of the complexes more directly by similar assays, we prepared ␣ /  -tubulin heterodimer from cells expressing His 6 - Tub2p (see Materials and Methods). We isolated this complex on Ni-NTA beads and then monitored its dissociation by as- saying for loss of the ␣ -tubulin polypeptide. The rate of ␣ -tu- bulin loss from the beads is low, consistent with a half-life for the heterodimer of about 10 h (Fig. 5). Therefore, by using essentially the same method to assay the stabilities of the two complexes, it can be concluded that the ␣ /  -tubulin hetero- dimer dissociates much more slowly than does the Rbl2p–  - tubulin complex. That  -tubulin can interact specifically with a protein other than ␣ -tubulin suggests several possible functions for such a complex. The results presented above characterize the forma- tion and properties of the Rbl2p–  -tubulin complex. The results demonstrate that the formation in vitro of the Rbl2p–  -tubulin complex is dependent upon the conformation of  -tubulin. Although there may be conformational alter- ations of  -tubulin prior or subsequent to binding Rbl2p, the form of  -tubulin that binds Rbl2p is at least in equilibrium with the form that binds ␣ -tubulin. That both the in vivo and in vitro complexes are essentially devoid of ␣ -tubulin argues that Rbl2p competes with ␣ -tubulin for binding to  -tubulin, per- haps because both ligands bind to similar sites on  -tubulin. These characteristics of the Rbl2p–  -tubulin complex are con- sistent with the comparable efficiencies of Rbl2p and ␣ -tubulin in rescuing cells from  -tubulin lethality. In cell extracts, it is clear that much more  -tubulin is associated with ␣ -tubulin than with Rbl2p, reflecting not only relative stabilities but also the likelihood that there is much less Rbl2p than ␣ -tubulin in wild-type cells. We note that the overproduction of Rbl2p is modestly toxic (1), a phenotype that may be explained by the ability of high levels to compete successfully with ␣ -tubulin and so to sequester  -tubulin. Two results are consistent with a role for Rbl2p in the pathway leading to heterodimer formation. First, the in vitro data suggest that the ␣ /  -tubulin heterodimer is more stable than the Rbl2p–  -tubulin complex. Of course this comparison is of dissociation rates that need not reflect conditions in vivo. For example, that the tubulin heterodimer can have an alter- native fate to dissociation, i.e., polymerization, may affect its apparent stability in the cytoplasm. In addition, there may be effectors that modify the stability of either  -tubulin complex. Second, the pulse induction experiment shows that  -tubulin can ...
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... into solutions containing His 6 - Rbl2p. Conventional chaperone binding and folding assays often make use of substrates that are denatured by treatment with 6 M guanidine hydrochloride. Relative to the control reaction mixtures that were diluted but not exposed to denaturing agent, the amount of -tubulin bound to Rbl2p was only barely detectable (Fig. 4). In contrast, virtually no bound -tubulin was detected in either the denatured or untreated samples. Immunoblots of these samples with anti-Rbl2p demonstrated that the amounts of Rbl2p bound to beads were the same in the treated and untreated samples. This result is consistent with the failure of -tubulin denatured in this fashion to ...
Citations
... However, functional contributions of TBC components do not restrict to tubulin biogenesis ('iv' in Fig.1e): they have also been shown to play roles in tubulin dissociation (Bhamidipati et al., 2000;Martín et al., 2000), storage/recycling (Abruzzi et al., 2002;Archer et al., 1998;Fanarraga et al., 1999), and degradation (Keller and Lauring, 2005). In the following, we will first outline current knowledge of the single TBC components in the canonical pathway. ...
... In contrast, over-expression experiments with TBCA and Rbl2 A in other contexts failed to reveal clear phenotypes (Lopez-Fanarraga et al., 2001) suggesting that the primary role of TBCA is to maintain β-tubulin pools that can feed tubulin biogenesis (Lopez-Fanarraga et al., 2001) or tubulin recycling processes (see 2 nd to last section). In agreement with this notion, Rbl2 A reduces the toxicity of over-expressed β-tubulin through direct interaction (Abruzzi et al., 2002;Archer et al., 1998), and a similar buffering role was observed in mouse (Fanarraga et al., 1999). Alp31 A seems to share these fundamental functions, since essential loss-of-function mutant phenotypes can be rescued by Rbl2 A ; but unlike Rbl2 A , its over-expression is lethal and causes MT network fragmentation, suggesting additional roles not displayed by its orthologues (Radcliffe, Pippa A et al., 2000). ...
Axons are the narrow, up-to-meter long cellular processes of neurons that form the biological cables wiring our nervous system. Most axons must survive for an organism’s lifetime, i.e. up to a century in humans. Axonal maintenance depends on loose bundles of microtubules that run without interruption all along axons. The continued turn-over and the extension of microtubule bundles during developmental, regenerative or plastic growth requires the availability of α/β-tubulin heterodimers up to a meter away from the cell body. The underlying regulation in axons is poorly understood and hardly features in past and contemporary research. Here we discuss potential mechanisms, particularly focussing on the possibility of local tubulin biogenesis in axons. Current knowledge might suggest that local translation of tubulin takes place in axons, but far less is known about the post-translational machinery of tubulin biogenesis involving three chaperone complexes: prefoldin, CCT and TBC. We discuss functional understanding of these chaperones from a range of model organisms including yeast, plants, flies and mice, and explain what is known from human diseases. Microtubules across species depend on these chaperones, and they are clearly required in the nervous system. However, most chaperones display a high degree of functional pleiotropy, partly through independent functions of individual subunits outside their complexes, thus posing a challenge to experimental studies. Notably, we found hardly any studies that investigate their presence and function particularly in axons, thus highlighting an important gap in our understanding of axon biology and pathology.
... Wild-type and tub3Δ control cells are not affected by overexpression of TBCD/Cin1. We also tested the effect of overexpressing TBCA/Rbl2, which binds to β-tubulin in the heterodimer and monomer forms (Archer et al., 1998). TBCA/Rbl2 overexpression strongly inhibits tub1-ND tub3-ND mutants, but slightly suppresses the benomyl sensitivity of tub1-ND single mutants ( Figure 8B). ...
... Previous studies show that excess free β-tubulin, generated either by increasing its expression or by decreasing levels of α-tubulin, has toxic effects (Burke et al., 1989;Katz et al., 1990). This toxicity is suppressed by TBCA/Rbl2, which binds to β-tubulin monomer (Archer et al., 1995;Archer et al., 1998). We find that overexpressing TBCA/Rbl2 suppresses the sensitivity of tub1-ND single mutants to microtubule depolymerizing drugs, and that rbl2Δ null mutants are lethal when combined with tub1-ND single mutants ( Figure 8; Table 1). ...
Mutations in the microtubule cytoskeleton are linked to cognitive and locomotor defects during development, and neurodegeneration in adults. How these mutations impact microtubules, and how this alters function at the level of neurons is an important area of investigation. Using a forward genetic screen in mice, we identified a missense mutation in Tuba1a α-tubulin that disrupts cortical and motor neuron development. Homozygous mutant mice exhibit cortical dysgenesis reminiscent of human tubulinopathies. Motor neurons fail to innervate target muscles in the limbs and show synapse defects at proximal targets. To directly examine effects on tubulin function, we created analogous mutations in the α-tubulin isotypes in budding yeast. These mutations sensitize yeast cells to microtubule stresses including depolymerizing drugs and low temperatures. Furthermore, we find that mutant α-tubulin is depleted from the cell lysate and from microtubules, thereby altering ratios of α-tubulin isotypes. Tubulin-binding cofactors suppress the effects of the mutation, indicating an important role for these cofactors in regulating the quality of the α-tubulin pool. Together, our results give new insights into the functions of Tuba1a, mechanisms for regulating tubulin proteostasis, and how compromising these may lead to neural defects.
... By this assay, 93% of tubulin in wild-type cells elutes from the column as heterodimer. The 7% of the tubulin that elutes at the void volume, in the position of aggregated tubulin, likely represents an artifact of the sample preparation technique, since all of the a-tubulin co-immunoprecipitates with b-tubulin from extracts of wild-type cells (Archer et al. 1998). In contrast, pac10D, plp1D, and pac10D plp1D yap4D cells all show much larger and significant amounts of heterodimer in aggregates. ...
In budding yeast, the essential roles of microtubules include segregating chromosomes and positioning the nucleus during mitosis. Defects in these functions can lead to aneuploidy and cell death. To ensure proper mitotic spindle and cytoplasmic microtubule formation, the cell must maintain appropriate stoichiometries of alpha- and beta-tubulin, the basic subunits of microtubules. The experiments described here investigate the minimal levels of tubulin heterodimers needed for mitotic function. We have found a triple-mutant strain, pac10Delta plp1Delta yap4Delta, which has only 20% of wild-type tubulin heterodimer levels due to synthesis and folding defects. The anaphase spindles in these cells are approximately 64% the length of wild-type spindles. The mutant cells are viable and accurately segregate chromosomes in mitosis, but they do have specific defects in mitosis such as abnormal nuclear positioning. The results establish that cells with 20% of wild-type levels of tubulin heterodimers can perform essential cellular functions with a short spindle, but require higher tubulin heterodimer concentrations to attain normal spindle length and prevent mitotic defects.
... Dissociation of the heterodimer also occurs after treatment with lactoperoxidase, which forms complexes with the tubulin monomers (Wolff and Knipling, 1995) and during immunopurification in Tris buffer (Giraudel et al., 1998). At least three proteins, Rbl2p-β/cofactorA, Cin1p/cofactorD and the inner centromere protein have been shown by immunoprecipitation to interact with monomeric βtubulin, and not with α-tubulin (Archer et al., 1995;Archer et al., 1998;Fleming et al., 2000;Wheatley et al., 2001). ...
Huntington's disease results from an expansion of a series of glutamine repeats in the protein huntingtin. We have discovered from immunopurification studies that huntingtin combines specifically with the beta subunit of tubulin. This binding explains why huntingtin can be shown on assembled microtubules by electron microscopy. Immunostaining shows that most of the huntingtin in the cytoplasm is associated with microtubules. Huntingtin is particularly abundant in the perinuclear region, where it is also associated with microtubules and in the centrosomal region, where it co-localizes with gamma-tubulin. In Huntington's disease, inclusions are often nuclear or perinuclear. Since the perinuclear concentration of huntingtin does not depend on the number of its glutamine repeats, we propose that inclusions are found in perinuclear and intranuclear locations because the beta-tubulin binding property of huntingtin brings it to the perinuclear region, from which it readily gains access to the nucleus. The mutational glutamine expansion then promotes insolubility and results in an inclusion.
... Identified in a search for proteins that, when overexpressed, rescue cells from the toxicity of free -tubulin (5), Rbl2p binds monomeric -tubulin to form a heterodimer that excludes ␣-tubulin, both in vivo and in vitro (5). Pulse-labeling experiments demonstrate that Rbl2p can bind both newly synthesized -tubulin before it is incorporated into ␣/-tubulin heterodimers and -tubulin released by dissociation of heterodimers (4). However, the precise function of Rbl2p in vivo is not known. ...
... To examine protein levels in cells overexpressing RBL2 and -tubulin (data not shown), cells were grown overnight to early log phase (10 7 /ml) in selective raffinose medium and then induced with 2% galactose for 3 h. A total of 2 ϫ 10 9 cells in mid-log phase (2 ϫ 10 7 to 3 ϫ 10 7 cells/ml) were harvested in all cases, and cell lysates were prepared by using a glass bead smash protocol as previously described (4). Serial dilutions of both the cell lysates and the standard (see below) were resolved on sodium dodecyl sulfate-14% (Rbl2p) or -7.5% (-tubulin) polyacrylamide gels. ...
... Recombinant Rbl2p was generated as previously described (4). The concentration of the recombinant Rbl2p standard (1.3 g/l) was determined with reference to a bovine serum albumin standard (1 mg/ml) after staining with Ponceau Red. ...
Free beta-tubulin not in heterodimers with alpha-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free beta-tubulin. This study focused specifically on the function of Rbl2p, which, like alpha-tubulin, can rescue cells from free beta-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of beta-tubulin relative to alpha-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of beta-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to beta-tubulin. The data suggest that Rbl2p binds transiently to free beta-tubulin, which then passes into an aggregated form that is not toxic.
... Identified in a search for proteins that, when overexpressed, rescue cells from the toxicity of free -tubulin (5), Rbl2p binds monomeric -tubulin to form a heterodimer that excludes ␣-tubulin, both in vivo and in vitro (5). Pulse-labeling experiments demonstrate that Rbl2p can bind both newly synthesized -tubulin before it is incorporated into ␣/-tubulin heterodimers and -tubulin released by dissociation of heterodimers (4). However, the precise function of Rbl2p in vivo is not known. ...
... To examine protein levels in cells overexpressing RBL2 and -tubulin (data not shown), cells were grown overnight to early log phase (10 7 /ml) in selective raffinose medium and then induced with 2% galactose for 3 h. A total of 2 ϫ 10 9 cells in mid-log phase (2 ϫ 10 7 to 3 ϫ 10 7 cells/ml) were harvested in all cases, and cell lysates were prepared by using a glass bead smash protocol as previously described (4). Serial dilutions of both the cell lysates and the standard (see below) were resolved on sodium dodecyl sulfate-14% (Rbl2p) or -7.5% (-tubulin) polyacrylamide gels. ...
... Recombinant Rbl2p was generated as previously described (4). The concentration of the recombinant Rbl2p standard (1.3 g/l) was determined with reference to a bovine serum albumin standard (1 mg/ml) after staining with Ponceau Red. ...
Free β-tubulin not in heterodimers with α-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free β-tubulin. This study focused specifically on the function of Rbl2p, which, like α-tubulin, can rescue cells from free β-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of β-tubulin relative to α-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of β-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to β-tubulin. The data suggest that Rbl2p binds transiently to free β-tubulin, which then passes into an aggregated form that is not toxic.
... The size of the p14/-tubulin complex determined by gel filtration analysis is about 60 kDa, in accordance with a 1:1 stoichiometry. As is the case for CoA/p14 (Llosa et al., 1996), Rbl2p has also been shown to form a 1:1 stoichiometric complex with -tubulin in vitro (Archer et al., 1998). Recently, the crystal structure of Rbl2p was obtained at a resolution of 2.2 Å. ...
The microtubule cytoskeleton consists of a highly organized network of microtubule polymers bound to their accessory proteins: microtubule-associated proteins, molecular motors, and microtubule-organizing proteins. The microtubule subunits are heterodimers composed of one alpha-tubulin polypeptide and one beta-tubulin polypeptide that should undergo a complex folding processing before they achieve a quaternary structure that will allow their incorporation into the polymer. Due to the extremely high protein concentration that exists at the cell cytoplasm, there are alpha- and beta-tubulin interacting proteins that prevent the unwanted interaction of these polypeptides with the surrounding protein pool during folding, thus allowing microtubule dynamics. Several years ago, the development of a nondenaturing electrophoretic technique made it possible to identify different tubulin intermediate complexes during tubulin biogenesis in vitro. By these means, the cytosolic chaperonin containing TCP-1 (CCT or TriC) and prefoldin have been demonstrated to intervene through tubulin and actin folding. Various other cofactors also identified along the alpha- and beta-tubulin postchaperonin folding route are now known to have additional roles in tubulin biogenesis such as participating in the synthesis, transport, and storage of alpha- and beta-tubulin. The future characterization of the tubulin-binding sites to these proteins, and perhaps other still unknown proteins, will help in the development of chemicals that could interfere with tubulin folding and thus modulating microtubule dynamics. In this paper, current knowledge of the above postchaperonin folding cofactors, which are in fact chaperones involved in tubulin heterodimer quaternary structure achievement, will be reviewed.
... cofactors) involved in tubulin heterodimer forma«on and microtubule assembly: e.g. Rbl2p, Rki 1 p, Pac I Op and a tubulin-bindir)g protein, Pac2p, that aids folding into an assembly competent state (Alvarei et al. 1998;Archer et al. 1998;Smith et al. 1998). Although these and other cofactors may not be required for cell viability, at least one CIN1 homologue from fission yeast, alp 1, appears to be essential and was shown to associate with microtubules in vivo (Hirata et al. 1998). ...
Microtubules are biochemically one of the simplest, yet functionally most important, cellular organelles in the plant and animal kingdoms. They are integral components of a dynamic, three-dimensional framework referred to as the cytoskeleton. In addition to a fundamental role in intracellular movement, the microtubule, microfilament and intermediate filament arrays of the cytoskeleton are networks upon which asymmetric distribution of subcellular constituents is established and from which these polarized regulatory molecules mediate morphogenesis. The functions of microtubules reflect this common theme of distribution and movement. In plants, four principal microtubular functions are recognized: determination of the division plane; translocation of chromosomes; cell plate/phragmoplast formation; and control of cell morphology. Obviously, from a biotechnological point of view, the ability to control and regulate these processes via modifications of the component parts will have a profound effect on plant growth and development. Microtubules are composed primarily of a single, repeating macromolecular unit — tubulin. Tubulin itself is a heterodimeric protein, composed of two similar subunits: alpha-(α) and beta-(β)tubulin. The α- and β-tubulins are typically encoded by gene families, and these give rise to various tubulin isotypes that are differentially expressed and modified during growth and development. In addition, a ubiquitous and diverse class of proteins that bind to microtubules, known as microtubule associated proteins (MAPs), are believed to be important in nucleation, stabilization and bundling of microtubules.
... Finally, the results we present here, in combination with the work of others, show that the interactions of the yeast cofactors with the tubulin proteins are the same as those of the mammalian cofactors. Alf1p and Pac2p associate with ␣-tubulin, and not -tubulin (see Fig. 1 B; Vega et al., 1998), whereas Rbl2p binds to -tubulin, and not ␣-tubulin (Archer et al., 1995(Archer et al., , 1998. We have also found that Cin1p interacts with -tubulin, and not ␣-tubulin (Feierbach, B., and T. Stearns, unpublished results) The similarity of these interactions with those of the mammalian proteins suggests that similar pathways are operating in both yeast and mammalian cells. ...
Tubulin is a heterodimer of alpha- and beta-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287-296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821-832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with alpha-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with alpha-tubulin. Mutations in alpha-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of alpha-tubulin; this domain is distinct from the region of interaction between alpha-tubulin and beta-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in alpha-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast alpha-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional alpha-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester alpha-tubulin from interaction with beta-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.
... Finally, the results we present here, in combination with the work of others, show that the interactions of the yeast cofactors with the tubulin proteins are the same as those of the mammalian cofactors. Alf1p and Pac2p associate with ␣-tubulin, and not -tubulin (see Fig. 1 B; Vega et al., 1998), whereas Rbl2p binds to -tubulin, and not ␣-tubulin (Archer et al., 1995(Archer et al., , 1998. We have also found that Cin1p interacts with -tubulin, and not ␣-tubulin (Feierbach, B., and T. Stearns, unpublished results) The similarity of these interactions with those of the mammalian proteins suggests that similar pathways are operating in both yeast and mammalian cells. ...
Tubulin is a heterodimer of α- and β-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287–296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821–832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with α-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with α-tubulin. Mutations in α-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of α-tubulin; this domain is distinct from the region of interaction between α-tubulin and β-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in α-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast α-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional α-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester α-tubulin from interaction with β-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.
