* Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210; and Central Laboratories for
Key Technology, Kirin Brewery Company, Ltd., Yokohama 236, Japan
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Abstract |
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Previous studies indicate that tubulin ring
complex (
TuRC) can nucleate microtubule assembly
and may be important in centrosome formation.
TuRC contains approximately eight subunits, which we refer to as Xenopus gamma ring proteins (Xgrips),
in addition to
tubulin. We found that one
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
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
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
TuRC and for the recruitment of
tubulin to the centrosome. Xgrip109,
therefore, is essential for the formation of a functional
centrosome.
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Introduction |
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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
-/
-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 tubulin as a suppressor of a
-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.
Tubulin
is highly conserved and has been found in all eukaryotes
examined (for review see Oakley, 1992
). Most
tubulins
share over 60% amino acid identity, with the exception of
Saccharomyces cerevisiae
tubulin, which is only ~40%
identical to the other
tubulins (Sobel and Synder, 1995
;
Marschall et al., 1996
; Spang et al., 1996
).
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
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
tubulin in microtubule nucleation at the centrosome.
Biochemical studies were also initiated to study how
tubulin is involved in microtubule nucleation at the
MTOCs. Human
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
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
-tubulin-containing complex has an estimated molecular mass of over 2,000 kD. This complex,
the
tubulin ring complex (
TuRC), has an open ring
structure and can nucleate microtubules in vitro. In addition to multiple
-tubulin molecules, the
TuRC contains
approximately eight additional polypeptides (Zheng et al.,
1995
). The S. cerevisiae
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
-tubulin complex
has a much smaller S value (6 S) (Geissler et al., 1996
;
Knop et al., 1997
) than that of the
TuRC (>25 S), it is not
clear what the functional relationship is between these
complexes or if the yeast
-tubulin complex is able to nucleate microtubules in vitro. Based on the structure and
function of the
TuRC, two models were proposed to explain how the
TuRC may nucleate microtubule assembly
(Zheng et al., 1995
; Erickson and Stoffler, 1996
). One
model suggests that the
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
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
TuRC.
In parallel structural studies, EM tomography has revealed hundreds of 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
tubulin (Moritz et al., 1995a
). The combined structural and biochemical studies of
tubulin led to
the hypothesis that the
TuRC is anchored within the PCM
where it acts to nucleate microtubules (Zheng et al., 1995
).
To better understand the relationship between the centrosomal -tubulin-containing rings and the cytosolic
TuRC,
we have begun to characterize the
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
-tubulin function.
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Materials and Methods |
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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 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
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-
-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
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 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 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 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 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
tubulin that
remained bound to Xgrip109 in high salt, the amount of
tubulin immunoprecipitated using XenC in low-salt buffer (this represents the total
tubulin) was compared with the amount of
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
tubulin and Xgrip109
interact with each other, the immunoprecipitation was carried out with an
excess of extract. Under this condition, the amount of
tubulin present in
the extract is in excess of the added
-tubulin antibodies and results in
cleaner immunoprecipitation.
To prepare 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-
-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 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 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
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-
-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--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.
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Results |
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Xgrip109 Is a Conserved Protein
We previously reported the purification and biochemical
characterization of TuRC (Zheng et al., 1995
).
TuRC
consists of approximately eight uncharacterized polypeptides in addition to
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.
|
We screened a 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.
|
|
Xgrip109 Is a Component of the 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 TuRC. First, we compared
the patterns of proteins coimmunoprecipitated by antibodies against Xgrip109 (109-2) and
tubulin (XenC, Fig.
3 B). Both XenC (an antibody raised against the COOH
terminus of
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
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
TuRC components are still readily identifiable (Fig. 3 B).
The presence of
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 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
tubulin. We
found that Xgrip109 and
tubulin cosediment (Fig. 3 C).
These results show that Xgrip109 is a component of the
TuRC.
Xgrip109 Is Localized to the Centrosomes
Since Xgrip109 is a component of the TuRC, we expected Xgrip109, like
tubulin, to localize to centrosomes.
Double label-immunofluorescence staining for
tubulin
and Xgrip109 in Xenopus kidney tissue culture cells
(XLK-WG) revealed that Xgrip109, like
tubulin, does
indeed localize to centrosomes (Fig. 4 A).
|
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
tubulin, Xgrip109 also
localized to these in vitro assembled centrosomes (Fig. 4 B).
To make sure that the
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 Tubulin in
the
TuRC
To determine if the interaction between Xgrip109 and tubulin is direct, we disrupted the
TuRC with high salt
and examined the sedimentation behavior of Xgrip109
and
tubulin by sucrose gradient sedimentation. The
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
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
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
tubulin and Xgrip109 to migrate as a smaller
complex (~11 S). On the other hand, the
TuRC remained intact (>19.4 S) when the 30% Asp was resuspended in the low-salt buffer (Fig. 5 A).
|
To determine whether the salt-dissociated Xgrip109 remained associated with any other TuRC components, we
immunoprecipitated Xgrip109 from the 30% Asp resuspended in either low-salt or high-salt buffers. We found
that
tubulin was the only
TuRC subunit that coimmunoprecipitated with Xgrip109 in high salt (Fig. 5 B, lane 2).
The presence of Xgrip109 and
tubulin in the immunoprecipitates was confirmed by Western analysis (data not
shown). These results suggest that Xgrip109 directly binds
to
tubulin in
TuRC. We estimated, based on densitometry scanning, that less than half of the total
tubulin in
the
TuRC remained associated with Xgrip109 in the
high-salt buffer (refer to Materials and Methods). As a control,
tubulin was also immunoprecipitated in parallel
using XenC. Since the XenC antibody did not bind to
tubulin well in high-salt buffers (Fig. 5 B, lane 1), we could
not determine whether the remaining
tubulin binds to
any other Xgrips under high salt conditions.
Immunodepletion of Xgrip109 Blocks
TuRC Reassembly
Since we were able to disrupt the TuRC with high salt,
we wanted to test whether removing the salt would allow
the
TuRC to reform. Furthermore, if the
TuRC could
reassemble, we wanted to examine whether Xgrip109 is
needed for
TuRC reformation. We used sucrose gradient
sedimentation to analyze what happened to the
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-
-tubulin and anti-Xgrip109 antibodies. The control, Fig. 6 E, shows that under low-salt conditions, the intact
TuRC sedimented as a
large particle (>19.4 S). On the other hand, the
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
tubulin and Xgrip109 were assembled into a complex that had the same S value as that
of the endogenous
TuRC (Fig. 6 C). When the salt was
removed from the Xgrip109-depleted sample, the remaining
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
tubulin. These results
suggest that 50% of
TuRC reassembled after desalting.
Furthermore, removing Xgrip109 and a small amount of
tubulin from the resuspended 30% Asp before lowering
the salt concentration blocked the reassembly.
|
A Complementation Assay for Centrosome Assembly
To study the role of Xgrip109 (or any other Xgrips) in the
recruitment of 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
TuRC that we can biochemically manipulate to selectively remove specific Xgrips. Since we
found that the
TuRC present in a 30% Asp can be dissociated with salt and reformed by desalting, we hope to use
this as a source of
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
TuRC from the egg extract blocked the
formation of a functional centrosome. We wanted to test
whether the
TuRC present in the 30% Asp can complement the
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
TuRC from the clarified extract (Fig. 7 A,
lane 3). Consistent with previous results (Felix et al.,
1994
), this
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).
|
To test whether the TuRC present in the 30% Asp can
complement the
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
TuRC-containing fraction restored the centrosome assembly activity of the
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
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
TuRC-depleted extract to form a functional centrosome
(Fig. 7). Since we know that the
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 -tubulin recruitment and
centrosome formation, we wanted to deplete the Xgrip109
from the salt-dissociated
TuRC present in the resuspended Asp, and ask whether the remaining
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
tubulin (refer to Fig. 5 B, lane 2), we estimated that more than half of
the total amount of
tubulin remained (Fig. 8 A, lanes 4-8).
Interestingly, this remaining
tubulin did not appear to be
recruited to the centrosome (Fig. 8 C,
-tubulin staining of
the centrosomes). These results suggest that Xgrip109 is
necessary for the recruitment of
tubulin to the centrosome, and for the formation of a functional centrosome.
|
![]() |
Discussion |
---|
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---|
Xgrip109 Is a Conserved Tubulin-Interacting Protein
The TuRC consists of approximately eight polypeptides
(Xgrips) in addition to
tubulin. In an effort to further understand the assembly and function of the
TuRC, we
have begun to characterize the
TuRC components. We
cloned and sequenced one of the Xgrips, Xgrip109. Sequence analysis revealed that Xgrip109 is homologous to
the yeast
tubulin-interacting protein Spc98 (Geissler et al.,
1996
) (refer to Fig. 1). Like Spc98, Xgrip109 also interacts with
tubulin (refer to Fig. 5). This suggests that Xgrip109 and Spc98 may have similar cellular functions. In Xenopus, Xgrip109 is a component of the
TuRC. The S. cerevisiae Spc98 is also a component of a
-tubulin-containing
complex, although the yeast
-tubulin complex has a much
smaller S value than that of the
TuRC (Geissler et al.,
1996
; Knop et al., 1997
). It will be interesting to study
whether the smaller yeast
-tubulin complex, like the
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
-tubulin-interacting proteins that are components of Drosophila
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
-tubulin interacting proteins that
participate in the formation of the
TuRC.
Xgrip109, Tubulin, and
TuRC Assembly
We found that the salt-dissociated 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
tubulin and Xgrip109 to that of the
TuRC in
our experiments. Because only ~50% of the total
TuRC
can reassemble, we did not attempt to purify the reassembled
TuRC due to the limited amount of starting material. Therefore, we did not carry out structural analysis to
compare the reassembled
TuRC with that of the endogenous
TuRC. However, we believe that the reassembled
TuRC is at least similar to the endogenous
TuRC for
two reasons. First, immunoprecipitation using anti-Xgrip109
and
-tubulin antibodies showed that the reassembled
TuRC contained the same set of proteins as that of the
endogenous
TuRC (data not shown). Second, the reassembled
TuRC can functionally replace the endogenous
TuRC in the centrosome formation assay (see below).
Using the TuRC reformation assay, we found that removing Xgrip109 and a small fraction of
tubulin before
desalting blocks the ability of the remaining
tubulin to
assemble into a
TuRC-sized complex after salt removal.
In fact, the remaining
tubulin has a similar S value before and after desalting. This suggests that Xgrip109 is required for
tubulin to assemble into larger complexes.
We only looked at TuRC dissociation and reassembly
using 30% ammonium sulfate-precipitated
TuRC. It will
be interesting to test whether the purified
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
TuRC, this type of experiment is currently impractical. Nevertheless, the fact that the dissociated
TuRC can be reassembled offers a useful assay to study
how
TuRC is assembled from its subunits.
Xgrip109 Is a Centrosome Component
-Tubulin-containing rings that have similar dimensions
to that of the purified
TuRC were found in the PCM of
purified centrosomes (Moritz et al., 1995a
,b). One important question is whether the
TuRC or only
tubulin in
the
TuRC is recruited to the PCM to act as a microtubule
nucleator. Because the purified
TuRC can nucleate microtubules in vitro, we propose that the
TuRC-like rings
in the centrosome contain at least some of the Xgrips
present in the
TuRC (Zheng et al., 1995
). The finding
that Xgrip109 is a centrosomal protein that interacts directly with
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 TuRC-depleted extract and a
30% Asp fraction that contains the
TuRC. Since the
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 TuRC in the Asp is the key activity
that complements the
TuRC-depleted extract, because
depleting Xgrip109 (Fig. 8) or
TuRC (our unpublished
observation) abolishes the activity. An obvious question is
whether the purified
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
TuRC concentrations for the
complementation assay.
Tubulin, Xgrip109,
TuRC, and
Centrosome Assembly
This study and previous work by Felix et al. (1994)
strongly suggest that
TuRC is essential for centrosome
assembly. An interesting question is how the
TuRC is
recognized by the centrosome assembly machinery. One
model is that the intact
TuRC is recognized by a recruiting factor(s) that assembles the whole
TuRC to the centrosome (Fig. 9 A). In this case, the observed centrosomal
-tubulin-containing rings (Moritz et al., 1995a
,b) would
be similar to the
TuRC. In an alternative model,
TuRC
is merely a cytoplasmic storage form of the centrosomal
ring components. Upon centrosome assembly,
TuRC is
disassembled and only
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
tubulin unless it
is in the
TuRC or associated with Xgrip109. When all of
the Xgrip109 and a small amount of
tubulin was removed, the remaining
tubulin did not reassemble into
the
TuRC and was not recruited to the centrosome (refer to Figs. 7 and 8). Since only ~50% of the total
tubulin
remained upon Xgrip 109 depletion, it is formally possible
that the failure of recruiting this remaining
tubulin is
merely due to insufficient
-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
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
tubulin to the centrosome should not be affected when the amount of
tubulin is only diluted onefold. We are currently testing the role of Xgrip109 in
-tubulin recruitment.
|
In conclusion, we have initiated molecular characterizations of the TuRC components. With the assays presented in this paper, we hope to determine the role of each
of the
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 initialThis 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 |
---|
TuRC,
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.
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