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© The Rockefeller University Press, 0021-9525/1999//721 $5.00
The Journal of Cell Biology, Volume 144, Number 4, , 1999 721-733

Characterization of Two Related Drosophila -tubulin Complexes that Differ in Their Ability to Nucleate Microtubules

Karen Oegema^*, Christiane Wiese, Ona C. Martin, Ronald A. Milligan, Akihiro Iwamatsu^||, Timothy J. Mitchison^*, and Yixian Zheng

^* Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; Department of Embryology, Carnegie Institute of Washington, Baltimore, Maryland 21210; Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037; and ^|| Central Laboratories for Key Technology, Kirin Brewery Company, Ltd., Yokohama 236, Japan

-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1– 12). Here, we report the purification and characterization of both complexes that we name -tubulin small complex (TuSC; 280,000 D) and Drosophila TuRC (2,200,000 D). In addition to -tubulin, the TuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The TuSC is a structural subunit of the TuRC, a larger complex containing about six additional polypeptides. Like the TuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), the Drosophila TuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with 13 radially arranged structural repeats. The TuSC also nucleates microtubules, but much less efficiently than the TuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the TuSC reveals that -tubulin binds preferentially to GDP over GTP, rendering -tubulin an unusual member of the tubulin superfamily.

Key Words: centrosome • cytoskeleton • nucleation • GTP • GDP

Abbreviations used in this paper: Dgrip, Drosophila gamma ring protein; TuRC, -tubulin ring complex; TuSC, -tubulin small complex; MT, microtubule; PCM, pericentriolar material; PEG, polyethylene glycol; Sc TuSC, Saccharomyces cerevisiae 6 S complex.

THE microtubule (MT)¹ cytoskeleton is essential for cell division and organization of the interphase cytoplasm. These functions are orchestrated by diverse and highly dynamic MT arrays generated by a variety of mechanisms including regulation of the polymerization dynamics of MTs, of proteins that interact with and organize MTs, and of MT nucleation (Desai and Mitchison, 1997). The latter mechanism is possible because the spontaneous nucleation of new tubulin polymers is kinetically limiting, both in vitro when the polymerization of pure tubulin is initiated, and in vivo (Alberts et al., 1994).

Evidence for a kinetic barrier to MT nucleation in vivo comes from analysis of repolymerization of MTs after cold treatment or treatment with anti-MT agents. In many animal cells, regrowth initiates from the pericentriolar material (PCM) that surrounds the centrioles (Frankel, 1976; Osborn and Weber, 1976; Keryer et al., 1984; Meads and Schroer, 1995), demonstrating that the PCM promotes MT nucleation. A major breakthrough in defining the molecular basis of the MT-nucleating activity of the PCM was the discovery of -tubulin (Oakley and Oakley, 1989). -tubulin is a member of the tubulin superfamily that localizes to MT organizing centers and is found in all eukaryotes (reviewed in Joshi, 1994; Pereira and Schiebel, 1997). Genetic studies have demonstrated that -tubulin is required for normal cytoplasmic and spindle MT formation in Aspergillus nidulans (Martin et al., 1997; Oakley et al., 1990), Schizosaccharomyces pombe (Horio et al., 1991), Drosophila melanogaster (Sunkel et al., 1995), and Saccharomyces cerevisiae (Sobel and Snyder, 1995; Marschall et al., 1996; Spang et al., 1996). Antibody inhibition experiments in vertebrates have also implicated -tubulin in MT nucleation by the centrosome (Joshi et al., 1992; Felix et al., 1994).

In higher eukaryotes, soluble -tubulin exists primarily in a large complex (between 25 and 32 S; Stearns and Kirschner, 1994; Meads and Schroer, 1995; Zheng et al., 1995; Detraves et al., 1997; Moritz et al., 1998; Murphy et al., 1998). Recently, this complex was purified from Xenopus egg extracts and shown to nucleate MTs in vitro (Zheng et al., 1995). This complex, called the TuRC (-tubulin ring complex), consists of about eight proteins in addition to -tubulin and has the appearance of an open ring with approximately the same diameter as a MT (Zheng et al., 1995). Rings of this diameter have also been observed in the PCM of centrosomes isolated from Drosophila embryos (Moritz et al., 1995a) and the surf clam, Spisula (Vogel et al., 1997). In Drosophila, immunoelectron microscopy has confirmed the presence of clusters of -tubulin in the ring structures and at the base of MTs nucleated by the PCM (Moritz et al., 1995b). Cumulatively, these results suggest that the TuRC is a highly conserved structure responsible for the MT-nucleating activity of the PCM.

The -tubulin in S. cerevisiae is the most divergent of all -tubulins. It is only 35–40% identical to the other known -tubulins, all of which are at least 65% identical to each other (Marschall et al., 1996). In S. cerevisiae, the only known soluble -tubulin–containing complex is 6 S and contains three proteins: -tubulin, and two related proteins, Spc97p and Spc98p (Geissler et al., 1996; Knop et al., 1997; Knop and Schiebel, 1997). Immunoprecipitation experiments with tagged proteins suggest that the S. cerevisiae complex contains one molecule of Spc97p, one molecule of Spc98p, and two or more molecules of -tubulin (Knop et al., 1997; Knop and Schiebel, 1997). The yeast -tubulin 6 S complex is thought to be anchored to the cytoplasmic side of the spindle pole body through the interaction of Spc97p and Spc98p with Spc72p (Knop and Schiebel, 1998), and to the nuclear side of the spindle pole body through interaction with the NH₂ terminus of Spc110p (Knop and Schiebel, 1997). To date, in vitro MT-nucleating activity for the yeast complex has not been demonstrated. Therefore, it remains unclear whether the yeast -tubulin complex nucleates MTs directly, or whether it assembles into a larger, perhaps TuRC-like structure at the spindle pole body. Interestingly, homologues of Spc97p and Spc98p in humans (hGCP2 and hGCP3/ HsSpc98; Murphy et al., 1998; Tassin et al., 1998) and in Xenopus (Xgrip109; Martin et al., 1998) colocalize with -tubulin at the centrosome and cosediment with -tubulin on sucrose gradients, indicating that they are components of the large -tubulin–containing complexes present in these organisms.

Understanding the role of -tubulin in MT nucleation is a challenging endeavor. Low cellular concentrations make purification from native sources difficult, and the complexity of the protein complexes that contain -tubulin limits expression-based studies. Analysis of MT nucleation is further complicated by the following: the complex structure of the MT lattice (Wade and Chretien, 1993), the large number of tubulin molecules potentially involved in the formation of a nucleus (Voter and Erickson, 1984; Fygenson et al., 1995), and the potential role of β-tubulin GTP hydrolysis in suppressing nucleation (Hyman et al., 1992). This difficulty is reflected by the fact that the mechanism of spontaneous nucleation of purified tubulin remains poorly understood (Voter and Erickson, 1984; Fygenson et al., 1995).

Central to understanding the mechanism of MT nucleation by -tubulin–containing complexes will be to understand the relationship between -tubulin and other members of the tubulin superfamily. One important aspect of this relationship is the nature of the contacts -tubulin makes with itself and with - or β-tubulin. A second important aspect is how -tubulin compares to other members of the tubulin family in its ability to bind and hydrolyze GTP. If -tubulin binds a guanine nucleotide, it will be important to determine whether nucleotide exchange and hydrolysis contribute to its ability to assemble, disassemble, nucleate, or release MTs, or whether the bound nucleotide has a structural role, as is the case for -tubulin.

In this paper, we begin to address the functional organization of the TuRC by purifying and analyzing -tubulin– containing complexes from Drosophila embryo extracts. In Drosophila, there are two related -tubulin–containing complexes. The larger complex can be collapsed into the smaller complex by treatment with high salt. This condensation suggests that the small complex is a structural subunit of the large complex (Moritz et al., 1998). We purify both complexes and show that the large Drosophila complex nucleates MTs much more potently than the small complex. We also show that, in contrast to - and β-tubulin which preferentially bind GTP, -tubulin in the small complex preferentially binds GDP.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Buffers and Reagents
HB: 50 mM K-Hepes, pH 7.6, 1 mM MgCl₂, 1 mM EGTA, 1 mM β-mercaptoethanol (β-ME) and protease inhibitor stock (1:200 final dilution; see below). HB100: HB plus 100 mM NaCl; HB200: HB plus 200 mM NaCl; and HB500: HB plus 500 mM NaCl. EB200: HB200 plus 100 µM GTP and 1 mg/ml DrosC17 peptide; EB500: HB500 plus 100 µM GTP and 1 mg/ml DrosC17 peptide; HB block: 50 mM K-Hepes, pH 7.6, 100 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mg/ml bovine serum albumin (fraction V; Sigma Chemical Co.). Homogenization buffer: HB100 plus 10% glycerol, 1 mM PMSF and protease inhibitor stock (1:100 final dilution). Protease inhibitor stock: 10 mM benzamidine-HCl, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin A in ethanol. LPC: 10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml chymostatin dissolved in DMSO. Mounting medium: 20 mM Tris-Cl, pH 9.0, 90% glycerol, and 0.1% p-phenylenediamine. BRB80: 80 mM K-PIPES, pH 6.8, 1 mM MgCl₂, 1 mM EGTA; 4x sample buffer: 250 mM Tris, pH 6.8, 12% SDS wt/vol, 20% β-ME vol/vol, and 40% glycerol vol/vol. GTP stock: 100 mM GTP (Boehringer Mannheim Corp.). Tubulin was purified from the bovine brain and labeled with tetramethylrhodamine as described (http://skye.med.harvard.edu). Drosophila embryo extract was prepared by homogenizing 0–3.5-h Drosophila embryos in homogenization buffer as described (Moritz et al., 1998). Clarified extract was prepared by centrifugation of crude extract for 10 min at 15,000 rpm (SS34 rotor; Sorvall) at 4°C. The supernatant was transferred to a new tube, and centrifuged a second time at 50,000 rpm in a rotor (50.2 Ti or SW55; Beckman) for 1 h at 4°C.

Sucrose Gradient Sedimentation and Gel Filtration Chromatography
Sucrose gradients (5–20 or 5–40%) were poured as step gradients (five steps of equal volume) in HB containing 100 or 500 mM NaCl plus nucleotide and allowed to diffuse into continuous gradients. Gradients were fractionated from the top by hand with cutoff pipet tips. Fractions from standards gradients run in parallel were separated by 10% PAGE and stained with Coomassie blue. Gels were scanned, band intensities were quantitated (Adobe Photoshop; Adobe Systems Inc.) and peak fractions were assigned (Kaleidagraph Synergy Software Inc.). Standard curves of peak fraction versus sedimentation coefficient (S_20,w) were used to estimate S values of protein complexes.

Gel filtration chromatography was carried out on a column (Superose-6; Pharmacia Biotech Sverige) in HB plus 100 µM GTP, and 100 or 500 mM NaCl as indicated. The column was calibrated with standards of known Stokes radii. Molecular weights and Stokes radii of protein complexes were estimated as described (Siegel and Monty, 1966). Fractions were separated by SDS-PAGE on 10% gels and -tubulin was detected by Western blotting.

Immunoisolation of -tubulin–containing Complexes from Drosophila Embryo Extract
PEG (polyethylene glycol P-2139; average mol wt = 8,000; Sigma Chemical Co.) was added to a final concentration of 2% (from a 30% stock in HB100) to clarified Drosophila embryo extract from 20-g embryos. The mixture was incubated on ice for 20 min, spun at 17,000 rpm for 10 min in a SS34 rotor and the supernatant was discarded. The pellets were resuspended in 20 ml of HB200 plus 0.05% NP-40, and 100 µM GTP by gentle Dounce homogenization and clarified at 35,000 rpm for 30 min in a 50.2 Ti rotor. -tubulin complexes were immunoprecipitated from the supernatant by adding 190 µg of DrosC17 antibody and incubating at 4°C for 1 h with gentle rotation. The immunoprecipitate was collected by slowly (over 1 h) pumping the antibody-extract mixture over a 350-µl column of protein A–agarose (GIBCO/BRL) in a disposable Bio-spin column housing (Bio-Rad). The column was washed with 15 ml of HB200 plus 0.05% NP-40 and 100 µM GTP, and 15 ml of the same buffer without NP-40. 400 µl of EB200 was loaded onto the column and the column was sealed with parafilm and incubated for 16–18 h at 4°C. -tubulin complexes were collected by loading an additional 400 µl of EB200 onto the column and collecting the flow through. For the sucrose gradient fractionation described in Fig. 2, 150 µl of isolated complexes was loaded onto a 2.1-ml 5–40% sucrose gradient, poured in HB100 plus 100 µM GTP, and sedimented at 50,000 rpm for 4 h in an TLS55 rotor at 4°C.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Purified Drosophila -tubulin complexes nucleate MTs in vitro. (A) Protein profile of peptide-eluted immunoisolated Drosophila -tubulin complexes after separation by 10% SDS-PAGE and Coomassie staining. (B) Nucleating activity is proportional to the concentration of -tubulin complexes in the reaction. The results shown here are the average of three independent experiments performed using the same preparation of peptide eluted complexes. At the highest concentrations tested, 370 nM or 0.02 mg/ml -tubulin, there were 94 times more MTs than in control reactions without -tubulin complexes. We estimate that the maximal concentration of MTs was 0.30 nM. Error bars represent the SEM.

Immunoisolation of the TuSC
PEG was added to 3% (from a 30% stock in HB100) to clarified extract corresponding to 40 g of embryos. The mixture was incubated on ice for 20 min, centrifuged at 17,000 rpm for 10 min in the SS34 rotor at 4°C, and the supernatant was discarded. Pellets were resuspended by gentle Dounce homogenization in 40 ml ice-cold HB500 plus 100 µM GTP and clarified at 35,000 rpm for 30 min at 4°C in the 50.2 Ti rotor. -tubulin small complex (TuSC) was immunoprecipitated by adding 1.46 mg of DrosC17 or DrosC12 antibody and incubating at 4°C for 1 h with gentle rotation. The immunoprecipitate was collected by pumping the antibody-extract mixture over a 500-µl column of protein A–agarose preequilibrated in HB500. The column was washed with 60 ml HB500 plus 100 µM GTP. Alternatively, the immunoprecipitate was collected by mixing the resin in a batch with the antibody-extract mixture at 4°C for 1 h. Afterwards, the resin was washed six times in batches with 5 ml of HB500 plus 100 µM GTP and loaded into a Bio-spin column. 500 µl of EB500 was loaded onto the column, the column was sealed with parafilm, and incubated for 4 h at 4°C. The TuSC was collected by loading an additional 550 µl of EB500 onto the column and collecting the flow through. 500 µl of this eluate was fractionated on a 4.5-ml 5–20% sucrose gradient in HB500 plus 100 µM GTP at 45,000 rpm in an SW55 rotor for 10 h at 4°C. 300-µl fractions were collected from the top. For coverslip and solution nucleation assays, 100 µl of each fraction was dialyzed against HB100 plus 100 µM GTP at 4°C for 6 h. The concentration of -tubulin was estimated after dialysis and, in general, was not significantly altered.

Solution Nucleation Assays to Quantitate Microtubule Nucleation
5-µl reactions of identical final buffer composition (0.5x BRB80, 0.5x EB200, 500 µM GTP) containing 4 mg/ml tubulin and varying concentrations of peptide-eluted complexes were incubated at 37°C for 4 min and fixed at room temperature for 3 min by addition of 45 µl of 1% glutaraldehyde in BRB80. 10 µl was removed to a new tube and diluted by addition of 1 ml ice-cold BRB80. MT spindowns and tubulin immunofluorescence were performed as described (http://skye.med.harvard.edu). Varying amounts of each sample were pelleted depending on the concentration of -tubulin in the reaction. 20 random fields were photographed with a 60x objective (1.4 NA; Nikon Corp.) using a cooled CCD camera (Princeton Instruments) and the MTs were counted. The fraction of total -tubulin in the peptide-eluted complexes present as the TuRC was determined by densitometry of Coomassie-stained gels after sucrose gradient fractionation.

To compare nucleating activity, peptide-eluted -tubulin complexes containing 0.65 µM -tubulin in the TuRC (0.87 µM total -tubulin) and isolated TuSC containing 0.74 µM -tubulin were assayed in parallel as above. The following exceptions were made: the incubation at 37°C was for 3 min; the final buffer composition of the TuSC was 0.5x BRB80, 0.5x HB100 plus 100 µM GTP, 500 µM GTP; after fixation, instead of sedimentation, samples were diluted with 200 µl of BRB80 + 70% glycerol and 3 µl were squashed and sealed under 18-mm square coverslips.

Coverslip Nucleation Assay
Polylysine-coated 12-mm diameter coverslips were placed on parafilm inside a humidified Petri dish kept in a 30°C water bath. The coverslips were rinsed 2x with filtered water and blocked for 5 min with 60 µl HB block. The HB block was removed by aspiration and replaced with 20 µl of the sample. After 10 min the coverslips were washed 2x with 60 µl of BRB80 + 10 mg/ml BSA + 1 mM GTP, and incubated with 20 µl 6 mg/ml tubulin (1:4 rhodamine labeled/unlabeled) in BRB80 + 1 mM GTP. After 10 min the tubulin was removed by aspiration and replaced with 60 µl 1% glutaraldehyde in BRB80 (warmed to 30°C) for 3 min, followed by 3 min postfixation with –20°C methanol. The coverslips were rehydrated, mounted, and sealed with nail polish.

Cloning and Sequencing of Dgrip84 and Dgrip91
The Drosophila gamma ring proteins (Dgrips) were immunoisolated as described below and internal peptide sequences were obtained for Dgrip84 and Dgrip91. The following peptides were obtained for Dgrip84: KILRTGK, KDAQQLIIGLVRK, DRSLTH, DELPEHY, DIHTHL, DLVTQMS, DAEVLTYL, DEQIPSFLA, RHREFL, DFTMQ, ERRTYTLR, DTTPVVFVRRGP, DRHRE, DEYRTSLL, DEQIPSFLAKY, DVNSAAGSVPTTLAIAST, and DLVTQMSKIMKKEENXQAQ. For Dgrip91 the peptides obtained were: KDVVTGRF, KGVYGLTN, KTVSDH, KHMEFVLS, DIMVGPHK, DFNEYY, KLSELGYY, DATKMLP(OR L)E, DRVVKFS, DVIVQRPFNGG, EMIICIKGKQMPE, DVVTGRIFPY, ELSKIV, DATQSSIGLXKQSLPNY, DDPNLQLFGTR, DQSRFYK, and DVSTGFNAIG. For Dgrip84, degenerate primers corresponding to the forward peptide KDAQQL and reverse peptide DLVTQM (underlined above) specifically amplified a band of 700 bp. A second round of PCR was performed with a primer corresponding to the forward peptide QQLIIG and the same reverse primer. For Dgrip91, primers corresponding to the forward peptide IKGKQM and reverse peptide TGFNAI (underlined above) specifically amplified a band of 800 bp. A second round of PCR was performed with a primer corresponding to the forward peptide GKQMPE and the same reverse primer. Both PCR products were cloned, sequenced, and used to screen a Drosophila cDNA library.

Antibodies
Synthetic peptides (Stanford University Medical Center) corresponding to the COOH-terminal 12 (QWSPAVEASKAG; DrosC12) or 17 amino acids (QIDYPQWSPAVEASKAG; DrosC17) of the maternal form of Drosophila -tubulin (37°C; These data are available from GenBank/ EMBL/DDB3 under accession number P42271) were used to raise and affinity purify rabbit polyclonal antibodies (Field et al., 1998). Antibodies to Dgrip84 and Dgrip91 were raised in rabbits against fusions of glutathione-S-transferase with amino acids 89–199 and 29–143 of the two proteins, respectively. Specific antibodies were purified as described (Kellogg and Alberts, 1992).

Embryo Fixation and Immunofluorescence
Embryos were fixed in methanol and immunofluorescence was performed as described (Theurkauf, 1994). Embryos were double-labeled with mouse anti–-tubulin (Sigma Chemical Co.) and rabbit anti-p91 or anti-p84 followed by FITC anti–rabbit and Cy-5 anti–mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Three-dimensional images were obtained on a wide field microscope (DeltaVision; Applied Precision Inc.). 512 x 512 pixel optical sections were taken at 0.2-µm intervals using an Olympus 60x, 1.4 NA objective and deconvolved. Appropriate Z-sections were projected.

GTP Cross-linking Experiments
120-µl peptide-eluted complexes were isolated as above, except GTP was omitted from the column wash and elution buffers. The isolated complexes were loaded onto a 2-ml 5–40% sucrose gradient in HB100, centrifuged at 55,000 rpm in a TLS55 rotor for 4 h at 4°C, and fractionated into 17 130-µl fractions. In a 96-well plate, 30 µl of each fraction was incubated for 90 min on ice with 10 µ Ci of [-³²P]GTP (400 Ci/mmol; Amersham Pharmacia Biotech Inc.), cross-linked for 5 min on ice in a cross-linker (Stratalinker UV; Stratagene) at a distance of 10 cm, and analyzed by 10% SDS-PAGE followed by autoradiography. For competition experiments, the [-³²P]GTP was premixed with a 200-fold excess of cold nucleotide competitor before being added to the fraction.

Determination of TuSC Nucleotide Content
TuSC was prepared as described above with the following modifications: 1:1,000 LPC was used in place of 1:200 protease inhibitor stock; pellets from the PEG precipitation were resuspended in buffer containing 100 µM of either GTP or GDP; and the wash, elution buffers, as well as sucrose gradients contained 20 µM of either GTP or GDP. Control gradients loaded with EB500 were fractionated in parallel to generate control buffers. β-tubulin samples were prepared by diluting bovine brain tubulin into control buffers and incubating 1 h on ice before desalting. Free nucleotide was removed by rapid desalting into 20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM MgCl₂ on a 800-µl Fast Desalting column (PC 3.2/10) mounted on a SMART^TM system (Pharmacia Biotech, Inc.). 100 µl of sample was loaded per run and the column was eluted at a flow rate of 400 µl/ min. One 100-µl fraction containing the protein peak was collected from each desalting run. Two separate runs were pooled, generating a total desalted sample volume of 200 µl. Under these conditions, desalting was complete in 45 s and 99.9% of free nucleotide was removed. 20 µl of the desalted sample was used for protein quantitation (see below). The concentration of - or β-tubulin loaded onto the desalting column was usually between 0.5 and 1 µM; protein recoveries were 40–50%.

To extract nucleotide from the remaining desalted sample, 90 mg of solid urea was added and the sample was vortexed and heated to 50°C for 3 min to denature the protein and release bound nucleotide. The denatured protein was removed by filtration through a 10,000 mol wt cutoff filter (model UFC3LGC00, Millipore Corp.; Rosenblatt et al., 1995). The filter was washed with 150 µl of water; eluate and wash were combined and frozen in liquid nitrogen. Nucleotide content was determined by chromatography on a 100-µl Mono Q (PC 1.6/5; Pharmacia Biotech, Inc.) column mounted on the SMART^TM system. Nucleotides were eluted with a gradient of ammonium bicarbonate (0.1–1 M in 30 column vol), quantified by peak integration, and compared with standard curves generated by processing nucleotide standards of known concentration in an identical fashion. The amount of -tubulin and β-tubulin was quantified using 10% SDS-PAGE, Coomassie-staining, and densitometry of the 20-µl aliquot of sample reserved after desalting relative to a standard curve of β-tubulin dimer on the same gel.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Drosophila Contains Two Related -tubulin Complexes: The TuSC Is a Subunit of the TuRC
Drosophila embryo extracts contain two -tubulin–containing complexes that can be separated by gel filtration chromatography or sucrose gradient sedimentation. In the presence of 500 mM KCl or NaCl, -tubulin is found exclusively in the smaller complex, indicating that the larger complex has been disrupted, and that the smaller complex is likely to be a structural subunit of the larger complex (Moritz et al., 1998). We named the large complex, Drosophila TuRC (see below), and the small complex the TuSC. To obtain size estimates for each complex, we performed gel filtration and sucrose gradient sedimentation under low salt conditions in buffers that were supplemented with magnesium and GTP (Fig. 1) to reduce aggregation that occurs in nucleotide-free buffers. Under these conditions, the TuSC has an S value of 9.8 and a Stokes radius of 7.0 nm, while the TuRC has an S value of 35.5 S and a 15-nm Stokes radius. Based on these values, we estimate the molecular masses of the TuSC and TuRC to be 280,000 and 2,200,000 D (Table I), respectively.

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Hydrodynamic analysis of -tubulin in concentrated Drosophila embryo extracts. (Top) -tubulin immunoblots of Superose 6 gel filtration column fractions in buffer containing 100 µM GTP and 100 or 500 mM NaCl. Calibration standards for the Superose 6 column: bovine thyroglobulin (Stokes radius = 8.5 nm), horse spleen ferritin (6.1 nm), bovine liver catalase (5.22 nm), and bovine serum albumin (3.55 nm), as indicated with arrowheads. (Bottom) -tubulin immunoblots of 5–40% sucrose gradient fractions in buffer containing 100 µM GTP and 100 or 500 mM NaCl. Gradients were sedimented at 50,000 rpm for 4 h in an SW55 rotor at 4°C and fractionated from the top; gradient pellets are also shown (P). The peak locations of standards run on parallel gradients are indicated with arrowheads. Sucrose gradient standards: bovine serum albumin (4.3 S), rabbit muscle aldolase (7.35 S), bovine liver catalase (11.3 S), and porcine thyroglobulin (19.4S).

View this table: [in this window] [in a new window]   Table I Properties of Drosophila -tubulin–containing Complexes

Purified Drosophila -tubulin Complexes Nucleate Microtubules In Vitro

The Protein Profile of the Drosophila TuRC Is Similar to That of the Xenopus TuRC
To determine the protein compositions of the TuSC and TuRC, we fractionated the peptide-eluted complexes on a 5–40% sucrose gradient (Fig. 3 A). For clarity, TuSC and TuRC are shown side by side in Fig. 3 B. The protein profile of the Drosophila TuRC is reminiscent of the Xenopus TuRC (Fig. 3 B). Therefore, by analogy to the Xgrips (Martin et al., 1998), we name Drosophila TuRC proteins Dgrips and designate them by their apparent molecular weights. Like the Xenopus TuRC, the Drosophila TuRC is composed of two high molecular mass proteins (Dgrip163 and Dgrip128), two prominent proteins near 100 kD (Dgrip91 and Dgrip84), and a group of three or four proteins with molecular masses near 75 kD (Dgrip75s). The protein below -tubulin (between the 56- and 38.5-kD markers) has been identified as actin. It is not clear whether actin is a specific component of TuRC, or if it fortuitously copurifies. Depending on the purification protocol, varying amounts of - and β-tubulin copurify with Xenopus TuRC (Zheng et al., 1995; Y. Zheng, unpublished results). In contrast, we have been unable to detect - or β-tubulin copurifying with Drosophila TuRC. Consistent with the idea that TuSC is a structural subunit of TuRC, TuSC is composed of the three most prominent proteins in TuRC: -tubulin, Dgrip84, and Dgrip91 (Fig. 3 B).

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Characterization of -tubulin–containing complexes isolated from Drosophila embryo extracts. -tubulin–containing complexes were immunoisolated and fractionated by sucrose gradient sedimentation in 100 mM NaCl. (A) 75 µl of each sucrose gradient fraction was TCA precipitated and analyzed by 10% SDS-PAGE and Coomassie staining. The sucrose gradient load is also shown. Peak fractions for standards run on a parallel gradient were: BSA (4.3 S), fraction 2.7; aldolase (7.35 S), fraction 4.0; catalase (11.3 S), fraction 5.6; and bovine thyroglobulin (19.4 S), fraction 8.0. (B) Protein profiles of TuRC and TuSC. TuSC consists of the three most prominent bands in TuRC. The profile of Drosophila TuRC resembles that of Xenopus TuRC (Zheng et al., 1995). (C) Schematic of the coverslip nucleation assay. The coverslip is washed and blocked with a BSA-containing buffer, incubated with the sample to be tested, rinsed to remove unbound protein, incubated with a mixture of unlabeled and rhodamine-labeled tubulin, fixed, and viewed using fluorescence microscopy. (D) Analysis of sucrose gradient fractions in A using the coverslip assay. The top two rows are equivalent exposures for fractions 3–14. The bottom row shows longer exposures (either 40x or 5x longer, as indicated) for some fractions. Bar, 10 µm.

The Drosophila TuRC Nucleates MTs In Vitro

The results of a coverslip assay on the sucrose gradient fractions in Fig. 3 A are shown in Fig. 3 D. The top two rows are equivalent exposures for fractions 3–14. The bottom row shows longer exposures (either 40x or 5x longer) for the indicated fractions. We observe a clear peak of activity corresponding to the fractions that contain TuRC (fractions 10–14). Under these conditions, no activity is seen in the fractions containing TuSC (fractions 4–6). Similar to the peptide-eluted complexes, gel filtration or sucrose gradient–fractionated Drosophila embryo extracts tested in this assay show only one peak of activity, corresponding to the peak of TuRC (data not shown). To distinguish between nucleation and capture of spontaneously nucleated MTs, we performed the coverslip nucleation assay with peptide-eluted material and observed unfixed samples in real time using video enhanced DIC microscopy (data not shown). MTs initiated at the coverslip surface and elongated, while maintaining a fixed orientation with one end anchored to the coverslip. Capture of a MT from solution by the surface was never observed. Cumulatively, these results indicate that the Drosophila TuRC has MT nucleating activity.

Cryo-electron Microscopy of the TuRC Reveals a Modular Structure
Negative stain electron microscopy of the peptide-eluted complexes (Fig. 4 A), and of the TuRC after sucrose gradient sedimentation (Fig. 4 B) reveals an open ring structure with a diameter of 25 nm. In side-by-side pictures of comparable preparations, the structure of the Drosophila TuRC is indistinguishable from that of the Xenopus TuRC (Wiese, C., and Y. Zheng, unpublished observations). To get a more detailed view of the TuRC, we examined the structure of the purified Drosophila TuRC by cryo-electron microscopy. A gallery of cryo-EM images reveals a modular structure (Fig. 4 C). The TuRC appears to have 13 structural repeats arranged in a radial symmetric pattern with a diameter of 25 nm. Some internal structures are also visible. A more detailed view will require single particle reconstructions.

View larger version (62K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Structure of Drosophila TuRC. (A and B) Negative stain electron microscopy of the peptide-eluted complexes (A) or of Drosophila TuRC after isolation on a sucrose gradient (B). Bar, 100 nm. (C) A gallery of cryo-electron microscopy images of Drosophila TuRC. Cryo-electron microscopy reveals a modular structure with 13 structural repeats (arrows) in a radial pattern. The ring has a diameter of 25 nm. Some internal structures are also apparent (arrowhead). Bar, 25 nm.

TuRC Is a More Potent Microtubule Nucleator than TuSC

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Characterization of the TuSC. (A) Immunoisolated TuSC was fractionated on a 5–20% sucrose gradient in buffer containing 500 mM NaCl. Fractions were separated by SDS-PAGE on a 10% gel and stained with Coomassie blue. A standards gradient was run in parallel. Peak fractions for standards were: BSA (4.3 S), fraction 4.9; aldolase (7.35 S), fraction 7.5; and catalase (11.3 S), fraction 10.8. (B) Fractions from the gradient in A were dialyzed against buffer containing 100 mM NaCl and tested in the coverslip assay. Bar, 10 µm. (C) TuSC from the sucrose gradient in 500 mM NaCl was fractionated by Superose 6 gel filtration in 500 mM NaCl (top), or was first dialyzed against buffer containing 100 mM NaCl and then fractionated by Superose-6 gel filtration in buffer containing 100 mM NaCl (bottom). Standards of known Stokes radius were used to calibrate the column. The peak fractions for the gel filtration standards were: bovine thyroglobulin (8.5 nm), fraction 13.5; horse spleen ferritin (6.1 nm), fraction 15.4; bovine liver catalase (5.22 nm), fraction 16.9; aldolase (4.81 nm), fraction 17.1; ovalbumin (3.05 nm), fraction 18.0; and chymotrypsinogen (2.09 nm), fraction 20.0.

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Comparison of the nucleating activity of peptide-eluted complexes and isolated TuSC. (A) Representative fields from solution nucleation assays. Bar, 20 µm. (B) Quantitation of solution nucleation assays. MTs in 10 microscope fields were counted and the average number of MTs/field is plotted. Error bars represent the SEM.

Dgrip84 and Dgrip91 Are Homologous to the Spc97/Spc98 Family of Proteins
To characterize the molecular nature of TuSC, we cloned and sequenced its non–-tubulin components, Dgrip84 and Dgrip91. Dgrip84 and Dgrip91 are homologous to each other and to the Spc97/98p family of proteins identified in S. cerevisiae. This family also includes two proteins identified in humans, hGCP2 and hGCP3 (Murphy et al., 1998). The homology between the Drosophila proteins and the other members of this family extends over the entire length of the proteins (data not shown). In comparisons of Dgrip84 and Dgrip91 with the corresponding human proteins, a one-to-one correspondence emerges. Dgrip84 is 32% identical (46% similar) to hGCP2 and only 21% identical (32% similar) to hGCP3; in contrast, Dgrip91 is 31% identical (45% similar) to hGCP3 and only 24% identical (37% similar) to hGCP2. These results suggest that Dgrip84 and hGCP2, and Dgrip91 and hGCP3 may be functionally homologous pairs. The sequences of the Drosophila proteins are available from GenBank/EMBL/DDBJ under accession numbers AF118379 (Dgrip84) and AF118380 (Dgrip91).

Stoichiometry of Proteins in TuSC
To estimate the stoichiometry of TuSC proteins, we performed densitometry of Coomassie-stained SDS-PAGE gels of purified TuSC. After correcting for the predicted molecular weight of each protein, we estimated that the ratio of Dgrip91 to Dgrip84 to -tubulin in the TuSC is 1:1:2. Since our estimate of the molecular mass of purified TuSC from sucrose gradient sedimentation and gel filtration is 280,000 D (Table I), we suspect that TuSC contains 1 molecule of Dgrip91, 1 molecule of Dgrip84, and 2 molecules of -tubulin. Interestingly, this corresponds to estimates of the stoichiometry of proteins in the S. cerevisiae 6 S -tubulin complex (Knop et al., 1997; Knop and Schiebel, 1997). If we assume that TuRC contains only one molecule of each non-TuSC component, and use our estimates for the molecular weights of TuRC and TuSC, then TuRC would contain approximately six TuSCs.

Dgrip84 and Dgrip91 Cofractionate with -tubulin on Sucrose Gradients and Colocalize with -tubulin in Embryos
If -tubulin in Drosophila embryos primarily exists associated with Dgrip84 and Dgrip91 in either TuSC or TuRC, we would expect these three proteins to cofractionate on sucrose gradients of embryo extract and to colocalize in embryos. To test this hypothesis, we raised and affinity-purified rabbit polyclonal antibodies that recognize Dgrip84 and Dgrip91. Each antibody recognizes a band of the expected molecular weight on Western blots of embryo extract (Fig. 7 A, left). As expected, both Dgrip84 and Dgrip91 comigrate with -tubulin in TuSC and TuRC when embryo extract is fractionated on sucrose gradients (Fig. 7 A, right). In addition, the localizations of Dgrip84 and Dgrip91 in Drosophila embryos are indistinguishable from that of -tubulin. Each antibody recognizes the centrosome throughout the cell cycle and shows some spindle staining during mitosis with enrichment at the spindle poles (Fig. 7 B), regardless of its cognate antigen. We propose that Drosophila -tubulin is stably associated with Dgrip91/84. Interestingly, we found no evidence for a non–-tubulin associated pool of either Dgrip84 or Dgrip91.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Dgrip91 and Dgrip84 associate and colocalize with -tubulin. (A) Clarified Drosophila embryo extract was immunoblotted for Dgrip91, Dgrip84, and -tubulin after separation on 10% SDS-PAGE gels (left). Each antibody recognizes a band of the expected molecular weight. Both Dgrip91 and Dgrip84 comigrate with -tubulin on sucrose gradients of embryo extract (right). (B) Immunofluorescence of early Drosophila embryos with antibodies against Dgrip91, Dgrip84, and -tubulin. Both Dgrip84 (top) and Dgrip91 (bottom) localize to centrosomes and to the mitotic spindle in a fashion that is indistinguishable from the localization of -tubulin. Examples of embryos in interphase and mitosis are shown for each antibody. Bar, 15 µm.

-tubulin in TuRC and TuSC Can be Cross-linked to GTP

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Photo cross-linking of radiolabeled GTP to -tubulin in TuSC and TuRC. (A) GTP cross-linking of peptide-eluted complexes. -tubulin is the only protein that cross-links to GTP. (B) GTP cross-linking after sucrose gradient fractionation of the material in A, demonstrating that -tubulin in both TuSC and TuRC cross-links to GTP. (C) Competition of the GTP cross-link of the material in A with 200-fold excess of the indicated unlabeled nucleotides.

-tubulin in TuSC Preferentially Binds GDP

Each β-tubulin dimer has two guanine nucleotide binding sites, one on each tubulin subunit. Exclusively GTP is bound to -tubulin at the nonexchangeable or N-site; this nucleotide does not exchange with GTP/GDP in solution and does not undergo hydrolysis. In contrast, β-tubulin binds guanine nucleotide in an exchangeable fashion at the E-site. Both GTP and GDP bind to the E-site with GTP having a three- to fourfold higher affinity than GDP (Zeeberg and Caplow, 1979). GTP bound to the E-site does not undergo significant hydrolysis in the absence of polymerization but gets hydrolyzed soon after incorporation into the MT lattice, resulting in GDP that is locked in the lattice and can only exchange after depolymerization (reviewed in Desai and Mitchison, 1997). These properties predict that if β-tubulin dimer is isolated from buffers containing GDP, then there will be 1 mol GTP (N-site) and 1 mol GDP (E-site) per mole of β-tubulin dimer. In contrast, if β-tubulin dimer is isolated from buffers containing GTP, under conditions where there is no polymerization, then there will be 2 mol GTP (1 N-site GTP and 1 E-site GTP) per mole of β-tubulin dimer. Consistent with these predictions, we recovered 1.1 mol of GTP and 0.8 mol GDP per mole of β-tubulin dimer isolated from GDP buffer (Fig. 9, B and G, and Table II); in contrast, we recovered exclusively 2.0 mol of GTP per mole of β-tubulin dimer isolated from GTP buffer (Fig. 9, E and G, and Table II). These results establish the validity of our assay for comparing the nucleotide-binding properties of TuSC to those of β-tubulin dimer.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Analysis of nucleotide bound to -tubulin in TuSC, a comparison with β-tubulin dimer. Desalting was used to remove free nucleotide from samples of purified TuSC or β-tubulin. Bound nucleotide was released by urea treatment and analyzed on a mono Q column. Arrows indicate the elution positions of GDP and GTP. For samples containing protein (B, C, E, and F), a gel lane of the desalted sample is shown to the right of the UV trace. (A–C) Nucleotide analysis after isolation from buffer containing 20 µM GDP. (A) After desalting of control buffer no nucleotide is detected (the broad peak just to the left of the GDP arrow is a background peak). (B) Both GDP and GTP are bound to β-tubulin dimer (for quantitation see Table II: 20 µM GDP, experiment 3). (C) Exclusively, GDP binds to -tubulin in TuSC (for quantitation see Table II: 20 µM GDP, experiment 3). (D–F) Nucleotide analysis after isolation from buffer containing 20 µM GTP. (D) Although GTP is desalted slightly less efficiently than GDP (compare with A), >99.9% of free GTP is removed from control buffer. (E) Exclusively, GTP binds β-tubulin dimer (for quantitation see Table II: 20 µM GTP, experiment 1). (F) Small amounts of both GDP and GTP are detected bound to -tubulin in the TuSC (for quantitation see Table II: 20 µM GTP, experiment 1). (G) Summary of nucleotide analysis from three independent experiments (raw data shown in Table II). Bar graphs indicate the ratio of bound nucleotide per β-tubulin dimer, or -tubulin monomer in TuSC after isolation from buffers containing 20 µM GDP or 20 µM GTP. Error bars represent the SEM. The ratio of GDP/GTP recovered when we desalt β-tubulin dimer from buffer containing GDP is very reproducible (0.733 ± 0.015, n = 5) suggesting that the protein concentration determined by densitometry is the least accurate parameter in this analysis. This ratio also suggests that we are recovering 73% of the GDP bound to the β-tubulin E-site.

View this table: [in this window] [in a new window]   Table II Analysis of Nucleotide Bound to -tubulin in TuSC

To distinguish between these possibilities, we isolated TuSC from GTP-containing buffer. Surprisingly, we recovered a greatly reduced amount of nucleotide (Fig. 9 F). Only 0.2 mol guanine nucleotide was recovered per mole of -tubulin, indicating that 80% of the -tubulin was empty at its nucleotide binding site (Fig. 9 G and Table II). To ascertain that the GTP in the buffer had not been degraded, we removed an aliquot before desalting and analyzed its nucleotide content. This sample contained the expected amount of GTP and a trace amount of GDP (3% of total guanine nucleotide). This amount of GDP was also recovered from a similarly processed control buffer, indicating that it did not arise from hydrolysis by TuSC or a contaminating GTPase (not shown). The low recovery of guanine nucleotide bound to TuSC isolated from GTP buffer indicates that GDP is bound exchangeably to -tubulin in TuSC. This result also argues against the theory that the GDP bound to -tubulin in TuSC, isolated from GDP buffer, is being generated by earlier GTP hydrolysis. The recovery of nearly 1 mol GDP per mole of -tubulin from GDP buffer and the nearly equivalent amounts of GTP and GDP in the 0.2 mol nucleotide recovered per mole of -tubulin from GTP buffer, despite a GTP/GDP ratio 30 before desalting, strongly suggest that -tubulin in TuSC has an exchangeable guanine nucleotide binding site that has a much higher affinity for GDP than GTP. To test this further, we isolated TuSC in buffer containing 20 µM GTP and, 1 h before desalting, added 20 µM GDP. Consistent with our interpretation, we recovered 0.44 mol GDP and 0.02 mol GTP per -tubulin monomer (Table II).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
-tubulin Complexes in Eukaryotes
The -tubulin in Drosophila embryo extracts exists in two related complexes of 280,000 (TuSC) and 2,200,000 D (TuRC). In contrast, when extracts of Xenopus eggs (Stearns and Kirschner, 1994; Zheng et al., 1995), Xenopus XTC cells (Stearns and Kirschner, 1994), human 293 cells (Stearns and Kirschner, 1994), or mouse fibroblasts (Murphy et al., 1998) were fractionated, only one complex was observed sedimenting at 32 S (Murphy et al., 1998). The significance of this finding is not clear, but might reflect a difference between -tubulin–containing complexes isolated from different organisms and cell types. Interestingly, -tubulin in the polarized human intestinal epithelial cell line Caco-2 was present in both 10 S and 29 S complexes (Meads and Schroer, 1995). In Caco-2 cells, -tubulin localizes both to centrosomes and to a diffuse layer beneath the apical membrane (Meads and Schroer, 1995). However, it is not yet clear whether the presence of an apical layer of -tubulin correlates with the existence of a smaller 10 S complex. Further experiments will be required to determine if the differences in -tubulin–containing complexes present in cellular extracts are caused by different extraction conditions, or if they reflect real diversity between systems and cell types in the nature and function of -tubulin complexes in vivo.

The protein profile of the purified Drosophila TuRC is very similar to that of the previously purified Xenopus TuRC (Zheng et al., 1995). Indeed, the protein profiles of -tubulin complexes immunoprecipitated from a number of sources, including mouse cells after metabolic labeling (Murphy et al., 1998) and sheep brain tubulin preparations (Detraves et al., 1997), bear a strong resemblance. In addition to molecular similarities, the Drosophila TuRC also resembles the Xenopus TuRC in its structure (both complexes appear as open rings when visualized by negative stain electron microscopy).

TuSC: a Conserved Subcomplex of the TuRC
Drosophila TuRC can be converted to TuSC by treatment with high salt, suggesting that TuSC is a structural subunit of TuRC. A similar dissociation by high salt has been reported for human and Xenopus large -tubulin complexes (Stearns and Kirschner, 1994; Meads and Schroer, 1995; Zheng et al., 1995). The hypothesis that TuSC is a subunit of TuRC is supported by our finding that purified TuSC is composed of the three proteins most prominent in TuRC: -tubulin, Dgrip84, and Dgrip91. Dgrip84 and Dgrip91 are members of the Spc97/98p family of proteins. This family includes hGCP2 and hGCP3/ HsSpc98p from humans (Murphy et al., 1998; Tassin et al., 1998) and Xgrip109 from Xenopus (Martin et al., 1998). Homologous ESTs have also been identified in mouse, zebrafish, and rice (Martin et al., 1998; Tassin et al., 1998). Genetic evidence in S. cerevisiae suggests that this family of proteins interacts directly with -tubulin (Geissler et al., 1996; Knop et al., 1997). Based on molecular weight estimates and densitometry of Coomassie stained gels, we estimate that Drosophila TuSC is a heterotetrameric complex containing one Dgrip84, one Dgrip91, and two molecules of -tubulin. This stoichiometry is identical to the proposed composition of the S. cerevisiae 6 S complex (Sc TuSC). Immunoprecipitation experiments with tagged proteins indicate that Sc TuSC contains one Spc97p, one Spc98p, and two or more molecules of -tubulin (Knop et al., 1997; Knop and Schiebel, 1997). Together, these results support the hypothesis that the organization of -tubulin into TuSC is likely to be conserved among all organisms where -tubulin is found. One question of fundamental importance that needs to be addressed in the future is how the two -tubulin molecules within TuSC are arranged. Are they arranged in a head to tail dimer as would be suggested by the model proposing that TuRC is a protofilament of -tubulin (Erickson and Stoffler, 1996), or are they arranged in a side-by-side configuration (Zheng et al., 1995)?

In metazoa, TuSC is further assembled into TuRC. Based on our hydrodynamic analysis of Drosophila complexes, we estimate that TuRC contains approximately four to six TuSCs. An exact determination awaits a more accurate appraisal of the molecular weight of TuRC, currently being attempted using scanning transmission electron microscopy. It will also be interesting to identify the structural correlate of TuSC within TuRC. When viewed by cryo-electron microscopy, TuRC has a modular structure with 13 structural repeats organized in a radial symmetric pattern. Based on our current estimates, we speculate that one TuSC might correspond to two of the radial symmetric structural repeats visible by cryo-electron microscopy.

Nucleation Activity of TuSC and TuRC
An important issue with respect to the in vivo roles of TuSC and TuRC is their relative MT nucleating activity. The fact that S. cerevisiae does not appear to contain a TuRC-like complex raises the question of whether Sc TuSC has nucleating activity or whether it must assemble into a larger structure at the spindle pole body to become active. Conversely, in metazoa it is possible that TuRC is a storage form for -tubulin and it could be TuSC that nucleates MTs at centrosomes (Knop and Schiebel, 1997). To begin to address this question, we compared the nucleating activity of peptide-eluted complexes (in which 75% of -tubulin is TuRC) to isolated TuSC at similar concentrations of -tubulin. Using both solution and coverslip nucleation assays, we found that both preparations had nucleating activity. However, whereas the solution nucleating activity of the peptide-eluted complexes was robust, typically 80–100-fold above the level of spontaneous nucleation, under similar conditions the level of nucleation for isolated TuSC was only two- to threefold above background. Thus, per mole of -tubulin TuRC is 25 times more active than TuSC in promoting nucleation. Combining these data with our stoichiometry measurements, we estimate that per mole of complex TuRC is 150 times more active than TuSC, suggesting that organization of TuSC into TuRC facilitates MT nucleation activity.

We emphasize that the nature of the nucleating activity of TuSC is still unclear. TuSC may have intrinsic nucleating activity or it may need to assemble into larger TuRC-like complexes in order to nucleate MTs. In Xenopus extracts, high-salt dissociated TuRC components can be reassembled by desalting. This reassembly is blocked by depleting Xgrip109, suggesting that intact TuSC is required for assembly of -tubulin into a TuRC-like structure (Martin et al., 1998). Here we separate TuSC from the remaining components of TuRC and do not find any evidence for assembly of larger structures after desalting. This occurrence suggests that TuSC is required but not sufficient for assembly of a TuRC-like structure. We cannot exclude the possibility that TuSC, like β-tubulin dimer, assembles into larger complexes when the temperature is raised during nucleation assays. Because the level of activity of TuSC is very low compared to that of TuRC, we also cannot rule out the possibility that the nucleating activity of TuSC depends on trace levels of other TuRC components that contaminate our TuSC preps. Expression and purification of the TuSC will be necessary to further characterize TuSC activity.

Nucleotide Binding Properties of -tubulin
Cross-linking experiments showed that -tubulin in both TuSC and TuRC can bind guanine nucleotide. To investigate how -tubulin compares to other members of the tubulin family in its nucleotide binding properties, we compared the nucleotide bound to -tubulin in TuSC to that bound to β-tubulin dimer after desalting from buffers containing GDP or GTP. We found, like β-tubulin (Weisenberg et al., 1976), -tubulin in TuSC binds guanine nucleotide exchangeably. However, in contrast to -tubulin, which has about a threefold higher affinity for GTP than GDP (Zeeberg and Caplow, 1979), -tubulin in TuSC strongly prefers binding GDP to GTP. Our results suggest that the affinity of -tubulin for GTP is much lower than that of β-tubulin, based on nucleotide recovery after desalting under similar conditions (Zeeberg and Caplow, 1979). Determination of the absolute affinities of -tubulin for GDP and GTP will be important to know whether the affinities of - and β-tubulin for GDP are similar. If they are, this will suggest that the strong preference of -tubulin for GDP is primarily because of a reduction in its affinity for GTP relative to β-tubulin. These experiments will require a reliable supply of TuSC, currently limited by antibody availability for immunoisolation and by lack of an expressed source. A structural comparison of the nucleotide binding pocket of -tubulin to those of - and β-tubulin should also be revealing. Development of procedures to prepare more concentrated and highly purified TuRC should allow a comparison of nucleotide binding by -tubulin in TuRC to that in the TuSC. If the nucleotide binding properties of -tubulin in TuRC are similar to those of -tubulin in TuSC, it will suggest that GTP hydrolysis by -tubulin may not be important for its function in vivo.

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health to T.J. Mitchison and Y. Zheng (RO1-GM56312-01). Y. Zheng was also supported by the Pew Scholar's Award and C. Wiese by a postdoctoral fellowship from the American Cancer Society.

Submitted: 9 September 1998

Revised: 11 January 1999

Address correspondence to Karen Oegema, European Molecular Biology Laboratory, Cell Biology Program, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel.: 49-6221-387-337. Fax: 49-6221-387-512-306. E-mail: Karen.Oegema{at}EMBL-Heidelberg.DE or Yixian Zheng, Department of Embryology, Carnegie Institute of Washington, Baltimore, MD 21210. Tel.: 410-554-1232. Fax: 410-243-6311. E-mail: zheng{at}mail1.ciwemb.edu

We dedicate this work to Christine Mirzayan. The authors thank Mike Sepanski and Mei Lie Wong for the negative stain EM. We also thank Arshad Desai for much help during the course of this work and Arshad Desai and Doug Kellogg for critical reading of the manuscript.

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