Xgrip109: A gamma  Tubulin-Associated Protein with an Essential Role in gamma  Tubulin Ring Complex (gamma TuRC) Assembly and Centrosome Function

Ona C. Martin,* Ruwanthi N. Gunawardane,* Akihiro Iwamatsu,Dagger and Yixian Zheng*

* Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210; and Dagger  Central Laboratories for Key Technology, Kirin Brewery Company, Ltd., Yokohama 236, Japan

    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous studies indicate that gamma  tubulin ring complex (gamma TuRC) can nucleate microtubule assembly and may be important in centrosome formation. gamma TuRC contains approximately eight subunits, which we refer to as Xenopus gamma ring proteins (Xgrips), in addition to gamma  tubulin. We found that one gamma TuRC subunit, Xgrip109, is a highly conserved protein, with homologues present in yeast, rice, flies, zebrafish, mice, and humans. The yeast Xgrip109 homologue, Spc98, is a spindle-pole body component that interacts with gamma tubulin. In vertebrates, Xgrip109 identifies two families of related proteins. Xgrip109 and Spc98 have more homology to one family than the other. We show that Xgrip109 is a centrosomal protein that directly interacts with gamma  tubulin. We have developed a complementation assay for centrosome formation using demembranated Xenopus sperm and Xenopus egg extract. Using this assay, we show that Xgrip109 is necessary for the reassembly of salt-disrupted gamma TuRC and for the recruitment of gamma  tubulin to the centrosome. Xgrip109, therefore, is essential for the formation of a functional centrosome.

    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PROPERLY organized microtubule arrays are critical for many cellular functions including mitosis, cytokinesis, and axonal and intracellular transport (for review see Kellogg et al., 1994). Microtubules are dynamic polymers that assemble from alpha -/beta -tubulin heterodimers (Weisenberg, 1972; Mitchison and Kirschner, 1984). In vivo, microtubule dynamics are coordinately regulated by many cellular factors (for review see Desai and Mitchison, 1998). There are proteins that stabilize (for reviews see Vallee et al., 1984; Olmstead, 1986), destabilize (Endow et al., 1994; Belmont and Mitchison, 1996), or sever (McNally et al., 1996) microtubule polymers, as well as proteins that sort microtubules into different arrays. In addition, microtubule nucleation is temporally and spatially controlled within the cell, occurring primarily at structures called microtubule organizing centers (MTOCs)1 (Kellogg et al., 1994). The major MTOC in animal cells is the centrosome that consists of a pair of centrioles surrounded by an electron-dense cloud of pericentriolar material (PCM). The PCM is responsible for microtubule nucleation (Kellogg et al., 1994).

The discovery of gamma  tubulin as a suppressor of a beta -tubulin mutation in Aspergillus nidulans (Weil et al., 1986; Oakley and Oakley, 1989) was a major breakthrough in the study of microtubule nucleation at a molecular level. gamma  Tubulin is highly conserved and has been found in all eukaryotes examined (for review see Oakley, 1992). Most gamma  tubulins share over 60% amino acid identity, with the exception of Saccharomyces cerevisiae gamma  tubulin, which is only ~40% identical to the other gamma  tubulins (Sobel and Synder, 1995; Marschall et al., 1996; Spang et al., 1996). gamma  Tubulin is localized to all MTOCs such as the spindle-pole body (the major fungal MTOC) and the centrosome (Stearns et al., 1991; Zheng et al., 1991).

Genetic studies in Aspergillus (Oakley et al., 1990), Saccharomyces pombe (Horio et al., 1991), Saccharomyces cerevisiae (Sobel and Synder, 1995; Marschall et al., 1996; Spang et al., 1996), and Drosophila melanogaster (Sunkel et al., 1995; Tavosanis et al., 1997) have demonstrated that gamma  tubulin is an essential gene required for the assembly of a functional mitotic spindle. Antibody inhibition or depletion experiments performed in animal cells (Joshi et al., 1992) and in Xenopus egg extracts (Felix et al., 1994), respectively, further show the critical role of gamma  tubulin in microtubule nucleation at the centrosome.

Biochemical studies were also initiated to study how gamma  tubulin is involved in microtubule nucleation at the MTOCs. Human gamma tubulin translated in vitro is monomeric (Melki et al., 1993), and binds to microtubules in an end-specific manner (Li and Joshi, 1995). On the other hand, in animal cells, the gamma  tubulin that is not associated with the centrosome is found in large cytoplasmic complexes (Raff et al., 1993; Stearns and Kirschner, 1994). The purified Xenopus gamma -tubulin-containing complex has an estimated molecular mass of over 2,000 kD. This complex, the gamma  tubulin ring complex (gamma TuRC), has an open ring structure and can nucleate microtubules in vitro. In addition to multiple gamma -tubulin molecules, the gamma TuRC contains approximately eight additional polypeptides (Zheng et al., 1995). The S. cerevisiae gamma  tubulin also appears to form a complex with at least two other proteins: the spindle-pole body components Spc98 and Spc97 (Geissler et al., 1996; Knop et al., 1997). However, since this gamma -tubulin complex has a much smaller S value (6 S) (Geissler et al., 1996; Knop et al., 1997) than that of the gamma TuRC (>25 S), it is not clear what the functional relationship is between these complexes or if the yeast gamma -tubulin complex is able to nucleate microtubules in vitro. Based on the structure and function of the gamma TuRC, two models were proposed to explain how the gamma TuRC may nucleate microtubule assembly (Zheng et al., 1995; Erickson and Stoffler, 1996). One model suggests that the gamma TuRC acts as a seed, similar to the plus end of a microtubule, to nucleate microtubule assembly (Zheng et al., 1995). The other model proposes that the gamma TuRC unrolls into a filament, similar to a tubulin protofilament, to initiate microtubule polymerization (Erickson and Stoffler, 1996). Further biochemical and structural analyses are needed to study the mechanism of microtubule nucleation by the gamma TuRC.

In parallel structural studies, EM tomography has revealed hundreds of gamma TuRC-like rings embedded in the PCM of Drosophila and of the surf clam Spisula (Moritz et al., 1995b; Vogel et al., 1997); in Drosophila these rings are known to contain gamma  tubulin (Moritz et al., 1995a). The combined structural and biochemical studies of gamma  tubulin led to the hypothesis that the gamma TuRC is anchored within the PCM where it acts to nucleate microtubules (Zheng et al., 1995).

To better understand the relationship between the centrosomal gamma -tubulin-containing rings and the cytosolic gamma TuRC, we have begun to characterize the gamma TuRC subunits. Here we report the identity and function of one of the subunits, Xgrip109. We show that Xgrip109 is a highly conserved centrosomal protein that is essential for gamma -tubulin function.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Buffers

Buffers used were: Hepes 100 (50 mM Hepes, pH 8, 1 mM MgCl2, 1 mM EGTA, 100 mM KCl); Hepes 300, Hepes 500, and Hepes 1 M are the same as Hepes 100 except that the concentration of KCl is 300 mM, 500 mM, and 1 M, respectively; cytostatic factor (CSF)-XB (10 mM potassium Hepes, pH 7.7, 100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 50 mM sucrose, 5 mM EGTA, pH 7.7); BRB80 (80 mM potassium Pipes, pH 6.8, 1 mM MgCl2, 1 mM EGTA); and OR2 (2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM NaHPO4, 5 mM Hepes, final pH adjusted to 8.3).

Generating Mouse Polyclonal Ascites against the Xgrips

To obtain quantities of the Xgrips sufficient for mouse immunization, we performed a large-scale immunoprecipitation of the gamma TuRC from a 15- 25% ammonium sulfate cut of a concentrated Xenopus CSF-arrested egg extract (Murray, 1991). Briefly, ammonium sulfate was added to 15% (from a 100% ammonium sulfate stock solution) to ~100 ml of concentrated extract that had been clarified by centrifugation at 35,000 rpm in a rotor (model SW55; Beckman Instruments, Inc., Palo Alto, CA) for 2 h. The extract was then centrifuged at 10,000 rpm in another rotor (model SS34; Sorvall Inc., Newtown, CT) for 15 min. The supernatant from this spin was adjusted to 25% ammonium sulfate and then the pellet was collected by centrifugation as before. The 25% ammonium sulfate pellet (Asp), which contained over 90% of gamma TuRC, was resuspended in 75 ml of Hepes 100 and then clarified by centrifugation at 30,000 rpm in a rotor (model SW55; Beckman Instruments, Inc.) for 1 h. The supernatant was collected and 2.6 ml of anti-gamma -tubulin antibodies (XenC) (Zheng et al., 1995) at a concentration of 2 mg/ml were added. After a 1-h incubation at 4°C, 2.7 ml of settled protein A-agarose beads (Life Technologies, Inc., Gaithersburg, MD) was added and then the incubation was continued for another hour. The protein A-agarose beads were then washed batchwise three times with 10 vol each of Hepes 100, Hepes 300, and Hepes 100. The gamma TuRC was eluted batchwise using 1.4 M MgCl2. The protein sample was precipitated by addition of trichloroacetic acid to 10%, neutralized, dissolved in SDS sample buffer, and then separated using preparative 10% SDS-PAGE. Gel slices containing either Xgrip195, 133, a mixture of 110 and 109, or a mixture of 75 kD (Zheng et al., 1995) proteins were homogenized and used to immunize mice according to Harlow and Lane (1988). We estimate that between 1 and 10 µg of the total protein was injected into each animal. Ascites fluid was induced after the fifth injection in mice that gave a positive response by Western analysis.

Internal Peptide Sequencing of the Xgrips

To obtain internal peptide sequences, the Xgrips were prepared as described above and separated on preparative 10% SDS-PAGE. After blotting to polyvinylidine difluoride membranes (Applied Biosystems, Inc., Foster City, CA), proteins were visualized by staining with Ponceau S. The membrane-immobilized proteins were reduced, S-carboxymethylated, and then digested in situ with Achromobacter protease I and endoproteinase Asp-N (Iwamatsu and Yoshida-Kubomura, 1996). Digested peptides were separated by reverse-phase HPLC using an Å column (model Wakosil-II AR C18 300, 2.0 × 150 mm; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Amino acid sequencing was carried out with a gas-phase sequencer (model PPSQ-10; Shimadzu Corp., Tokyo, Japan).

DNA Cloning and Sequencing

The mouse polyclonal ascites that recognized Xgrip109 by Western blot analysis were used to screen a lambda ZAP® cDNA library of Xenopus oocytes (Stratagene, La Jolla, CA) according to Sambrook et al. (1989), with modifications as described (Hirano and Mitchison, 1994). We screened 7 × 105 plaques and isolated two positive clones (clone 3-1 and clone 3-4) that had overlapping sequences. Because neither clone had a complete 5' coding region, we rescreened the library using clone 3-4 as probe. 20 positives were obtained. One clone (p109-14) was longer than clone 3-4, but it still lacked a complete 5' coding region.

To obtain the missing 5' end, we carried out 5' rapid amplification of cDNA ends (RACE) using a 5'/3' RACE Kit (Boehringer Mannheim Biochemicals, Indianapolis, IN). Three partially overlapping primers corresponding to the 5' region of the known sequence of p109-14 were made: (a) gsp1 (5'-ACTAATCCCACTGCTGCCGAT-3'), (b) gsp2 (5'-CGGAATTCGATGCTTGCAGCATTCTGTGC-3'), and (c) gsp3 (5'-CGGAATTCTGCACACTGCACATTCCGATC-3') for the initial reverse transcription of Xenopus mRNA (gsp1) and two rounds of PCR (gsp2 and gsp3). Pfu DNA polymerase (Stratagene) was used in both rounds of PCR reactions. A 650-bp-amplified DNA fragment was cloned, sequenced, and contained the complete 5' coding region of Xgrip109 since the open reading frame (ORF) began with a methionine, which was preceded by an in-frame stop codon 48-bp upstream. The race product contained the 131 amino acids that were missing from the 5' end of p109-14.

Expression of Recombinant Fusion Proteins, Production of Rabbit Antibodies, and Immunoblotting

We produced six fusion proteins between glutathione S-transferase (GST) and six different fragments of the Xgrip109. Only the fusion between GST and the 134-244-amino acid fragment of Xgrip109, p109-2, elicited rabbit antibodies that worked for Western analysis, immunofluorescence, and immunodepletion. The primers 5'-CGGGATCCCACGAGGCCAGGCCACAGAGC-3' and 5'-CGGAATTCTTCAACAGATCCACTTGAGTC-3' were used to PCR-amplify this fragment of Xgrip109 using p10-4 as template. The resulting fragment was subcloned into the BamHI and EcoRI sites of pGEX-2TK (Pharmacia Biotech., Inc., Piscataway, NJ). To purify the p109-2 fusion protein, 1 liter of bacterial culture (BL21 lys.S) expressing p109-2 was pelleted and lysed in ~20 ml of Hepes 100 containing 0.1 mM of PMSF. After centrifugation, the clarified extract was incubated with ~5 ml of prewashed glutathione agarose (Sigma Chemical Co.) for 1 h at 4°C. The glutathione agarose was then packed into a PD-10 column (Pharmacia Biotech., Inc.), washed sequentially with 10 column volumes each of Hepes 100, Hepes 500, and Hepes 100, and then eluted with 10 mM of reduced glutathione (Sigma Chemical Co.) in Hepes 100. Each of these wash and elution buffers contained 0.1 mM PMSF.

The p109-2 fusion protein and a synthetic peptide (C)RLRVSMGTRGRRSFHV, corresponding to the COOH-terminal 16 amino acids of the Xgrip109 were used to immunize separate rabbits (Spring Valley Laboratories, Inc., Sykesville, MD). An NH2-terminal cysteine was added to the peptide for sulfhydryl coupling. Peptide conjugation and antibody affinity purification were performed as described (Harlow and Lane, 1988). The antibodies against p109-2 fusion protein and the synthetic peptide are referred to as 109-2 and 109-c, respectively.

The XenC antibodies used in this paper were raised against a synthetic peptide corresponding to the last 15 amino acids of the Xenopus gamma  tubulin (Zheng et al., 1995). Immunoblotting was carried out using affinity-purified antibodies at a concentration of ~1 µg/ml using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech., Inc., Piscataway, NJ).

Preparation of Xenopus Egg Extracts and Demembranated Xenopus Sperm

Concentrated extracts made from CSF-arrested Xenopus eggs according to Murray (1991) were further clarified in a rotor (model TLS55; Beckman Instruments, Inc.) at 50,000 rpm, 4°C for 1 h. 20× energy mix (150 mM creatine phosphate, 20 mM ATP, 2 mM EGTA, pH 7.7, 20 mM MgCl2) was added to the supernatant to a final concentration of 1×. This extract was frozen in liquid N2 and then stored at -80°C as 100-µl aliquots. All centrosome assembly assays were performed using the clarified and frozen extract. Demembranated Xenopus sperm was prepared as described (Sawin and Mitchison, 1991).

Centrosome Assembly Assay and Quantitation

1-µl of Xenopus sperm (~1.5 × 104 sperm/ml) that had been diluted 10-fold in CSF-XB was added to 10 µl of thawed and clarified Xenopus egg extract. After addition of 1 µl of 4 mg/ml rhodamine-tubulin (Hyman et al., 1991), the mixture was incubated at room temperature for 10 min. The reaction was stopped by diluting with 1 ml of BRB80 containing 30% glycerol (vol/vol) and then layered in a Corex (Corning GlassWorks, Corning, NY) tube onto 2 ml of BRB80 containing 30% glycerol, followed by spinning in a rotor (model HB-6; Sorvall Inc.) at 10,000 rpm for 20-30 min at 20°C onto a glass coverslip placed at the bottom of the tube (Evans et al., 1985). The sperm nuclei and microtubule asters were fixed in -20°C methanol for 5 min followed by hydration in BRB80 containing 0.1% Triton X-100 for 5 min. The samples were then stained with 0.2 µg/ml of 4',6-diamidino-2-phenylindole (DAPI) dissolved in BRB80 containing 0.1% Triton X-100 for 2 min followed by mounting in antifade (1 mg/ml p-phenylenediamine, 0.5× PBS, pH 9, 50% glycerol, 0.02% NaN3).

A total of 100 sperm nuclei were scored in random fields. These 100 sperm nuclei were divided into three groups: (a) sperm nuclei with a microtubule aster attached at the tip, "centrosome"; (b) sperm nuclei with a few disorganized microtubules attached at the tip, "disorganized centrosome"; or (c) sperm nuclei alone. Because the microtubule asters break away from the tips of the sperm nuclei at a low frequency, we also counted the free microtubule asters while scoring the sperm nuclei. The number of the free microtubule asters was subtracted from the sperm nuclei alone group and added to the sperm nuclei with a microtubule aster centrosome group.

30% Ammonium Sulfate Fractionation, Immunodepletion, and Complementation Assays

To fractionate the clarified egg extract with 30% ammonium sulfate, 43 µl of 100% ammonium sulfate was added to 100 µl of extract and then incubated on ice for 15 min, followed by centrifugation in a rotor (model SS34; Sorvall Inc.) at 10,000 rpm for 10 min at 4°C. The pellet, which contained over 90% of the total gamma TuRC and only ~20% of the total extract protein, was resuspended in 100 µl of either Hepes 100 or Hepes 1 M containing 1 mM GTP and then incubated on ice for 80 min. This mixture was either analyzed by sucrose gradient sedimentation (see below) directly or after desalting using a 1-ml P6 Bio-Spin column (Bio-Rad Laboratories, Hercules, CA), equilibrated in Hepes 100 containing 1 mM of GTP. The eluate was either analyzed by sucrose gradient sedimentation or concentrated ~20-fold by placing in a collodion bag (Sartorius, Göttingen, Germany) that was kept on a bed of Sephadex G-50 resin (Pharmacia Biotech., Inc.) at 4°C. The concentrated eluate was then used in complementation assays (see below).

To immunodeplete Xgrip109 and gamma  tubulin from the resuspended 30% Asp in either Hepes 100 or Hepes 1 M, 10 µl of 109-2 IgG (~0.25 mg/ml), 4.5 µl of XenC IgG (~1.6 mg/ml), or 2.5 µl of random IgG (~3 mg/ml) were bound to 10-20 µl of settled protein A beads. The beads were washed with either Hepes 1 M or Hepes 100 and then added to the appropriate resuspended pellets that were incubated on ice for 20 min. After rotating for 1 h at 4°C, the protein A beads were collected by pelleting and then washed. The immunoprecipitated proteins were analyzed by SDS-PAGE followed by Coomassie blue staining or Western analysis, probing with XenC and 109-2. To estimate the percentage of total gamma  tubulin that remained bound to Xgrip109 in high salt, the amount of gamma  tubulin immunoprecipitated using XenC in low-salt buffer (this represents the total gamma  tubulin) was compared with the amount of gamma  tubulin immunoprecipitated using 109-2 in high salt buffer by densitometry scanning. The supernatants from the 109-2 and random IgG immunodepletion reactions were desalted and concentrated ~20-fold as described above and then used for complementation assays (see below). To assess whether gamma  tubulin and Xgrip109 interact with each other, the immunoprecipitation was carried out with an excess of extract. Under this condition, the amount of gamma  tubulin present in the extract is in excess of the added gamma -tubulin antibodies and results in cleaner immunoprecipitation.

To prepare gamma TuRC-depleted egg extract, either 4.5 µl of XenC antibodies (~1.6 mg/ml), or 2.5 µl of control random IgG (~3 mg/ml) were bound to 10-20 µl of settled Affi-Prep Protein A Support (Bio-Rad Laboratories). 100 µl of the thawed Xenopus egg extract was added to the CSF-XB-washed protein A beads and then incubated with rotation at 4°C for 1 h. The protein A beads were then pelleted and the supernatant was used for centrosome assembly assays.

To determine whether the random IgG- or XenC IgG-depleted extracts were functional in centrosome assembly, 7.5 µl of each extract was combined with 2.5 µl of Hepes 100 containing 1 mM of GTP, 1 µl of rhodamine-tubulin, and 1 µl of Xenopus sperm (see above). The centrosome formation assay was carried out as described above. To complement the XenC-depleted extract, 2.5 µl of the concentrated Asp that was resuspended in Hepes 100, Hepes 1 M, Hepes 1 M depleted with random IgG, or Hepes 1 M depleted with 109-2 IgG was added to 7.5 µl of XenC-depleted extract instead of buffer. Centrosome formation was quantitated as described above.

Cell Culture and Cell Extract

Cell line XLK-WG, derived from a primary culture of Xenopus kidney cells, was provided by Z. Wu and J.G. Gall (both from Carnegie Institution of Washington, Baltimore, MD). The cells were grown at 32°C in a water-saturated atmosphere of 5% CO2 in air in medium containing 60% RPMI medium 1640 (Life Technologies, Inc.), 20% heat-inactivated FBS (Life Technologies, Inc.), 100 U/ml each of penicillin and streptomycin, and 2 g/liter of NaHCO3.

To make XLK-WG cell extract, 5 plates (100-mm-diam) of 80% confluent XLK-WG cells were first rinsed with Hepes 100 and then harvested by scraping. The cell pellet was resuspended in 1 ml Hepes 100 containing 0.1% Triton X-100, 1 mM 2-mercaptoethanol, 1 mM PMSF, 0.01 mM benzamidine HCl, 0.001 mg/ml phenanthroline, and 0.01 mg/ml each of aprotinin, leupeptin, and pepstatin A. The cells were homogenized in a Dounce homogenizer, frozen in liquid N2, and then stored at -80°C.

Immunofluorescence Staining

For indirect immunofluorescence staining, XLK-WG cells were rinsed with OR2, fixed in -20°C methanol for 5 min, followed by hydrating in OR2 for 5 min. After permeabilizing with OR2 containing 0.1% Triton X-100 for 1 min, the cells were rinsed with OR2, blocked in OR2 containing 2% BSA for 30 min, and then incubated for 1 h with monoclonal anti- gamma -tubulin antibody diluted 200-fold (clone number GTU-88; Sigma Chemical Co.) and 109-2 or 109-c diluted 400-fold. After three 5-min washes, the cells were incubated for 1 h with FITC-conjugated goat anti-mouse and Cy3-conjugated goat anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 400-fold. After three 5-min washes, the cells were stained with DAPI and then mounted in antifade.

To localize gamma  tubulin and Xgrip109 in the sperm nuclei and microtubule asters, we carried out the centrosome assembly assay as described above but in the absence of rhodamine tubulin. The sperm nuclei and microtubule asters were centrifuged onto round glass coverslips (refer to above) and the same immunofluorescence staining steps as for the XLK-WG cells were performed. Photomicrographs were obtained using a cooled charge-coupled device camera (Princeton Scientific Instruments, Inc., Princeton, NJ) on a microscope (model E800; Nikon Corp., Tokyo, Japan). Images were handled digitally using Adobe Photoshop (Adobe Systems Inc., Mountain View, CA).

Sucrose Gradient Sedimentation

To analyze the sedimentation behavior of gamma  tubulin and Xgrip109 in XLK-WG cell extracts and Xenopus egg extracts, 5 ml of 5-40% sucrose step gradients were poured and allowed to diffuse overnight. Each step (950 µl) contained 5, 10, 20, 30, or 40% sucrose in Hepes 100. The thawed XLK-WG cell extract was clarified at top speed in a microfuge (Eppendorf 5417C; Hamburg, Germany) for 10 min, and 300 µl of this supernatant or 300 µl of clarified egg extract was directly loaded onto the sucrose gradients. Another 300 µl of the supernatant or clarified egg extract was precipitated with 2.5% polyethylene glycol (PEG), mol wt of 8,000 (Sigma Chemical Co.). The pellet, which contained over 90% of the total cellular gamma TuRC, was resuspended in 300 µl of Hepes 100 and then loaded onto a separate gradient. Centrifugation was performed in an SW55 rotor (model SW55; Beckman Instruments, Inc.) at 50,000 rpm for 4.5 h at 4°C. 300-µl fractions were collected from the top of each gradient. The fractions were separated by 10% SDS-PAGE and then analyzed by Western analysis, probing with anti-gamma -tubulin and anti-Xgrip109 antibodies.

We used 2 ml of 5-40% sucrose gradients to analyze 100 µl of the Asp that was resuspended in either Hepes 100 or Hepes 1 M (see above). The gradients were poured as described above except that each of the five steps was 400 µl. The standards, BSA (4.4 S), bovine liver catalase (11.3 S), and porcine thyroglobulin (19.4 S), were dissolved in either Hepes 100 or Hepes 1 M and run on identical sucrose gradients as the samples. The gradients were centrifuged in a rotor (model TLS55; Beckman Instruments, Inc.) at 55,000 rpm for 2 h at 4°C. 130-µl fractions were collected from the top of each gradient and analyzed by SDS-PAGE followed by Western analysis, probing with anti-gamma -tubulin and anti-Xgrip109 antibodies. Because the Hepes 1 M resuspended samples were heavier than the 5% sucrose at the top of the gradient, standards dissolved in Hepes 1 M appeared to sediment faster than the same standards dissolved in Hepes 100. 

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Xgrip109 Is a Conserved Protein

We previously reported the purification and biochemical characterization of gamma TuRC (Zheng et al., 1995). gamma TuRC consists of approximately eight uncharacterized polypeptides in addition to gamma  tubulin. We refer to each polypeptide as Xgrip followed by the respective apparent molecular mass of that polypeptide (see Fig. 3 B). We took two approaches to identify the Xgrips. First, we raised mouse polyclonal antibodies against gel-purified Xgrips. Xgrip109 and Xgrip195 both elicited antibody responses in mice. Second, we sequenced the purified Xgrips that were immobilized on polyvinylidine difluoride membrane. We obtained internal peptide sequences for Xgrip109, Xgrip110, and the Xgrip75s. Since we could not resolve Xgrip109 and Xgrip110 well enough on the gel to sequence them separately, the peptide sequences obtained are derived from both proteins, and the same is true for the Xgrip75 group of proteins.


View larger version (57K):
[in this window]
[in a new window]
 


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 3.   Xgrip109 is a component of gamma TuRC. (A) Xgrip109 antibodies. Lanes 1 and 2 show Western analysis of Xenopus egg extracts separated by 10% SDS-PAGE followed by immunoblotting with affinity-purified antibodies 109-2 and 109-c, respectively. Both antibodies specifically recognize a protein of 109 kD in the Xenopus egg extract. (B) Antibodies against Xgrip109 (109-2) and gamma  tubulin (XenC) both immunoprecipitated gamma TuRC components. Lanes 1 and 3 are immunoprecipitations carried out from the clarified egg extracts. Lanes 2 and 4 are immunoprecipitations carried out from 30% ammonium sulfate precipitates that were resuspended in low-salt buffer (refer to Materials and Methods). The immunoprecipitated proteins were separated by 10% SDS-PAGE and stained with Coomassie blue. Each of the gamma TuRC components (Xgrips) is indicated by its respective molecular mass. Immunoprecipitating antibodies are indicated at the bottom of the lanes. (C) Xgrip109 cosediments with gamma  tubulin. Xenopus egg extracts or extracts made from a Xenopus kidney cell line (XKL-WG cell line) were precipitated with 2.5% PEG, a treatment that precipitates more than 90% of the gamma  tubulin (data not shown). The pellet was resuspended in Hepes 100 and then sedimented on a 5-40% sucrose density gradient. Fractions were collected from the top of the gradients and then analyzed by Western blotting, probing with XenC and 109-2. The last fraction (fraction 17) in each sedimentation experiment included the pellet.

We screened a lambda ZAP Xenopus oocyte cDNA library using the mouse polyclonal antibodies against Xgrip109 and cloned overlapping cDNAs that contained a single ORF (refer to Materials and Methods). We found that half of the peptide sequences we obtained had perfect matches in the ORF, confirming that we cloned the correct Xgrip109 cDNA. We believed that the peptide sequences that do not match Xgrip109 are derived from Xgrip110. The longest cDNA clone has a stop codon followed by a poly A tail at its 3' end, but it lacks a complete 5' coding sequence. Using 5' RACE, we cloned the missing 5' end.

The complete Xgrip109 cDNA (GenBank/EMBL/DDBJ accession number AF052663) encodes a protein of 906 amino acids with a predicted molecular mass of 103.6 kD. Searches of the protein databases reveal that Xgrip109 is most closely related to Spc98, a S. cerevisiae spindle-pole body component (Geissler et al., 1996). Although overall Xgrip109 is only ~21% identical and 46% similar to Spc98, the stretch of amino acids (~180) in the middle of both proteins share 28% amino acid identity (Fig. 1). When we searched the expressed sequence tag (EST) databases using the Xgrip109 sequence, we found two groups of conserved human, mouse, and zebrafish ESTs that both share homology with a region of Xgrip109 between amino acids 512-684. The first group of ESTs (Fig. 2 A) are over 85% identical to Xgrip109 over the entire region between amino acids 512-684. The second group of ESTs, consisting of one human EST and one mouse EST that are over 85% identical to each other, shares more limited homology (~30% identity) with Xgrip109 over a smaller region between amino acids 534-612. Furthermore, database searches with the entire Xgrip109 sequence also identified homologous ESTs in rice (~52% amino acid identity, accession number C26482) and Drosophila (36% amino acid identity, accession number AA246343). These sequence analyses suggest that Xgrip109 is a conserved protein that, at least in vertebrates, may share homology with two families of related proteins.


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1.   Xgrip109 is homologous to the yeast gamma -tubulin-interacting protein Spc98. Sequence comparison of Xgrip109 and Spc98. Vertical lines, identical amino acids; two dots, conserved amino acid changes. The overall amino acid identity shared between the two sequences is ~21%. The underlined region shares ~28% amino acid identity. The sequences in Xgrip109 that match the internal peptide are boxed.


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 2.   There may be two protein families in vertebrates that share homology with Xgrip109. (A) Sequence comparisons among one group of human (GenBank/EMBL/DDBJ accession number T55505), mouse (accession number AA152700), zebrafish (accession number AA495279) ESTs, and Xgrip109 (from amino acid 512 to 684). These sequences share over 85% amino acid identity. (B) Sequence comparisons among another group of human (accession number R13714) and mouse (accession number AA543491) ESTs, and Xgrip109 (from amino acid 534 to 612). Although the two EST sequences share over 85% amino acid identity with each other, Xgrip109 is only ~38% identical to the two EST sequences.

Xgrip109 Is a Component of the gamma TuRC

To study the function of Xgrip109, we raised antibodies against a GST fusion protein with amino acids 134-244 of Xgrip109, as well as a synthetic peptide corresponding to the COOH terminus of Xgrip109 (refer to Materials and Methods). The two affinity-purified antibodies, which we refer to as 109-2 and 109-c, respectively, specifically recognize a protein of 109 kD when used to probe Western blots of Xenopus egg extracts (Fig. 3 A).

Two approaches were used to confirm that Xgrip109 is indeed a component of the gamma TuRC. First, we compared the patterns of proteins coimmunoprecipitated by antibodies against Xgrip109 (109-2) and gamma  tubulin (XenC, Fig. 3 B). Both XenC (an antibody raised against the COOH terminus of gamma  tubulin (Zheng et al., 1995) and 109-2 antibodies immunoprecipitated the same set of proteins. Moreover, this protein profile is identical to that of the gamma TuRC that we identified previously (Zheng et al., 1995). Although immunoprecipitations from clarified egg extracts gave a much higher background than immunoprecipitations from resuspended, clarified 30% Asps, the gamma TuRC components are still readily identifiable (Fig. 3 B). The presence of gamma  tubulin and Xgrip109 in the immunoprecipitates was confirmed by Western analysis using XenC and 109-2, respectively (data not shown).

Second, we compared the sedimentation behavior of Xgrip109 and gamma  tubulin on sucrose gradients. Xenopus egg extracts or cell extracts made from a Xenopus kidney cell line were either directly sedimented (data not shown) on 5-40% sucrose gradients, or first precipitated with 2.5% PEG and then sedimented (Fig. 3 C). The fractions from these gradients were analyzed by Western blotting, probing with antibodies against Xgrip109 and gamma  tubulin. We found that Xgrip109 and gamma  tubulin cosediment (Fig. 3 C). These results show that Xgrip109 is a component of the gamma TuRC.

Xgrip109 Is Localized to the Centrosomes

Since Xgrip109 is a component of the gamma TuRC, we expected Xgrip109, like gamma  tubulin, to localize to centrosomes. Double label-immunofluorescence staining for gamma  tubulin and Xgrip109 in Xenopus kidney tissue culture cells (XLK-WG) revealed that Xgrip109, like gamma  tubulin, does indeed localize to centrosomes (Fig. 4 A).


View larger version (60K):
[in this window]
[in a new window]
 


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 4.   Xgrip109 is localized to centrosomes. (A) Xgrip109 and gamma  tubulin colocalized to the centrosomes in XLK-WG cells. gamma  tubulin, gamma -tubulin localization revealed by fluorescein secondary antibody; Xgrip109, Xgrip109 localization revealed by Cy3 secondary antibody; DAPI, DNA staining with DAPI; Nom, Nomarski images of the cells. (B) Xgrip109 is localized to the in vitro-assembled centrosomes. The in vitro-assembled centrosomes were spun onto glass coverslips and indirect immunofluorescence staining was carried out using anti-gamma -tubulin and anti-Xgrip109 antibodies (refer to Materials and Methods). gamma  tubulin, gamma  tubulin was localized to the tip of the sperm nucleus; Xgrip109, Xgrip109 was also localized to the tip of the sperm nucleus; DAPI, the two-sperm nuclei that were stained with either anti-gamma -tubulin antibody (GTU-88) or anti-Xgrip109 antibodies (109-2) were stained with DAPI. The images are a combination of fluorescence and phase images. Arrows, microtubule asters at the tips of the two-sperm nuclei. Bars: (A) 20 µm; (B) 10 µm.

We next assembled centrosomes in vitro by incubating demembranated Xenopus sperm with Xenopus egg extracts. In the presence of an ATP-containing energy regenerating system, the pair of centrioles located at the tips of the sperm nuclei can recruit proteins to form a functional centrosome that nucleates a microtubule aster (Felix et al., 1994; Stearns and Kirschner, 1994). Using immunofluorescence staining, we found that, like gamma  tubulin, Xgrip109 also localized to these in vitro assembled centrosomes (Fig. 4 B). To make sure that the gamma  tubulin and Xgrip109 staining would not bleed through due to microtubule staining, we omitted the microtubule label and visualized aster formation by phase-contrast microscopy. The extensive microtubule arrays appear clearly in the phase image (Fig. 4 B, arrows).

Xgrip109 Directly Interacts with gamma  Tubulin in the gamma TuRC

To determine if the interaction between Xgrip109 and gamma  tubulin is direct, we disrupted the gamma TuRC with high salt and examined the sedimentation behavior of Xgrip109 and gamma  tubulin by sucrose gradient sedimentation. The gamma TuRC was precipitated from the Xenopus egg extract with 30% ammonium sulfate and resuspended in either low-salt or high-salt buffers and then incubated on ice for 60-80 min to allow complete dissociation of gamma TuRC in the presence of high salt. Both samples were then separated on 5-40% sucrose gradients followed by Western analysis, probing with antibodies against Xgrip109 and gamma  tubulin. Protein standards BSA, catalase, and thyroglobulin were dissolved in either low-salt or high-salt buffers and then run on identical gradients. Fig. 5 A shows that high salt caused both gamma  tubulin and Xgrip109 to migrate as a smaller complex (~11 S). On the other hand, the gamma TuRC remained intact (>19.4 S) when the 30% Asp was resuspended in the low-salt buffer (Fig. 5 A).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5.   Xgrip109 interacts directly with gamma  tubulin. (A) gamma TuRC is dissociated in high salt. Clarified Xenopus egg extracts were first precipitated with 30% ammonium sulfate. The pellet (30% Asp) was resuspended in either Hepes 1 M (refer to Materials and Methods) or Hepes 100. The resuspended proteins were fractionated on 5-40% sucrose gradients. The sucrose gradient standards used were bovine serum albumin (4.4S), bovine liver catalase (11.3S), and bovine thyroglobulin (19.4S). The protein standards used (indicated at the bottom of each panel in A) were dissolved in either Hepes 1 M or Hepes 100 and then fractionated under identical conditions. Gradient fractions were collected from the top and each fraction was analyzed by SDS-PAGE followed by Western blotting with XenC and 109-2 antibodies. (B) A fraction of the gamma  tubulin remains associated with Xgrip109 in high salt. Immunoprecipitations with XenC (lanes 1 and 3) or 109-2 antibodies (lanes 2 and 4) were carried out using 30% Asp resuspended in either Hepes 1 M (lanes 1 and 2) or Hepes 100 (lanes 3 and 4). The precipitated proteins were separated by SDS-PAGE and then stained by Coomassie blue. The gamma TuRC components (Xgrips) are indicated by their respective molecular masses.

To determine whether the salt-dissociated Xgrip109 remained associated with any other gamma TuRC components, we immunoprecipitated Xgrip109 from the 30% Asp resuspended in either low-salt or high-salt buffers. We found that gamma  tubulin was the only gamma TuRC subunit that coimmunoprecipitated with Xgrip109 in high salt (Fig. 5 B, lane 2). The presence of Xgrip109 and gamma  tubulin in the immunoprecipitates was confirmed by Western analysis (data not shown). These results suggest that Xgrip109 directly binds to gamma  tubulin in gamma TuRC. We estimated, based on densitometry scanning, that less than half of the total gamma  tubulin in the gamma TuRC remained associated with Xgrip109 in the high-salt buffer (refer to Materials and Methods). As a control, gamma  tubulin was also immunoprecipitated in parallel using XenC. Since the XenC antibody did not bind to gamma  tubulin well in high-salt buffers (Fig. 5 B, lane 1), we could not determine whether the remaining gamma  tubulin binds to any other Xgrips under high salt conditions.

Immunodepletion of Xgrip109 Blocks gamma TuRC Reassembly

Since we were able to disrupt the gamma TuRC with high salt, we wanted to test whether removing the salt would allow the gamma TuRC to reform. Furthermore, if the gamma TuRC could reassemble, we wanted to examine whether Xgrip109 is needed for gamma TuRC reformation. We used sucrose gradient sedimentation to analyze what happened to the gamma TuRC after salt treatment and immunodepletion with either random IgG or Xgrip109 IgG. A 30% Asp was resuspended in high-salt buffer and then immunodepleted with either random IgG or Xgrip109 antibodies (109-2). The immunodepletion supernatants were either directly loaded on 5-40% sucrose gradients, or desalted, and then loaded on sucrose gradients. We analyzed the sucrose gradient fractions by Western, probing with anti-gamma -tubulin and anti-Xgrip109 antibodies. The control, Fig. 6 E, shows that under low-salt conditions, the intact gamma TuRC sedimented as a large particle (>19.4 S). On the other hand, the gamma TuRC was completely dissociated when the 30% Asp was resuspended in high-salt buffer and then immunodepleted with either random IgG (Fig. 6 A) or Xgrip109 IgG (Fig. 6 B). When the salt was removed from the random IgG-depleted sample, at least 50% of gamma  tubulin and Xgrip109 were assembled into a complex that had the same S value as that of the endogenous gamma TuRC (Fig. 6 C). When the salt was removed from the Xgrip109-depleted sample, the remaining gamma  tubulin had a similar S value to that in high-salt (Fig. 6 D). We analyzed the proteins that were removed by 109-2 IgG and random IgG by both Coomassie blue staining and Western, probing with 109-2 and XenC (Fig. 6, F and G). It is clear that, compared with random IgG, 109-2 IgG specifically removed Xgrip109 and gamma  tubulin. These results suggest that 50% of gamma TuRC reassembled after desalting. Furthermore, removing Xgrip109 and a small amount of gamma  tubulin from the resuspended 30% Asp before lowering the salt concentration blocked the reassembly.


View larger version (73K):
[in this window]
[in a new window]
 
Fig. 6.   Xgrip109 is required for the reformation of salt-disrupted gamma TuRC. Clarified Xenopus egg extracts were precipitated with 30% ammonium sulfate. The pellet fraction (30% Asp) was resuspended in either Hepes 100 as control or in Hepes 1 M. Sucrose gradients in A-E are all 5-40%. (A) 30% Asp resuspended in Hepes 1M was immunoprecipitated with random IgG and then analyzed on a sucrose gradient. (B) The same as in A, except that the anti-Xgrip109 antibody, 109-2, was used in the immunoprecipitation. (C) The same as in A, except that after immunoprecipitation, a desalting step was included before the sucrose gradient sedimentation. (D) The same as in B, except that after immunoprecipitation, a desalting step was included before the sucrose gradient sedimentation. (E) Control, 30% Asp resuspended in Hepes 100, desalted, and fractionated on a sucrose gradient. (F) SDS-PAGE separation followed by Coomassie blue staining of proteins that were immunoprecipitated with random IgG (lane 1) and 109-2 IgG (lane 2). (G) The same protein samples in F were analyzed by Western probing with XenC and 109-2. Because samples in A and B contained higher amounts of salt than that of the samples in C, D, and E (refer to Materials and Methods), the sucrose gradient fractions of A and B cannot be compared directly to that of C, D, and E. The molecular weight standards for A and B are indicated at the bottom of A; C, D, and E are indicated at the bottom of E. Standards used are BSA (4.4S), bovine liver catalase (11.3S), and bovine thyroglobulin (19.4S).

A Complementation Assay for Centrosome Assembly

To study the role of Xgrip109 (or any other Xgrips) in the recruitment of gamma  tubulin to the centrosome and/or the formation of a functional centrosome, we needed to develop a centrosome formation assay where the activity is dependent on a source of gamma TuRC that we can biochemically manipulate to selectively remove specific Xgrips. Since we found that the gamma TuRC present in a 30% Asp can be dissociated with salt and reformed by desalting, we hope to use this as a source of gamma TuRC.

We took advantage of the existing centrosome formation assay using Xenopus egg extracts and sperm (Felix et al., 1994; Stearns and Kirschner, 1994). This centrosome formation assay was based on the original observation of Lohka and Masui (1983) who showed that the demembranated sperm and Xenopus egg extract can be used to study various cellular processes such as DNA replication, chromosome condensation, and centrosome and spindle formation in vitro. The centrosome formation assay is essentially an assay for PCM assembly around the sperm centrioles located at the tip of the sperm nucleus. When the sperm is incubated with the egg extract, PCM assembles around the centriole pair, resulting in the formation of a functional centrosome that can nucleate microtubule asters. Using this assay, Felix et al. (1994) showed that immunodepleting gamma TuRC from the egg extract blocked the formation of a functional centrosome. We wanted to test whether the gamma TuRC present in the 30% Asp can complement the gamma TuRC-depleted extract to form a functional centrosome. We have previously found that the pellet alone did not support centrosome formation (data not shown).

Felix et al. (1994) used crude Xenopus egg extract for their centrosome formation assays. We found that clarifying the crude egg extract by centrifugation did not affect centrosome formation activity. Using XenC, we immunodepleted the gamma TuRC from the clarified extract (Fig. 7 A, lane 3). Consistent with previous results (Felix et al., 1994), this gamma TuRC-depleted extract was not able to assemble a functional centrosome judging by the absence of a microtubule aster (Fig. 7 B). As expected, Xgrip109 was also absent from the centrosome (Fig. 7 C). The control extract that was depleted with random IgG, on the other hand, assembled a functional centrosome around the sperm centrioles, and Xgrip109 was present at the centrosome (Fig. 7, B and C).


View larger version (62K):
[in this window]
[in a new window]
 


View larger version (45K):
[in this window]
[in a new window]
 


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7.   The complementation assay for centrosome formation. (A) Western analysis of the immunodepleted extracts, 30% ammonium supernatant, the pellet was probed with 109-2 and XenC. Lanes 1-4: clarified extract; clarified extract immunodepleted using random IgG; clarified extract immunodepleted using XenC; 30% ammonium sulfate supernatant (there is no detectable gamma  tubulin or Xgrip109), respectively. Lanes 5 and 6: 30% Asp resuspended in Hepes 100 (lane 5) or Hepes 1 M (lane 6), desalted, and then concentrated ~20-fold. (B) Quantitation of the complementation assays. Red columns, percentage of sperm with centrosomes that nucleated astral microtubules; blue columns, percentage of sperm without any microtubule nucleation from the tip of the sperm; green columns, percentage of sperm with centrosomes that nucleated a few disorganized microtubules; Ran. IgG, depletion with random IgG allowed over 80% of sperm centrioles to assemble into centrosomes; XenC IgG, depletion with XenC IgG completely abolished the centrosome assembly activity in the extract; +ASP.100mM, addition of 30% Asp resuspended in Hepes 100 to the gamma TuRC- depleted extract resulted in over 80% of sperm centrioles to assemble into centrosomes; +ASP.1M, addition of 1 M salt-treated and desalted 30% Asp to the gamma TuRC-depleted extract resulted in over 70% of sperm centrioles to assemble into centrosomes. The error bars were determined from four independent assays in each case. (C) Representative sperm nuclei with or without a microtubule aster from the assays in B are shown. The microtubules were labeled by the addition of a small amount of rhodamine- tubulin in the assays. Xgrip109 was detected using 109-c antibodies and a fluorescein conjugated goat anti-rabbit secondary antibody. The sperm DNA was stained by DAPI. Bar, 10 µm.

To test whether the gamma TuRC present in the 30% Asp can complement the gamma TuRC-depleted extract to form a centrosome, we resuspended this pellet in low-salt buffer, desalted and concentrated it ~20-fold. We found that this gamma TuRC-containing fraction restored the centrosome assembly activity of the gamma TuRC-depleted extract (Fig. 7). We next tested whether an Asp that was resuspended in high salt still retains the complementing activity after salt removal. The pellet was resuspended in high-salt buffer and incubated on ice to allow complete dissociation of gamma TuRC. The fraction was desalted, concentrated ~20-fold, and then tested in the complementation assay. We found that this salt-treated 30% Asp also complemented the gamma TuRC-depleted extract to form a functional centrosome (Fig. 7). Since we know that the gamma TuRC in this pellet is dissociated by the high-salt incubation (see above), this complementation assay allows us to study the effect of selective immunodepletion of Xgrips on centrosome assembly.

Immunodepletion of Xgrip109 Abolishes the Complementing Activity

To study the role of Xgrip109 in gamma -tubulin recruitment and centrosome formation, we wanted to deplete the Xgrip109 from the salt-dissociated gamma TuRC present in the resuspended Asp, and ask whether the remaining gamma TuRC components still function in the complementation assay described above. Xgrip109 was immunodepleted from the pellet resuspended in high-salt buffers (Fig. 8 A, lanes 4-8). Control reactions were immunodepleted with random IgG. The two antibody-depleted samples were desalted into low-salt buffers and then concentrated ~20-fold (Fig. 8 A, lanes 5-8 for the extent of immunodepletion). We found that immunodepleting Xgrip109 abolished the complementing activity present in the resuspended pellet (Fig. 8, B and C), whereas the random IgG control retained the complementing activity (Fig. 8, B and C). Although immunodepleting Xgrip109 removed a fraction of gamma  tubulin (refer to Fig. 5 B, lane 2), we estimated that more than half of the total amount of gamma  tubulin remained (Fig. 8 A, lanes 4-8). Interestingly, this remaining gamma  tubulin did not appear to be recruited to the centrosome (Fig. 8 C, gamma -tubulin staining of the centrosomes). These results suggest that Xgrip109 is necessary for the recruitment of gamma  tubulin to the centrosome, and for the formation of a functional centrosome.


View larger version (66K):
[in this window]
[in a new window]
 


View larger version (43K):
[in this window]
[in a new window]
 


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 8.   Xgrip109 is essential for the formation of a functional centrosome. (A) Western analysis of the immunodepleted extract, 30% Asp, and 30% ammonium sulfate supernatant probed with XenC and 109-2. Lanes 1 and 2: clarified extract immunodepleted with either random IgG (lane 1) or XenC (lane 2). Judging by the absence of gamma  tubulin and Xgrip109, XenC depleted the gamma TuRC (lane 2). Lane 3, 30% ammonium sulfate supernatant that does not contain detectable gamma TuRC as expected. Lane 4, 30% Asp resuspended in Hepes 1 M. Lanes 5 and 6, 30% Asp resuspended in Hepes 1 M and then immunodepleted using either random IgG (lane 5) or 109-2 (lane 6). Lanes 7 and 8 are the same as lanes 5 and 6, respectively, except that the proteins were desalted into Hepes 100, and concentrated ~20-fold. Lanes 5-8 show that immunodepletion of Xgrip109 in 1 M KCl removed only a fraction of gamma  tubulin (compare gamma -tubulin signals in lanes 5 and 6), whereas Xgrip109 is completely depleted. (B) Quantitation of the complementation assay. Red columns, percentages of sperm with microtubule asters nucleated from the assembled centrosomes; blue columns, percentages of sperm without microtubule asters; white columns, percentages of sperm with assembled centrosomes that nucleated only a few disorganized microtubules; Ran. IgG, centrosome formation assays carried out with clarified extracts that were immunodepleted with random IgG. Over 80% of the sperm centrioles assembled into centrosomes. XenC. IgG, centrosome formation assays carried out with clarified extracts that were immunodepleted of gamma TuRC using XenC IgG. The centrosome assembly activity was abolished. +Asp Ran. IgG, 30% Asp that was resuspended in Hepes 1 M and immunodepleted with random IgG complemented the gamma TuRC-depleted extract to assemble centrosomes. +Asp Xgrip109, 30% Asp that was resuspended in Hepes 1M and immunodepleted of Xgrip109 did not complement the gamma TuRC-depleted extract to assemble centrosomes. (C) Representative sperm nuclei with or without a microtubule aster from the assays in B are shown. The microtubules were labeled by the addition of a small amount of rhodamine-tubulin in the assays. gamma  Tubulin was detected using an anti-gamma -tubulin monoclonal antibody GTU-88 (Sigma Chemical Co.) and a fluorescein-conjugated goat anti-mouse secondary antibody. gamma  Tubulin is not recruited to the centrosome in the absence of Xgrip109. The sperm DNA was stained by DAPI. Bar, 10 µm.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Xgrip109 Is a Conserved gamma  Tubulin-Interacting Protein

The gamma TuRC consists of approximately eight polypeptides (Xgrips) in addition to gamma  tubulin. In an effort to further understand the assembly and function of the gamma TuRC, we have begun to characterize the gamma TuRC components. We cloned and sequenced one of the Xgrips, Xgrip109. Sequence analysis revealed that Xgrip109 is homologous to the yeast gamma  tubulin-interacting protein Spc98 (Geissler et al., 1996) (refer to Fig. 1). Like Spc98, Xgrip109 also interacts with gamma  tubulin (refer to Fig. 5). This suggests that Xgrip109 and Spc98 may have similar cellular functions. In Xenopus, Xgrip109 is a component of the gamma TuRC. The S. cerevisiae Spc98 is also a component of a gamma -tubulin-containing complex, although the yeast gamma -tubulin complex has a much smaller S value than that of the gamma TuRC (Geissler et al., 1996; Knop et al., 1997). It will be interesting to study whether the smaller yeast gamma -tubulin complex, like the gamma TuRC, can nucleate microtubules in vitro.

EST database searches shows that Xgrip109 is highly conserved among humans, mice, fish, rice, and flies. The Xgrip109 EST homologues in vertebrates can be divided into two groups that share sequence identities with a similar region of Xgrip109 (refer to Fig. 2). One group of ESTs shares over 85% amino acid identity with Xgrip109 and with each other in the 140-amino acid overlapping sequences. The other group of ESTs shares over 30% amino acid identity with Xgrip109, and over 85% amino acid identity with each other in the 80-amino acid overlapping sequences (refer to Fig. 2). Approximately 30% of amino acid identity is shared between the two groups of ESTs. This suggests that Xgrip109 is highly conserved in vertebrates and that it belongs to one of the two families of related proteins. We have recently identified two related gamma -tubulin-interacting proteins that are components of Drosophila gamma TuRC and found that each shares sequence homology with one of the two families of proteins (our unpublished data). We suggest that there are two related families of conserved gamma -tubulin interacting proteins that participate in the formation of the gamma TuRC.

Xgrip109, gamma  Tubulin, and gamma TuRC Assembly

We found that the salt-dissociated gamma TuRC present in a resuspended 30% Asp can reassemble after desalting (refer to Fig. 6). The reassembly was judged by the shifting in the S value of gamma tubulin and Xgrip109 to that of the gamma TuRC in our experiments. Because only ~50% of the total gamma TuRC can reassemble, we did not attempt to purify the reassembled gamma TuRC due to the limited amount of starting material. Therefore, we did not carry out structural analysis to compare the reassembled gamma TuRC with that of the endogenous gamma TuRC. However, we believe that the reassembled gamma TuRC is at least similar to the endogenous gamma TuRC for two reasons. First, immunoprecipitation using anti-Xgrip109 and gamma -tubulin antibodies showed that the reassembled gamma TuRC contained the same set of proteins as that of the endogenous gamma TuRC (data not shown). Second, the reassembled gamma TuRC can functionally replace the endogenous gamma TuRC in the centrosome formation assay (see below).

Using the gamma TuRC reformation assay, we found that removing Xgrip109 and a small fraction of gamma  tubulin before desalting blocks the ability of the remaining gamma  tubulin to assemble into a gamma TuRC-sized complex after salt removal. In fact, the remaining gamma  tubulin has a similar S value before and after desalting. This suggests that Xgrip109 is required for gamma  tubulin to assemble into larger complexes.

We only looked at gamma TuRC dissociation and reassembly using 30% ammonium sulfate-precipitated gamma TuRC. It will be interesting to test whether the purified gamma TuRC can also reassemble after dissociation with high salt, or whether accessory factors present in the 30% Asp fraction are required. Unfortunately, since we can only purify a few micrograms of gamma TuRC, this type of experiment is currently impractical. Nevertheless, the fact that the dissociated gamma TuRC can be reassembled offers a useful assay to study how gamma TuRC is assembled from its subunits.

Xgrip109 Is a Centrosome Component

gamma -Tubulin-containing rings that have similar dimensions to that of the purified gamma TuRC were found in the PCM of purified centrosomes (Moritz et al., 1995a,b). One important question is whether the gamma TuRC or only gamma  tubulin in the gamma TuRC is recruited to the PCM to act as a microtubule nucleator. Because the purified gamma TuRC can nucleate microtubules in vitro, we propose that the gamma TuRC-like rings in the centrosome contain at least some of the Xgrips present in the gamma TuRC (Zheng et al., 1995). The finding that Xgrip109 is a centrosomal protein that interacts directly with gamma  tubulin suggests that Xgrip109 is likely a component of the centrosomal rings.

The Complementation Assay for Centrosome Assembly

To study how each of the Xgrips is involved in the assembly of a functional centrosome, we developed a complementation assay for centrosome formation. In this assay the centrosome formation activity depends on the combination of two fractions: a gamma TuRC-depleted extract and a 30% Asp fraction that contains the gamma TuRC. Since the gamma TuRC in the pellet can be dissociated with high salt and reassembled by removing the salt, we can remove specific subunit(s) by immunodepletion in high salt, remove the salt, and then study whether the remaining components reform a complex that can function in centrosome assembly. We believe that this type of assay will also be useful in studying other centrosomal components.

We know that the gamma TuRC in the Asp is the key activity that complements the gamma TuRC-depleted extract, because depleting Xgrip109 (Fig. 8) or gamma TuRC (our unpublished observation) abolishes the activity. An obvious question is whether the purified gamma TuRC is sufficient for the complementation. To address this question in a meaningful way, we are currently improving our purification methods to achieve sufficiently high gamma TuRC concentrations for the complementation assay.

gamma Tubulin, Xgrip109, gamma TuRC, and Centrosome Assembly

This study and previous work by Felix et al. (1994) strongly suggest that gamma TuRC is essential for centrosome assembly. An interesting question is how the gamma TuRC is recognized by the centrosome assembly machinery. One model is that the intact gamma TuRC is recognized by a recruiting factor(s) that assembles the whole gamma TuRC to the centrosome (Fig. 9 A). In this case, the observed centrosomal gamma -tubulin-containing rings (Moritz et al., 1995a,b) would be similar to the gamma TuRC. In an alternative model, gamma TuRC is merely a cytoplasmic storage form of the centrosomal ring components. Upon centrosome assembly, gamma TuRC is disassembled and only gamma  tubulin, and possibly some of the Xgrips, such as Xgrip109, are recognized and recruited to the centrosome to form the centrosomal ring (Fig. 9 B). Although we cannot yet differentiate between these two models, our findings suggest that the centrosome assembly machinery cannot recognize and recruit gamma  tubulin unless it is in the gamma TuRC or associated with Xgrip109. When all of the Xgrip109 and a small amount of gamma  tubulin was removed, the remaining gamma  tubulin did not reassemble into the gamma TuRC and was not recruited to the centrosome (refer to Figs. 7 and 8). Since only ~50% of the total gamma  tubulin remained upon Xgrip 109 depletion, it is formally possible that the failure of recruiting this remaining gamma  tubulin is merely due to insufficient gamma -tubulin concentration. However, we believe that this is an unlikely possibility for the following reasons. First, in our centrosome formation assays, the extract was always diluted to 60% of the original concentration (refer to Materials and Methods) and the centrosome formation and gamma TuRC recruiting were not significantly affected. Furthermore, Felix et al. (1994) showed that the extract can be diluted threefold without drastically affecting the centrosome formation activity. These observations suggest that the recruitment of gamma  tubulin to the centrosome should not be affected when the amount of gamma  tubulin is only diluted onefold. We are currently testing the role of Xgrip109 in gamma -tubulin recruitment.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 9.   Recruitment models. (A) Direct recruitment model. gamma TuRC directly binds to a hypothetical recruitment factor(s) and assembles to the centrosome to act as a microtubule nucleator. (B) Subunit recruitment model. gamma TuRC is first disassembled to subunits. Only some subunits are recruited to the centrosome by the recruitment factor.

In conclusion, we have initiated molecular characterizations of the gamma TuRC components. With the assays presented in this paper, we hope to determine the role of each of the gamma TuRC subunits in centrosome formation and microtubule nucleation.

    Footnotes

Received for publication 2 February 1998 and in revised form 11 March 1998.

   The cloning of Xgrip109 was initiated in the Alberts lab (University of California, San Francisco, CA). We thank T. Hirano (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) for his advice on the library screen and Z. Wu and J. Gall (both from Carnegie Institution of Washington, Baltimore, MD) for the Xenopus tissue culture cell line. We are grateful to T. Mitchison (Harvard University, Cambridge, MA) for his advice and help with the initial gamma TuRC purification and characterization. We thank K. Oogema (Harvard University, Cambridge, MA) C. Wiese, C.-M. Fan, O. Cohen-Fix, and D. Koshland (all four from Carnegie Institution of Washington) for their critical comments on the manuscript.
   Address all correspondence to Yixian Zheng, Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210. Tel.: (410) 554-1232. Fax: (410) 243-6311. E-mail: zheng{at}mail1.ciwemb.edu

This work was supported by grants from National Institutes of Health (RO1- GM56312-01) and the Pew Scholar's Award to Y. Zheng.

    Abbreviations used in this paper

gamma TuRC, gamma tubulin ring complex; Xgrip, Xenopus gamma ring protein; Asp, ammonium sulfate pellet; CSF, cytostatic factor; DAPI, 4',6-diamidino-2-phenylindole; EST, expressed sequence tag; GST, glutathione S-transferase; MTOC, microtubule organizing center; ORF, open reading frame; PCM, pericentriolar material; PEG, polyethylene glycol.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Belmont, L., and T. Mitchison. 1996. Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell 84: 623-631
2. Desai, A., and T.J. Mitchison. 1998. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol 13: 83-117 .
3. Endow, S., S. Kang, L. Satterwhite, M. Rose, V. Skeen, and E. Salmon. 1994. Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends. EMBO (Eur. Mol. Biol. Organ.) J 13: 2708-2713 [Abstract].
4. Erickson, H., and D. Stoffler. 1996. Tubulin rings are universal polymers of the tubulin family - alpha/beta, gamma, and FtsZ. J. Cell Biol 135: 5-8
5. Evans, L., T. Mitchison, and M. Kirschner. 1985. Influence of the centrosome on the structure of nucleated microtubules. J. Cell Biol. 100: 1185-1191 [Abstract].
6. Felix, M.-A., C. Antony, M. Wright, and B. Maro. 1994. Centrosome assembly in vitro: Role of gamma -tubulin recruitment in Xenopus sperm aster formation. J. Cell Biol. 124: 19-31 [Abstract].
7. Geissler, S., G. Pereira, A. Spang, M. Knop, S. Soues, J. Kilmartin, and E. Schiebel. 1996. The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO (Eur. Mol. Biol. Organ.) J 15: 3899-3911 [Abstract].
8. Harlow, E., and D. Lane. 1988. In Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 726 pp.
9. Hirano, T., and T. Mitchison. 1994. A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell 79: 449-458
10. Horio, T., S. Uzawa, M.K. Jung, B.R. Oakley, K. Tanaka, and M. Yanagida. 1991. The fission yeast gamma-tubulin is essential for mitosis and is localized at microtubule organizing centers. J. Cell Sci. 99: 693-700 [Abstract].
11. Hyman, A., D. Drechsel, D. Kellogg, S. Salser, K. Sawin, P. Steffen, L. Wordeman, and T. Mitchison. 1991. Preparation of modified tubulins. Methods Enzymol. 196: 478-487
12. Iwamatsu, A., and N. Yoshida-Kubomura. 1996. Systematic peptide fragmentation of polyvinylidene difluoride(PVDF)-immobilized proteins prior to microsequencing. J. Biochem. (Tokyo) 120: 29-34 [Abstract].
13. Joshi, H.C., M.J. Palacios, L. McNamara, and D.W. Cleveland. 1992. Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature 356: 80-83
14. Kellogg, D.R., M. Moritz, and B.M. Alberts. 1994. The centrosome and cellular organization. Annu. Rev. Biochem. 63: 639-674
15. Knop, M., G. Pereira, S. Geissler, K. Brein, and E. Schiebel. 1997. The spindle pole body component Spc97p interacts with the gamma tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO (Eur. Mol. Biol. Organ.) J. 16: 1550-1564 [Abstract/Free Full Text].
16. Li, Q., and H. Joshi. 1995. Gamma-tubulin is a minus-end-specific microtubule binding protein. J. Cell Biol 131: 207-214 [Abstract].
17. Lohka, M.J., and Y. Masui. 1983. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220: 719-721
18. Marschall, L., R. Jeng, J. Mulholland, and T. Stearns. 1996. Analysis of Tub4p, a yeast gamma-tubulin-like protein: Implications for microtubule organizing center function. J. Cell Biol 134: 443-454 [Abstract].
19. McNally, F., K. Okawa, A. Iwamatsu, and R. Vale. 1996. Katanin, the microtubule-severing ATPase, is concentrated at centrosomes. J. Cell Sci 109: 561-567 [Abstract/Free Full Text].
20. Melki, R., I. Vainberg, R. Chow, and N. Cowan. 1993. Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin. J. Cell Biol 122: 1301-1310 [Abstract].
21. Mitchison, T.J., and M.W. Kirschner. 1984. Dynamic instability of microtubule growth. Nature 312: 237-242
22. Moritz, M., M. Braunfeld, J. Sedat, B. Alberts, and D. Agard. 1995a. Gamma- tubulin-containing rings in the centrosome. Nature 378: 638-640
23. Moritz, M., M.B. Braunfeld, J.C. Fung, J.W. Sedat, B.M. Alberts, and D.A. Agard. 1995b. 3D structural characterization of centrosomes from early Drosophila embryos. J. Cell Biol. 130: 1149-1159 [Abstract].
24. Murray, A.W.. 1991. Cell cycle extracts. Methods Cell Biol. 36: 58-605 .
25. Oakley, B.R.. 1992. Gamma-tubulin: The microtubule organizer? Trends Cell Biol. 2: 1-5 .
26. Oakley, C.E., and B.R. Oakley. 1989. Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. Nature 338: 662-664
27. Oakley, B.R., C.E. Oakley, Y. Yoon, and M.K. Jung. 1990. Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell 61: 1289-1301
28. Olmstead, J.. 1986. Microtubule-associated proteins. Annu. Rev. Cell Biol. 2: 421-457 .
29. Raff, J.W., D.R. Kellogg, and B.M. Alberts. 1993. Drosophila gamma tubulin is part of a complex containing two previously identified centrosomal MAPs. J. Cell Biol. 121: 823-835 [Abstract].
30. Sambrook, J., E. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. C. Nolan, editor. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 545 pp.
31. Sawin, K., and T. Mitchison. 1991. Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol 112: 925-940 [Abstract].
32. Sobel, S., and M. Synder. 1995. A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae. J. Cell Biol 131: 1775-1788 [Abstract].
33. Spang, A., S. Geissler, K. Grein, and E. Schiebel. 1996. Gamma-tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle-pole body substructures that organize microtubules and is required for mitotic spindle formation. J. Cell Biol 134: 429-441 [Abstract].
34. Stearns, T., and M. Kirschner. 1994. In vitro reconstitution of centrosome assembly and function: The role of g-tubulin. Cell 76: 623-637
35. Stearns, T., L. Evans, and M. Kirschner. 1991. Gamma-tubulin is a highly conserved component of the centrosome. Cell 65: 825-836
36. Sunkel, C., R. Gomes, P. Sampaio, J. Perdigao, and C. Gonzalez. 1995. Gamma-tubulin is required for the structure and function of the microtubule organizing centre in Drosophila neuroblasts. EMBO (Eur. Mol. Biol. Organ.) J. 14: 28-36 [Abstract].
37. Tavosanis, G., S. Llamazares, G. Goulielmos, and C. Gonzalez. 1997. Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila. EMBO (Eur. Mol. Biol. Organ.) J 16: 1809-1819 [Abstract/Free Full Text].
38. Vallee, R., G. Bloom, and W. Theurkauf. 1984. Microtubule-associated proteins: Subunits of the cytomatrix. J Cell Biol 99: 38s-44s [Free Full Text].
39. Vogel, J., T. Stearns, C. Rieder, and R. Palazzo. 1997. Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin. J Cell Biol 137: 193-202 [Abstract/Free Full Text].
40. Weil, C., C. Oakley, and B. Oakley. 1986. Isolation of mip (microtubule-interacting protein) mutations of Aspergillus nidulans. Mol. Cell. Biol. 6: 2963-2968
41. Weisenberg, R.. 1972. Microtubule formation in vitro in solutions containing low calcium concentrations. Science 177: 1104-1105
42. Zheng, Y., M.K. Jung, and B.R. Oakley. 1991. Gamma-tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with the centrosome. Cell 65: 817-823
43. Zheng, Y., M. Wong, B. Alberts, and T. Mitchison. 1995. Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature 378: 578-583

Copyright © 1998 by The Rockefeller University Press.
0021-9525/98/05/675/13 $2.00