Tubulin Folding Cofactors as GTPase-activating Proteins

Microtubules are highly dynamic structures, composed of ␣/␤-tubulin heterodimers. Biosynthesis of the functional dimer involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, 2). We show that cofactor D is also a centrosomal protein and that overexpression of either the fulllength protein or either of two centrosome localization domains leads to the loss of anchoring of the ␥-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor D by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor D activity produced a change in the levels of ␣-or ␤-tubulin, we conclude that these newly discovered functions for cofactor D are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor D at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.Centrosomes are the major microtubule-organizing center in animal cells and are composed of two centrioles (barrelshaped structures composed of ␣/␤-tubulin heterodimers) surrounded by a dense fibrillar network of proteins called the pericentriolar material (PCM).2 A key step in the initiation of new microtubule growth is the regulated recruitment to the PCM of ␥-tubulin ring complexes (␥-TuRCs), consisting of ␥-tubulin and ␥-complex proteins (GCPs) (3-7), that promote the polymerization of ␣/␤-tubulin heterodimers. In addition to nucleating new microtubule growth, centrosomes also organize cytosolic microtubules during interphase, spindle microtubules during mitosis, and axonemes in ciliogenesis (8, 9).Formation of ␣/␤ tubulin heterodimers is promoted by five tubulin-specific co-chaperones, termed cofactors A-E, that act on ␣-and ␤-tubulin folding intermediates in a stepwise process that generates polymerizable heterodimers (1). Demonstration of the roles for these five cofactors in folding tubulin heterodimers comes from in vitro folding assays, which also allowed purification of the five co-chaperones (1, 10). This function is consistent with genetic studies in Saccharomyces cerevisiae (11,12), Schizosaccharomyces pombe (13-15), Arabidopsis thaliana (16,17), and Caenorhabditis elegans (18) in which mutations in cofactor D (CoD) yielded defects in microtubule-dependent processes, including maintenance of chromosome number.That the tubulin-specific cofactors may play additional roles in regulating microtubule stability or dynamicity was first proposed by Tian et al. (2), who found that overexpression of bovine CoD in HeLa cells led to the loss of microtubules and decreased levels of ␣-tubulin in cells. With additional in vitro data showing that purified bovine CoD disrupts the tubulin heterodimer, the authors proposed a role for CoD in heterodimer destruction, with a secondary loss of microtubules (19,20). A regulated destructive pathway was suggested by the observations that increa...

Section: Resultsmentioning

confidence: 94%

Cofactor D Functions as a Centrosomal Protein and Is Required for the Recruitment of the γ-Tubulin Ring Complex at Centrosomes and Organization of the Mitotic Spindle

Cunningham

Kahn

2008

“…1 legend). Release of radiolabeled inorganic phosphate was measured at 1 min intervals as described (10).…”

Section: Methodsmentioning

confidence: 99%

“…Each step in this pathway has been deduced from in vitro reconstitution experiments using purified components (7)(8)(9). The pathway hinges on the formation of a supercomplex containing ␣Ϫ and ␤-tubulin and cofactors C, D, and E. The hydrolysis of GTP by tubulin, stimulated by cofactors C and D, is part of the heterodimer assembly reaction; the stimulated hydrolysis of GTP by ␤-tubulin acts as a switch for the release from the supercomplex of native, newly made tubulin heterodimers (10). Cofactor C and D in combination have also been shown to be a GTPase activator (GAP) for native tubulin; cofactor E enhances this tubulin-GAP activity (10).…”

mentioning

confidence: 99%

“…The pathway hinges on the formation of a supercomplex containing ␣Ϫ and ␤-tubulin and cofactors C, D, and E. The hydrolysis of GTP by tubulin, stimulated by cofactors C and D, is part of the heterodimer assembly reaction; the stimulated hydrolysis of GTP by ␤-tubulin acts as a switch for the release from the supercomplex of native, newly made tubulin heterodimers (10). Cofactor C and D in combination have also been shown to be a GTPase activator (GAP) for native tubulin; cofactor E enhances this tubulin-GAP activity (10). These biochemical data are consistent with the wealth of genetic evidence on cofactor homologs in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (11)(12)(13)(14).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Functional Overlap between Retinitis Pigmentosa 2 Protein and the Tubulin-specific Chaperone Cofactor C

Bartolini¹,

Bhamidipati²,

Thomas³

et al. 2002

Self Cite

101

102

Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells. The retinitis pigmentosa 2 protein (RP2) is similar in sequence to the tubulin-specific chaperone cofactor C. Together with cofactors D and E, cofactor C stimulates the GTPase activity of native tubulin, a reaction regulated by ADP-ribosylation factor-like 2 protein. Here we show that in the presence of cofactor D, RP2 protein also stimulates the GTPase activity of tubulin. We find that this function is abolished by mutation in an arginine residue that is conserved in both cofactor C and RP2. Notably, mutations that alter this arginine codon cause familial retinitis pigmentosa. Our data imply that this residue acts as an "arginine finger" to trigger the tubulin GTPase activity and suggest that loss of this function in RP2 contributes to retinal degeneration. We also show that in Saccharomyces cerevisiae, both cofactor C and RP2 partially complement the microtubule phenotype resulting from deletion of the cofactor C homolog, demonstrating their functional overlap in vivo. Finally, we find that RP2 interacts with GTP-bound ADP ribosylation factor-like 3 protein, providing a link between RP2 and several retinal-specific proteins, mutations in which also cause retinitis pigmentosa.Retinitis pigmentosa is a degenerative disease and the major cause of heritable blindness (1). It is caused by mutations in a dizzying variety of genes; some are structural proteins of photoreceptors, many are involved in photoreception and transduction, and a few are seemingly unrelated ubiquitously expressed proteins (see RetNet, www.sph.uth.tmc.edu/retnet/). Most (80 -90%) cases of X-linked retinitis pigmentosa are caused by mutations in two genes, XRP2 and XRP3 (2). These were isolated by positional cloning and shown to encode, respectively, the RP2 1 protein (3) and the retinitis pigmentosa GTPase regulator (RP3) (4, 5). The function of RP2 is unknown, and the gene is expressed at a low level in all tissues examined; it is targeted to the plasma membrane by myristoylation and palmitoylation of its amino terminus (6).RP2 has amino acid sequence similarity over half of its length to the tubulin-specific chaperone protein cofactor C (3). It has been shown that cofactor C acts in a pathway together with four other tubulin-specific chaperone proteins (cofactors A, B, D, and E) to chaperone quasi-native ␣-and ␤-tubulin subunits released from the cytosolic chaperonin CCT and assemble the ␣/␤-tubulin heterodimer. Each step in this pathway has been deduced from in vitro reconstitution experiments using purified components (7-9). The pathway hinges on the formation of a supercomplex containing ␣Ϫ and ␤-tubulin and cofactors C, D, and E. The hydrolysis of GTP by tubulin, stimulated by cofactors C and D, is part of the heterodimer assembly reaction; the stimulated hydrolysis of GTP by ␤-tubulin acts as a switch for the release from the supercomplex of native, newly made tubulin heterodimers (10). Cofactor C and D in combination have also b...

“…The biological significance of this process lies in the fact that the ability of ␤-tubulin to hydrolyze GTP is an essential property for the proper functioning of microtubule dynamics. The same reaction also serves to regulate microtubule dynamics by modulating the availability of GTP-bound heterodimers (14,15). Direct, biochemical evidence for a role of Arl2 in the complex process of the regulation of the action of one of these cofactors, cofactor D, was provided by Bhamidipati et al (16).…”

mentioning

confidence: 99%

Cytosolic Arl2 Is Complexed with Cofactor D and Protein Phosphatase 2A

Shern

Sharer

Pallas

et al. 2003

Self Cite

Arl2 is a member of the ADP-ribosylation factor family of 20-kDa GTPases that is highly conserved in eukaryotes. Recent results revealed that a portion of cellular Arl2 and its binding partner, BART, localize to mitochondria. Because ϳ90% of cellular Arl2 is cytosolic, we investigated properties of the soluble protein and found that it is stably bound in a complex that migrates in gel filtration medium with a predicted molecular mass of ϳ300 kDa. This complex was purified ϳ500-fold from the soluble fraction of bovine brain. Protein components were identified by mass spectroscopy and revealed the presence of four other proteins that include the tubulin folding cochaperone cofactor D and all three subunits of at least two protein phosphatase 2A (PP2A) protein phosphatase trimers. The presence of more than one PP2A B-type subunit and the low stoichiometry of Arl2 indicate that the purified preparation still contains a mixture of complexes that cannot currently be completely resolved. Thus, although all the soluble Arl2 in bovine brain is in high molecular mass complexes, only a portion of the total cellular cofactor D and PP2A are associated with the Arl2. We further show that the Arl2 in the complex cannot bind GTP and that complexed cofactor D does not efficiently participate in tubulin refolding reactions in a manner comparable with free cofactor D. Our data suggest functional roles for the cytosolic Arl2 complex in modulating tubulin and microtubule behavior as well as a possible role in apoptosis.