* Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; Department of Embryology, Carnegie
Institute of Washington, Baltimore, Maryland 21210; § Department of Cell Biology, The Scripps Research Institute, La Jolla,
California 92037; and
Central Laboratories for Key Technology, Kirin Brewery Company, Ltd., Yokohama 236, Japan
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Abstract |
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-tubulin exists in two related complexes in
Drosophila embryo extracts (Moritz, M., Y. Zheng,
B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1-
12). Here, we report the purification and characterization of both complexes that we name
-tubulin small
complex (
TuSC; ~280,000 D) and Drosophila
TuRC
(~2,200,000 D). In addition to
-tubulin, the
TuSC
contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The
TuSC is a
structural subunit of the
TuRC, a larger complex containing about six additional polypeptides. Like the
TuRC isolated from Xenopus egg extracts (Zheng, Y.,
M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature.
378:578-583), the Drosophila
TuRC can nucleate microtubules in vitro and has an open ring structure with a
diameter of 25 nm. Cryo-electron microscopy reveals a
modular structure with ~13 radially arranged structural
repeats. The
TuSC also nucleates microtubules, but
much less efficiently than the
TuRC, suggesting that
assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the
TuSC
reveals that
-tubulin binds preferentially to GDP over GTP, rendering
-tubulin an unusual member of the tubulin superfamily.
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Introduction |
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THE microtubule (MT)1 cytoskeleton is essential for
cell division and organization of the interphase cytoplasm. These functions are orchestrated by diverse and highly dynamic MT arrays generated by a variety
of mechanisms including regulation of the polymerization dynamics of MTs, of proteins that interact with and organize MTs, and of MT nucleation (Desai and Mitchison,
1997). The latter mechanism is possible because the spontaneous nucleation of new tubulin polymers is kinetically
limiting, both in vitro when the polymerization of pure tubulin is initiated, and in vivo (Alberts et al., 1994
).
Evidence for a kinetic barrier to MT nucleation in vivo
comes from analysis of repolymerization of MTs after cold
treatment or treatment with anti-MT agents. In many animal cells, regrowth initiates from the pericentriolar material (PCM) that surrounds the centrioles (Frankel, 1976;
Osborn and Weber, 1976
; Keryer et al., 1984
; Meads and
Schroer, 1995
), demonstrating that the PCM promotes MT
nucleation. A major breakthrough in defining the molecular basis of the MT-nucleating activity of the PCM was the discovery of
-tubulin (Oakley and Oakley, 1989
).
-tubulin is a member of the tubulin superfamily that localizes to
MT organizing centers and is found in all eukaryotes (reviewed in Joshi, 1994
; Pereira and Schiebel, 1997
). Genetic
studies have demonstrated that
-tubulin is required for
normal cytoplasmic and spindle MT formation in Aspergillus nidulans (Martin et al., 1997
; Oakley et al.,
1990
), Schizosaccharomyces pombe (Horio et al., 1991
), Drosophila melanogaster (Sunkel et al., 1995
), and Saccharomyces cerevisiae (Sobel and Snyder, 1995
; Marschall et
al., 1996
; Spang et al., 1996
). Antibody inhibition experiments in vertebrates have also implicated
-tubulin in MT
nucleation by the centrosome (Joshi et al., 1992
; Felix et
al., 1994
).
In higher eukaryotes, soluble -tubulin exists primarily
in a large complex (between 25 and 32 S; Stearns and Kirschner, 1994
; Meads and Schroer, 1995
; Zheng et al., 1995
;
Detraves et al., 1997
; Moritz et al., 1998
; Murphy et al.,
1998
). Recently, this complex was purified from Xenopus
egg extracts and shown to nucleate MTs in vitro (Zheng
et al., 1995
). This complex, called the
TuRC (
-tubulin
ring complex), consists of about eight proteins in addition
to
-tubulin and has the appearance of an open ring with approximately the same diameter as a MT (Zheng et al.,
1995
). Rings of this diameter have also been observed in
the PCM of centrosomes isolated from Drosophila embryos (Moritz et al., 1995a
) and the surf clam, Spisula (Vogel et al., 1997
). In Drosophila, immunoelectron microscopy has confirmed the presence of clusters of
-tubulin in
the ring structures and at the base of MTs nucleated by the PCM (Moritz et al., 1995b
). Cumulatively, these results
suggest that the
TuRC is a highly conserved structure responsible for the MT-nucleating activity of the PCM.
The -tubulin in S. cerevisiae is the most divergent of all
-tubulins. It is only ~35-40% identical to the other
known
-tubulins, all of which are at least 65% identical to
each other (Marschall et al., 1996
). In S. cerevisiae, the
only known soluble
-tubulin-containing complex is ~6 S
and contains three proteins:
-tubulin, and two related
proteins, Spc97p and Spc98p (Geissler et al., 1996
; Knop et al., 1997
; Knop and Schiebel, 1997
). Immunoprecipitation experiments with tagged proteins suggest that the S. cerevisiae complex contains one molecule of Spc97p, one
molecule of Spc98p, and two or more molecules of
-tubulin (Knop et al., 1997
; Knop and Schiebel, 1997
). The yeast
-tubulin 6 S complex is thought to be anchored to the cytoplasmic side of the spindle pole body through the interaction of Spc97p and Spc98p with Spc72p (Knop and
Schiebel, 1998
), and to the nuclear side of the spindle
pole body through interaction with the NH2 terminus of
Spc110p (Knop and Schiebel, 1997
). To date, in vitro MT-nucleating activity for the yeast complex has not been
demonstrated. Therefore, it remains unclear whether the yeast
-tubulin complex nucleates MTs directly, or whether
it assembles into a larger, perhaps
TuRC-like structure
at the spindle pole body. Interestingly, homologues of
Spc97p and Spc98p in humans (hGCP2 and hGCP3/
HsSpc98; Murphy et al., 1998
; Tassin et al., 1998
) and in
Xenopus (Xgrip109; Martin et al., 1998
) colocalize with
-tubulin at the centrosome and cosediment with
-tubulin on sucrose gradients, indicating that they are components
of the large
-tubulin-containing complexes present in these organisms.
Understanding the role of -tubulin in MT nucleation is
a challenging endeavor. Low cellular concentrations make
purification from native sources difficult, and the complexity of the protein complexes that contain
-tubulin limits
expression-based studies. Analysis of MT nucleation is
further complicated by the following: the complex structure of the MT lattice (Wade and Chretien, 1993
), the
large number of tubulin molecules potentially involved in the formation of a nucleus (Voter and Erickson, 1984
; Fygenson et al., 1995
), and the potential role of
-tubulin
GTP hydrolysis in suppressing nucleation (Hyman et al.,
1992
). This difficulty is reflected by the fact that the mechanism of spontaneous nucleation of purified tubulin remains poorly understood (Voter and Erickson, 1984
; Fygenson et al., 1995
).
Central to understanding the mechanism of MT nucleation by -tubulin-containing complexes will be to understand the relationship between
-tubulin and other members of the tubulin superfamily. One important aspect of
this relationship is the nature of the contacts
-tubulin
makes with itself and with
- or
-tubulin. A second important aspect is how
-tubulin compares to other members of the tubulin family in its ability to bind and hydrolyze GTP. If
-tubulin binds a guanine nucleotide, it will
be important to determine whether nucleotide exchange
and hydrolysis contribute to its ability to assemble, disassemble, nucleate, or release MTs, or whether the bound
nucleotide has a structural role, as is the case for
-tubulin.
In this paper, we begin to address the functional organization of the TuRC by purifying and analyzing
-tubulin-
containing complexes from Drosophila embryo extracts.
In Drosophila, there are two related
-tubulin-containing
complexes. The larger complex can be collapsed into the
smaller complex by treatment with high salt. This condensation suggests that the small complex is a structural subunit of the large complex (Moritz et al., 1998
). We purify both complexes and show that the large Drosophila complex nucleates MTs much more potently than the small
complex. We also show that, in contrast to
- and
-tubulin which preferentially bind GTP,
-tubulin in the small
complex preferentially binds GDP.
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Materials and Methods |
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Buffers and Reagents
HB: 50 mM K-Hepes, pH 7.6, 1 mM MgCl2, 1 mM EGTA, 1 mM -mercaptoethanol (
-ME) and protease inhibitor stock (1:200 final dilution;
see below). HB100: HB plus 100 mM NaCl; HB200: HB plus 200 mM
NaCl; and HB500: HB plus 500 mM NaCl. EB200: HB200 plus 100 µM
GTP and 1 mg/ml DrosC17 peptide; EB500: HB500 plus 100 µM GTP and
1 mg/ml DrosC17 peptide; HB block: 50 mM K-Hepes, pH 7.6, 100 mM
KCl, 1 mM MgCl2, 1 mM EGTA, 10 mg/ml bovine serum albumin (fraction V; Sigma Chemical Co.). Homogenization buffer: HB100 plus 10%
glycerol, 1 mM PMSF and protease inhibitor stock (1:100 final dilution).
Protease inhibitor stock: 10 mM benzamidine-HCl, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin A in ethanol. LPC: 10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml chymostatin dissolved in
DMSO. Mounting medium: 20 mM Tris-Cl, pH 9.0, 90% glycerol, and
0.1% p-phenylenediamine. BRB80: 80 mM K-PIPES, pH 6.8, 1 mM
MgCl2, 1 mM EGTA; 4× sample buffer: 250 mM Tris, pH 6.8, 12% SDS
wt/vol, 20%
-ME vol/vol, and 40% glycerol vol/vol. GTP stock: 100 mM
GTP (Boehringer Mannheim Corp.). Tubulin was purified from the bovine brain and labeled with tetramethylrhodamine as described (http://skye.med.harvard.edu). Drosophila embryo extract was prepared by homogenizing 0-3.5-h Drosophila embryos in homogenization buffer as described (Moritz et al., 1998
). Clarified extract was prepared by centrifugation of crude extract for 10 min at 15,000 rpm (SS34 rotor; Sorvall) at 4°C.
The supernatant was transferred to a new tube, and centrifuged a second
time at 50,000 rpm in a rotor (50.2 Ti or SW55; Beckman) for 1 h at 4°C.
Sucrose Gradient Sedimentation and Gel Filtration Chromatography
Sucrose gradients (5-20 or 5-40%) were poured as step gradients (five steps of equal volume) in HB containing 100 or 500 mM NaCl plus nucleotide and allowed to diffuse into continuous gradients. Gradients were fractionated from the top by hand with cutoff pipet tips. Fractions from standards gradients run in parallel were separated by 10% PAGE and stained with Coomassie blue. Gels were scanned, band intensities were quantitated (Adobe Photoshop; Adobe Systems Inc.) and peak fractions were assigned (Kaleidagraph Synergy Software Inc.). Standard curves of peak fraction versus sedimentation coefficient (S20,w) were used to estimate S values of protein complexes.
Gel filtration chromatography was carried out on a column (Superose-6;
Pharmacia Biotech Sverige) in HB plus 100 µM GTP, and 100 or 500 mM
NaCl as indicated. The column was calibrated with standards of known
Stokes radii. Molecular weights and Stokes radii of protein complexes
were estimated as described (Siegel and Monty, 1966). Fractions were separated by SDS-PAGE on 10% gels and
-tubulin was detected by Western blotting.
Immunoisolation of -tubulin-containing Complexes
from Drosophila Embryo Extract
PEG (polyethylene glycol P-2139; average mol wt = 8,000; Sigma Chemical Co.) was added to a final concentration of 2% (from a 30% stock in
HB100) to clarified Drosophila embryo extract from 20-g embryos. The
mixture was incubated on ice for 20 min, spun at 17,000 rpm for 10 min in
a SS34 rotor and the supernatant was discarded. The pellets were resuspended in 20 ml of HB200 plus 0.05% NP-40, and 100 µM GTP by gentle
Dounce homogenization and clarified at 35,000 rpm for 30 min in a 50.2 Ti
rotor. -tubulin complexes were immunoprecipitated from the supernatant
by adding 190 µg of DrosC17 antibody and incubating at 4°C for 1 h with gentle rotation. The immunoprecipitate was collected by slowly (over 1 h)
pumping the antibody-extract mixture over a 350-µl column of protein
A-agarose (GIBCO/BRL) in a disposable Bio-spin column housing (Bio-Rad). The column was washed with 15 ml of HB200 plus 0.05% NP-40 and
100 µM GTP, and 15 ml of the same buffer without NP-40. 400 µl of
EB200 was loaded onto the column and the column was sealed with parafilm and incubated for 16-18 h at 4°C.
-tubulin complexes were collected
by loading an additional 400 µl of EB200 onto the column and collecting
the flow through. For the sucrose gradient fractionation described in Fig.
2, 150 µl of isolated complexes was loaded onto a 2.1-ml 5-40% sucrose gradient, poured in HB100 plus 100 µM GTP, and sedimented at 50,000 rpm for 4 h in an TLS55 rotor at 4°C.
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Electron Microscopy
Negative stain electron microscopy of peptide-eluted complexes and sucrose gradient purified TuRC was performed as described (Zheng et al.,
1995
), except that grids of sucrose gradient fractions were rinsed with water before staining. For cryo-electron microscopy fresh peptide-eluted
-tubulin complexes were applied to glow discharged, holey carbon films
supported on copper EM grids. Excess liquid was removed by blotting and
the resulting thin film was rapidly frozen by plunging into liquid ethane
slush. Frozen grids were stored in liquid nitrogen. Grids were examined
using a Gatan cryo-transfer holder in an electron microscope (CM120;
Philips Electron Optics). During transfer, examination, and imaging, the grid was maintained at
180°C. Underfocused images (
1.2 to
2.0 µm) of layers of frozen solution spanning holes in the support film were
recorded using low dose methods (Kodak SO163 film). Positive prints
were made for further examination of the complexes.
Immunoisolation of the TuSC
PEG was added to 3% (from a 30% stock in HB100) to clarified extract
corresponding to 40 g of embryos. The mixture was incubated on ice for
20 min, centrifuged at 17,000 rpm for 10 min in the SS34 rotor at 4°C, and
the supernatant was discarded. Pellets were resuspended by gentle
Dounce homogenization in 40 ml ice-cold HB500 plus 100 µM GTP and
clarified at 35,000 rpm for 30 min at 4°C in the 50.2 Ti rotor. -tubulin
small complex (
TuSC) was immunoprecipitated by adding 1.46 mg of
DrosC17 or DrosC12 antibody and incubating at 4°C for 1 h with gentle
rotation. The immunoprecipitate was collected by pumping the antibody-extract mixture over a 500-µl column of protein A-agarose preequilibrated in HB500. The column was washed with 60 ml HB500 plus 100 µM
GTP. Alternatively, the immunoprecipitate was collected by mixing the
resin in a batch with the antibody-extract mixture at 4°C for 1 h. Afterwards, the resin was washed six times in batches with 5 ml of HB500 plus 100 µM GTP and loaded into a Bio-spin column. 500 µl of EB500 was
loaded onto the column, the column was sealed with parafilm, and incubated for 4 h at 4°C. The
TuSC was collected by loading an additional
550 µl of EB500 onto the column and collecting the flow through. 500 µl
of this eluate was fractionated on a 4.5-ml 5-20% sucrose gradient in
HB500 plus 100 µM GTP at 45,000 rpm in an SW55 rotor for 10 h at 4°C.
300-µl fractions were collected from the top. For coverslip and solution
nucleation assays, 100 µl of each fraction was dialyzed against HB100 plus
100 µM GTP at 4°C for 6 h. The concentration of
-tubulin was estimated
after dialysis and, in general, was not significantly altered.
Solution Nucleation Assays to Quantitate Microtubule Nucleation
5-µl reactions of identical final buffer composition (0.5× BRB80, 0.5×
EB200, 500 µM GTP) containing 4 mg/ml tubulin and varying concentrations of peptide-eluted complexes were incubated at 37°C for 4 min and
fixed at room temperature for 3 min by addition of 45 µl of 1% glutaraldehyde in BRB80. 10 µl was removed to a new tube and diluted by addition
of 1 ml ice-cold BRB80. MT spindowns and tubulin immunofluorescence
were performed as described (http://skye.med.harvard.edu). Varying
amounts of each sample were pelleted depending on the concentration of
-tubulin in the reaction. 20 random fields were photographed with a 60×
objective (1.4 NA; Nikon Corp.) using a cooled CCD camera (Princeton Instruments) and the MTs were counted. The fraction of total
-tubulin in the
peptide-eluted complexes present as the
TuRC was determined by densitometry of Coomassie-stained gels after sucrose gradient fractionation.
To compare nucleating activity, peptide-eluted -tubulin complexes
containing 0.65 µM
-tubulin in the
TuRC (0.87 µM total
-tubulin) and
isolated
TuSC containing 0.74 µM
-tubulin were assayed in parallel as
above. The following exceptions were made: the incubation at 37°C was
for 3 min; the final buffer composition of the
TuSC was 0.5× BRB80,
0.5× HB100 plus 100 µM GTP, 500 µM GTP; after fixation, instead of
sedimentation, samples were diluted with 200 µl of BRB80 + 70% glycerol and 3 µl were squashed and sealed under 18-mm square coverslips.
Coverslip Nucleation Assay
Polylysine-coated 12-mm diameter coverslips were placed on parafilm inside a humidified Petri dish kept in a 30°C water bath. The coverslips were
rinsed 2× with filtered water and blocked for 5 min with 60 µl HB block.
The HB block was removed by aspiration and replaced with 20 µl of the
sample. After 10 min the coverslips were washed 2× with 60 µl of BRB80 + 10 mg/ml BSA + 1 mM GTP, and incubated with 20 µl 6 mg/ml tubulin
(1:4 rhodamine labeled/unlabeled) in BRB80 + 1 mM GTP. After 10 min
the tubulin was removed by aspiration and replaced with 60 µl 1% glutaraldehyde in BRB80 (warmed to 30°C) for 3 min, followed by 3 min
postfixation with 20°C methanol. The coverslips were rehydrated,
mounted, and sealed with nail polish.
Cloning and Sequencing of Dgrip84 and Dgrip91
The Drosophila gamma ring proteins (Dgrips) were immunoisolated as described below and internal peptide sequences were obtained for Dgrip84 and Dgrip91. The following peptides were obtained for Dgrip84: KILRTGK, KDAQQLIIGLVRK, DRSLTH, DELPEHY, DIHTHL, DLVTQMS, DAEVLTYL, DEQIPSFLA, RHREFL, DFTMQ, ERRTYTLR, DTTPVVFVRRGP, DRHRE, DEYRTSLL, DEQIPSFLAKY, DVNSAAGSVPTTLAIAST, and DLVTQMSKIMKKEENXQAQ. For Dgrip91 the peptides obtained were: KDVVTGRF, KGVYGLTN, KTVSDH, KHMEFVLS, DIMVGPHK, DFNEYY, KLSELGYY, DATKMLP(OR L)E, DRVVKFS, DVIVQRPFNGG, EMIICIKGKQMPE, DVVTGRIFPY, ELSKIV, DATQSSIGLXKQSLPNY, DDPNLQLFGTR, DQSRFYK, and DVSTGFNAIG. For Dgrip84, degenerate primers corresponding to the forward peptide KDAQQL and reverse peptide DLVTQM (underlined above) specifically amplified a band of ~700 bp. A second round of PCR was performed with a primer corresponding to the forward peptide QQLIIG and the same reverse primer. For Dgrip91, primers corresponding to the forward peptide IKGKQM and reverse peptide TGFNAI (underlined above) specifically amplified a band of ~800 bp. A second round of PCR was performed with a primer corresponding to the forward peptide GKQMPE and the same reverse primer. Both PCR products were cloned, sequenced, and used to screen a Drosophila cDNA library.
Antibodies
Synthetic peptides (Stanford University Medical Center) corresponding
to the COOH-terminal 12 (QWSPAVEASKAG; DrosC12) or 17 amino
acids (QIDYPQWSPAVEASKAG; DrosC17) of the maternal form of
Drosophila -tubulin (37°C; These data are available from GenBank/ EMBL/DDB3 under accession number P42271) were used to raise and affinity purify rabbit polyclonal antibodies (Field et al., 1998
). Antibodies to
Dgrip84 and Dgrip91 were raised in rabbits against fusions of glutathione-S-transferase with amino acids 89-199 and 29-143 of the two proteins, respectively. Specific antibodies were purified as described (Kellogg and Alberts, 1992
).
Embryo Fixation and Immunofluorescence
Embryos were fixed in methanol and immunofluorescence was performed
as described (Theurkauf, 1994). Embryos were double-labeled with
mouse anti-
-tubulin (Sigma Chemical Co.) and rabbit anti-p91 or anti-p84 followed by FITC anti-rabbit and Cy-5 anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Three-dimensional images were obtained on a wide field microscope (DeltaVision; Applied
Precision Inc.). 512 × 512 pixel optical sections were taken at 0.2-µm intervals using an Olympus 60×, 1.4 NA objective and deconvolved. Appropriate Z-sections were projected.
GTP Cross-linking Experiments
120-µl peptide-eluted complexes were isolated as above, except GTP was
omitted from the column wash and elution buffers. The isolated complexes were loaded onto a 2-ml 5-40% sucrose gradient in HB100, centrifuged at 55,000 rpm in a TLS55 rotor for 4 h at 4°C, and fractionated into
17 130-µl fractions. In a 96-well plate, 30 µl of each fraction was incubated
for 90 min on ice with 10 µ Ci of [-32P]GTP (400 Ci/mmol; Amersham
Pharmacia Biotech Inc.), cross-linked for 5 min on ice in a cross-linker
(Stratalinker UV; Stratagene) at a distance of 10 cm, and analyzed by 10%
SDS-PAGE followed by autoradiography. For competition experiments,
the [
-32P]GTP was premixed with a 200-fold excess of cold nucleotide
competitor before being added to the fraction.
Determination of TuSC Nucleotide Content
TuSC was prepared as described above with the following modifications:
1:1,000 LPC was used in place of 1:200 protease inhibitor stock; pellets
from the PEG precipitation were resuspended in buffer containing 100 µM
of either GTP or GDP; and the wash, elution buffers, as well as sucrose
gradients contained 20 µM of either GTP or GDP. Control gradients
loaded with EB500 were fractionated in parallel to generate control buffers.
-tubulin samples were prepared by diluting bovine brain tubulin
into control buffers and incubating 1 h on ice before desalting. Free nucleotide was removed by rapid desalting into 20 mM Tris, pH 7.5, 100 mM
NaCl, 1 mM MgCl2 on a 800-µl Fast Desalting column (PC 3.2/10)
mounted on a SMARTTM system (Pharmacia Biotech, Inc.). 100 µl of sample was loaded per run and the column was eluted at a flow rate of 400 µl/
min. One 100-µl fraction containing the protein peak was collected from
each desalting run. Two separate runs were pooled, generating a total desalted sample volume of 200 µl. Under these conditions, desalting was
complete in ~45 s and
99.9% of free nucleotide was removed. 20 µl of
the desalted sample was used for protein quantitation (see below). The
concentration of
- or
-tubulin loaded onto the desalting column was
usually between 0.5 and 1 µM; protein recoveries were ~40-50%.
To extract nucleotide from the remaining desalted sample, 90 mg of
solid urea was added and the sample was vortexed and heated to 50°C for
3 min to denature the protein and release bound nucleotide. The denatured protein was removed by filtration through a 10,000 mol wt cutoff filter (model UFC3LGC00, Millipore Corp.; Rosenblatt et al., 1995). The filter was washed with 150 µl of water; eluate and wash were combined and
frozen in liquid nitrogen. Nucleotide content was determined by chromatography on a 100-µl Mono Q (PC 1.6/5; Pharmacia Biotech, Inc.) column
mounted on the SMARTTM system. Nucleotides were eluted with a gradient of ammonium bicarbonate (0.1-1 M in 30 column vol), quantified by
peak integration, and compared with standard curves generated by processing nucleotide standards of known concentration in an identical fashion. The amount of
-tubulin and
-tubulin was quantified using 10%
SDS-PAGE, Coomassie-staining, and densitometry of the 20-µl aliquot of
sample reserved after desalting relative to a standard curve of
-tubulin dimer on the same gel.
![]() |
Results |
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Drosophila Contains Two Related -tubulin
Complexes: The
TuSC Is a Subunit of the
TuRC
Drosophila embryo extracts contain two -tubulin-containing complexes that can be separated by gel filtration
chromatography or sucrose gradient sedimentation. In the
presence of 500 mM KCl or NaCl,
-tubulin is found exclusively in the smaller complex, indicating that the larger
complex has been disrupted, and that the smaller complex
is likely to be a structural subunit of the larger complex
(Moritz et al., 1998
). We named the large complex, Drosophila
TuRC (see below), and the small complex the
TuSC. To obtain size estimates for each complex, we performed gel filtration and sucrose gradient sedimentation
under low salt conditions in buffers that were supplemented with magnesium and GTP (Fig. 1) to reduce aggregation that occurs in nucleotide-free buffers. Under
these conditions, the
TuSC has an S value of 9.8 and a Stokes radius of 7.0 nm, while the
TuRC has an S value
of 35.5 S and a 15-nm Stokes radius. Based on these values, we estimate the molecular masses of the
TuSC and
TuRC to be 280,000 and 2,200,000 D (Table I), respectively.
|
|
Purified Drosophila -tubulin Complexes Nucleate
Microtubules In Vitro
To purify Drosophila -tubulin complexes, we used an immunoaffinity strategy based on antibodies raised against a
COOH-terminal peptide (Zheng et al., 1995
) of Drosophila
-tubulin. Immunoprecipitated protein complexes
were eluted from the antibody with buffers containing competing peptide (Fig. 2 A). The peptide-eluted mixture
of
TuRC and
TuSC nucleates MTs in solution (Fig. 2
B). The number of MTs nucleated is directly proportional
to the concentration of
-tubulin complexes (Fig. 2 B). At
the highest concentrations tested (370 nM or ~0.02 mg/ml
-tubulin),
-tubulin complex-containing reactions nucleated ~100-fold more MTs than control reactions. The relative proportions of
TuRC and
TuSC vary between
preps. For the experiments shown in Fig. 2, 67% of the
-tubulin was present as
TuRC, 22% was present as
TuSC, and 11% was in complexes intermediate in size.
The Protein Profile of the Drosophila TuRC Is Similar
to That of the Xenopus
TuRC
To determine the protein compositions of the TuSC and
TuRC, we fractionated the peptide-eluted complexes on
a 5-40% sucrose gradient (Fig. 3 A). For clarity,
TuSC
and
TuRC are shown side by side in Fig. 3 B. The protein
profile of the Drosophila
TuRC is reminiscent of the Xenopus
TuRC (Fig. 3 B). Therefore, by analogy to the
Xgrips (Martin et al., 1998
), we name Drosophila
TuRC
proteins Dgrips and designate them by their apparent molecular weights. Like the Xenopus
TuRC, the Drosophila
TuRC is composed of two high molecular mass proteins
(Dgrip163 and Dgrip128), two prominent proteins near
100 kD (Dgrip91 and Dgrip84), and a group of three or
four proteins with molecular masses near 75 kD (Dgrip75s). The protein below
-tubulin (between the 56- and 38.5-kD markers) has been identified as actin. It is not clear
whether actin is a specific component of
TuRC, or if it
fortuitously copurifies. Depending on the purification protocol, varying amounts of
- and
-tubulin copurify with
Xenopus
TuRC (Zheng et al., 1995
; Y. Zheng, unpublished results). In contrast, we have been unable to detect
- or
-tubulin copurifying with Drosophila
TuRC.
Consistent with the idea that
TuSC is a structural subunit
of
TuRC,
TuSC is composed of the three most prominent proteins in
TuRC:
-tubulin, Dgrip84, and Dgrip91
(Fig. 3 B).
|
The Drosophila TuRC Nucleates MTs In Vitro
To determine which of the Drosophila -tubulin complexes were able to nucleate MTs, we separated them by
sucrose gradient sedimentation and tested them using a
coverslip nucleation assay (outlined in Fig. 3 C). In this assay, the sample to be tested is allowed to bind to a preblocked coverslip, unbound protein is washed away, and
the coverslip is incubated with purified bovine brain tubulin containing a small amount of rhodamine-labeled tubulin. Unincorporated tubulin and spontaneously nucleated
MTs are removed by aspiration, whereas MTs nucleated
and tethered to the coverslip by the
-tubulin complexes
remain and are fixed and viewed by fluorescence microscopy. Although this assay is not quantitative, it has two advantages over the conventional solution nucleation assay:
(a) the background of spontaneously nucleated MTs is removed by aspiration, allowing detection of very low levels
of
-tubulin dependent nucleation; (b) buffer components
are washed away before exposure to tubulin. The latter is
useful when directly assaying sucrose gradient fractions
because of the strong interfering effects of varying sucrose
concentrations on tubulin nucleation and elongation.
The results of a coverslip assay on the sucrose gradient
fractions in Fig. 3 A are shown in Fig. 3 D. The top two
rows are equivalent exposures for fractions 3-14. The bottom row shows longer exposures (either 40× or 5× longer)
for the indicated fractions. We observe a clear peak of activity corresponding to the fractions that contain TuRC
(fractions 10-14). Under these conditions, no activity is
seen in the fractions containing
TuSC (fractions 4-6).
Similar to the peptide-eluted complexes, gel filtration or
sucrose gradient-fractionated Drosophila embryo extracts
tested in this assay show only one peak of activity, corresponding to the peak of
TuRC (data not shown). To distinguish between nucleation and capture of spontaneously
nucleated MTs, we performed the coverslip nucleation assay with peptide-eluted material and observed unfixed samples in real time using video enhanced DIC microscopy (data not shown). MTs initiated at the coverslip surface and elongated, while maintaining a fixed orientation
with one end anchored to the coverslip. Capture of a MT
from solution by the surface was never observed. Cumulatively, these results indicate that the Drosophila
TuRC
has MT nucleating activity.
Cryo-electron Microscopy of the TuRC Reveals a
Modular Structure
Negative stain electron microscopy of the peptide-eluted
complexes (Fig. 4 A), and of the TuRC after sucrose gradient sedimentation (Fig. 4 B) reveals an open ring structure with a diameter of ~25 nm. In side-by-side pictures of
comparable preparations, the structure of the Drosophila
TuRC is indistinguishable from that of the Xenopus
TuRC (Wiese, C., and Y. Zheng, unpublished observations). To get a more detailed view of the
TuRC, we examined the structure of the purified Drosophila
TuRC by
cryo-electron microscopy. A gallery of cryo-EM images
reveals a modular structure (Fig. 4 C). The
TuRC appears to have ~13 structural repeats arranged in a radial
symmetric pattern with a diameter of 25 nm. Some internal structures are also visible. A more detailed view will
require single particle reconstructions.
|
TuRC Is a More Potent Microtubule Nucleator
than
TuSC
The absence of nucleation activity of sucrose gradient-isolated TuSC in the coverslip assay (Fig. 3 D) can be explained in several ways:
TuSC (a) might not have nucleating activity; (b) might not bind to the coverslip under the
assay conditions; (c) might become inactivated upon binding to the coverslip, or (d) might be too dilute to exhibit
activity. To distinguish between these possibilities we developed a protocol to prepare more concentrated
TuSC,
taking advantage of the disruption of
TuRC into
TuSC by high salt (Fig. 1).
TuRC was disrupted by isolating
-tubulin-containing complexes in the presence of 500 mM NaCl. The resulting
TuSC was eluted with peptide
containing buffer in 500 mM NaCl. The peptide-eluted
material was further fractionated on a 5-20% sucrose gradient in 500 mM NaCl (Fig. 5 A). This gradient separated
TuSC from residual larger complexes and from non-
TuSC components of
TuRC. This resulted in highly
concentrated, relatively pure
TuSC (15 µl of each fraction was loaded on the gel in Fig. 5 A compared with 50 µl
in Fig. 3 A). Inclusion of 500 mM salt in the sucrose gradient was important to prevent any reassociation of
TuSC
with non
TuSC components of
TuRC. Typically, the
peak
TuSC sucrose gradient fraction contained ~700
nM
-tubulin (as judged by densitometry of Coomassie-stained bands relative to
-tubulin standards). For
comparison, after sucrose gradient fractionation, the
peak
TuSC-containing sucrose gradient fraction in the
mixed complex preparation (Fig. 3 A) contained ~70 nM
-tubulin.
|
Concentrated TuSC fractions were dialyzed to remove
salt and tested for nucleating activity. In contrast to the robust activity of the peptide-eluted complexes, nucleation
by isolated
TuSC in solution was weak and slightly variable between preparations. A direct comparison between
the nucleating activity of the peptide-eluted complexes
(containing 0.87 µM total
-tubulin, 0.65 µM
-tubulin in
TuRC) and isolated
TuSC (0.74 µM
-tubulin) in a solution nucleation assay is shown in Fig. 6. In nucleation reactions containing between 350-450 nM
-tubulin and 4 mg/ml tubulin, the nucleating activity of the peptide-eluted complexes was typically 80-100-fold above the level
of spontaneous nucleation; under these conditions the
level of nucleation for isolated
TuSC was only two- to
threefold above background (see Fig. 6 B). Therefore, it is
likely that the nucleating activity of the peptide-eluted
complexes is due to primarily the activity of
-tubulin in
TuRC. Based on those data, we estimate that per mole of
-tubulin
TuRC is ~25 times more active than
TuSC in
promoting nucleation. Since there are ~12
-tubulin molecules in
TuRC and only 2 in
TuSC (see below for stoichiometry estimates),
TuRC is ~150 times more active than
TuSC per mole of complex. We also tested the nucleating activity of
TuSC in the coverslip nucleation assay. The concentrated
TuSC was also able to nucleate
MTs in this assay (Fig. 5 B).
|
The activity of TuSC could be explained at least two
ways: (a)
TuSC might reassemble into a
TuRC-like
higher-order structure, or (b)
TuSC itself might have intrinsic nucleating activity. To begin distinguishing between
these possibilities, we examined dialyzed
TuSC for evidence of formation of higher-order structures using gel filtration chromatography. Undialyzed
TuSC migrated in a
position typical of that for the smaller complex (Fig. 5 C,
top). The dialyzed sample migrated in the same position
(Fig. 5 C, bottom), suggesting that dialysis did not induce
detectable assembly of
TuSC into larger structures.
Dgrip84 and Dgrip91 Are Homologous to the Spc97/Spc98 Family of Proteins
To characterize the molecular nature of TuSC, we cloned
and sequenced its non-
-tubulin components, Dgrip84
and Dgrip91. Dgrip84 and Dgrip91 are homologous to
each other and to the Spc97/98p family of proteins identified in S. cerevisiae. This family also includes two proteins
identified in humans, hGCP2 and hGCP3 (Murphy et al.,
1998
). The homology between the Drosophila proteins
and the other members of this family extends over the entire length of the proteins (data not shown). In comparisons of Dgrip84 and Dgrip91 with the corresponding
human proteins, a one-to-one correspondence emerges.
Dgrip84 is 32% identical (46% similar) to hGCP2 and
only 21% identical (32% similar) to hGCP3; in contrast,
Dgrip91 is 31% identical (45% similar) to hGCP3 and
only 24% identical (37% similar) to hGCP2. These results suggest that Dgrip84 and hGCP2, and Dgrip91 and
hGCP3 may be functionally homologous pairs. The sequences of the Drosophila proteins are available from GenBank/EMBL/DDBJ under accession numbers AF118379
(Dgrip84) and AF118380 (Dgrip91).
Stoichiometry of Proteins in TuSC
To estimate the stoichiometry of TuSC proteins, we performed densitometry of Coomassie-stained SDS-PAGE
gels of purified
TuSC. After correcting for the predicted
molecular weight of each protein, we estimated that the
ratio of Dgrip91 to Dgrip84 to
-tubulin in the
TuSC is
1:1:2. Since our estimate of the molecular mass of purified
TuSC from sucrose gradient sedimentation and gel filtration is 280,000 D (Table I), we suspect that
TuSC contains 1 molecule of Dgrip91, 1 molecule of Dgrip84, and 2 molecules of
-tubulin. Interestingly, this corresponds to
estimates of the stoichiometry of proteins in the S. cerevisiae 6 S
-tubulin complex (Knop et al., 1997
; Knop and
Schiebel, 1997
). If we assume that
TuRC contains only
one molecule of each non-
TuSC component, and use our
estimates for the molecular weights of
TuRC and
TuSC, then
TuRC would contain approximately six
TuSCs.
Dgrip84 and Dgrip91 Cofractionate with
-tubulin on Sucrose Gradients and Colocalize
with
-tubulin in Embryos
If -tubulin in Drosophila embryos primarily exists associated with Dgrip84 and Dgrip91 in either
TuSC or
TuRC, we would expect these three proteins to cofractionate on sucrose gradients of embryo extract and to
colocalize in embryos. To test this hypothesis, we raised
and affinity-purified rabbit polyclonal antibodies that recognize Dgrip84 and Dgrip91. Each antibody recognizes a
band of the expected molecular weight on Western blots
of embryo extract (Fig. 7 A, left). As expected, both
Dgrip84 and Dgrip91 comigrate with
-tubulin in
TuSC
and
TuRC when embryo extract is fractionated on sucrose gradients (Fig. 7 A, right). In addition, the localizations of Dgrip84 and Dgrip91 in Drosophila embryos are
indistinguishable from that of
-tubulin. Each antibody
recognizes the centrosome throughout the cell cycle and
shows some spindle staining during mitosis with enrichment at the spindle poles (Fig. 7 B), regardless of its cognate antigen. We propose that Drosophila
-tubulin is stably associated with Dgrip91/84. Interestingly, we found no evidence for a non-
-tubulin associated pool of either
Dgrip84 or Dgrip91.
|
-tubulin in
TuRC and
TuSC Can be Cross-linked
to GTP
The homology between -,
-, and
-tubulins extends into
domains that are involved in GTP binding by
- and
-tubulin (Burns, 1995
). Thus, it is tempting to speculate
that
-tubulin can bind, and possibly hydrolyze, GTP. To
determine if
-tubulin binds guanine nucleotide, we immunoisolated
-tubulin-containing complexes in the absence
of GTP. The isolated complexes, either before or after sucrose gradient sedimentation, were incubated with
[
-32P]GTP and UV cross-linked. In the peptide-eluted
complexes,
-tubulin is the only protein that cross-links
to GTP (Fig. 8 A). Furthermore,
-tubulin in both the
TuRC and
TuSC cross-links to GTP (Fig. 8 B). Competition experiments showed that the cross-link can be competed by addition of excess cold GTP, GDP, and GTP
S
but not GMP-PNP, ATP, or CTP (Fig. 8 C).
|
-tubulin in
TuSC Preferentially Binds GDP
To characterize the nucleotide binding properties of
-tubulin, we compared the nucleotide content of
-tubulin in
TuSC to that of similarly treated
-tubulin dimer.
TuSC was isolated in buffers containing either 20 µM
GDP or 20 µM GTP. To remove free nucleotide, we used
a rapid (within 45 s) microscale desalting procedure. For
comparison, pure
-tubulin dimer was diluted into a
buffer identical to that containing
TuSC and desalted in
parallel. Nucleotide was extracted from the desalted samples and analyzed by mono Q chromatography. To estimate the stoichiometry of bound nucleotide to protein,
the amount of
- and
-tubulin in each desalted sample
was quantitated by densitometry of Coomassie-stained gel
bands relative to
-tubulin standards, and the nucleotide concentration was determined by peak integration and
comparison with nucleotide standards processed in an
identical fashion.
Each -tubulin dimer has two guanine nucleotide
binding sites, one on each tubulin subunit. Exclusively
GTP is bound to
-tubulin at the nonexchangeable or
N-site; this nucleotide does not exchange with GTP/GDP
in solution and does not undergo hydrolysis. In contrast,
-tubulin binds guanine nucleotide in an exchangeable
fashion at the E-site. Both GTP and GDP bind to the
E-site with GTP having a three- to fourfold higher affinity
than GDP (Zeeberg and Caplow, 1979
). GTP bound to
the E-site does not undergo significant hydrolysis in the
absence of polymerization but gets hydrolyzed soon after incorporation into the MT lattice, resulting in GDP that is
locked in the lattice and can only exchange after depolymerization (reviewed in Desai and Mitchison, 1997
).
These properties predict that if
-tubulin dimer is isolated from buffers containing GDP, then there will be 1 mol GTP (N-site) and 1 mol GDP (E-site) per mole of
-tubulin dimer. In contrast, if
-tubulin dimer is isolated from buffers containing GTP, under conditions where
there is no polymerization, then there will be 2 mol GTP
(1 N-site GTP and 1 E-site GTP) per mole of
-tubulin
dimer. Consistent with these predictions, we recovered 1.1 mol of GTP and 0.8 mol GDP per mole of
-tubulin
dimer isolated from GDP buffer (Fig. 9, B and G, and Table II); in contrast, we recovered exclusively 2.0 mol of GTP per mole of
-tubulin dimer isolated from GTP
buffer (Fig. 9, E and G, and Table II). These results establish the validity of our assay for comparing the nucleotide-binding properties of
TuSC to those of
-tubulin dimer.
|
|
When TuSC was similarly analyzed, the nucleotide recovered from
TuSC incubated in GDP buffers was exclusively GDP (Fig. 9 C). Approximately 0.7 mol GDP was
recovered per mole of
-tubulin (Fig. 9 G and Table II).
The exclusive presence of GDP could be explained at least
three ways: (a) the guanine nucleotide binding site on
-tubulin subunits of
TuSC is freely exchangeable; (b) GDP is locked nonexchangeably into
-tubulin subunits of
TuSC, analogous to GTP bound at the N-site in
-tubulin; or (c) GDP is locked nonexchangeably into
TuSC as
the product of earlier GTP hydrolysis, much like
-tubulin
bound GDP within the body of a polymerizing MT.
To distinguish between these possibilities, we isolated
TuSC from GTP-containing buffer. Surprisingly, we recovered a greatly reduced amount of nucleotide (Fig. 9 F).
Only 0.2 mol guanine nucleotide was recovered per mole
of
-tubulin, indicating that ~80% of the
-tubulin was
empty at its nucleotide binding site (Fig. 9 G and Table II).
To ascertain that the GTP in the buffer had not been degraded, we removed an aliquot before desalting and analyzed its nucleotide content. This sample contained the expected amount of GTP and a trace amount of GDP (~3%
of total guanine nucleotide). This amount of GDP was also
recovered from a similarly processed control buffer, indicating that it did not arise from hydrolysis by
TuSC or a
contaminating GTPase (not shown). The low recovery of
guanine nucleotide bound to
TuSC isolated from GTP
buffer indicates that GDP is bound exchangeably to
-tubulin in
TuSC. This result also argues against the
theory that the GDP bound to
-tubulin in
TuSC, isolated from GDP buffer, is being generated by earlier GTP
hydrolysis. The recovery of nearly 1 mol GDP per mole
of
-tubulin from GDP buffer and the nearly equivalent
amounts of GTP and GDP in the 0.2 mol nucleotide recovered per mole of
-tubulin from GTP buffer, despite a
GTP/GDP ratio
30 before desalting, strongly suggest
that
-tubulin in
TuSC has an exchangeable guanine nucleotide binding site that has a much higher affinity for
GDP than GTP. To test this further, we isolated
TuSC
in buffer containing 20 µM GTP and, 1 h before desalting,
added 20 µM GDP. Consistent with our interpretation, we
recovered 0.44 mol GDP and 0.02 mol GTP per
-tubulin
monomer (Table II).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
-tubulin Complexes in Eukaryotes
The -tubulin in Drosophila embryo extracts exists in two
related complexes of ~280,000 (
TuSC) and 2,200,000 D
(
TuRC). In contrast, when extracts of Xenopus eggs
(Stearns and Kirschner, 1994
; Zheng et al., 1995
), Xenopus
XTC cells (Stearns and Kirschner, 1994
), human 293 cells
(Stearns and Kirschner, 1994
), or mouse fibroblasts (Murphy et al., 1998
) were fractionated, only one complex was
observed sedimenting at ~32 S (Murphy et al., 1998
). The
significance of this finding is not clear, but might reflect a
difference between
-tubulin-containing complexes isolated from different organisms and cell types. Interestingly,
-tubulin in the polarized human intestinal epithelial
cell line Caco-2 was present in both 10 S and 29 S complexes (Meads and Schroer, 1995
). In Caco-2 cells,
-tubulin localizes both to centrosomes and to a diffuse layer beneath the apical membrane (Meads and Schroer, 1995
).
However, it is not yet clear whether the presence of an apical layer of
-tubulin correlates with the existence of a
smaller 10 S complex. Further experiments will be required to determine if the differences in
-tubulin-containing complexes present in cellular extracts are caused
by different extraction conditions, or if they reflect real diversity between systems and cell types in the nature and
function of
-tubulin complexes in vivo.
The protein profile of the purified Drosophila TuRC is
very similar to that of the previously purified Xenopus
TuRC (Zheng et al., 1995
). Indeed, the protein profiles
of
-tubulin complexes immunoprecipitated from a number of sources, including mouse cells after metabolic labeling (Murphy et al., 1998
) and sheep brain tubulin preparations (Detraves et al., 1997
), bear a strong resemblance. In
addition to molecular similarities, the Drosophila
TuRC
also resembles the Xenopus
TuRC in its structure (both
complexes appear as open rings when visualized by negative stain electron microscopy).
TuSC: a Conserved Subcomplex of the
TuRC
Drosophila TuRC can be converted to
TuSC by treatment with high salt, suggesting that
TuSC is a structural
subunit of
TuRC. A similar dissociation by high salt
has been reported for human and Xenopus large
-tubulin complexes (Stearns and Kirschner, 1994
; Meads and
Schroer, 1995
; Zheng et al., 1995
). The hypothesis that
TuSC is a subunit of
TuRC is supported by our finding
that purified
TuSC is composed of the three proteins most
prominent in
TuRC:
-tubulin, Dgrip84, and Dgrip91.
Dgrip84 and Dgrip91 are members of the Spc97/98p family of proteins. This family includes hGCP2 and hGCP3/
HsSpc98p from humans (Murphy et al., 1998
; Tassin et al.,
1998
) and Xgrip109 from Xenopus (Martin et al., 1998
). Homologous ESTs have also been identified in mouse, zebrafish, and rice (Martin et al., 1998
; Tassin et al., 1998
). Genetic evidence in S. cerevisiae suggests that this family of
proteins interacts directly with
-tubulin (Geissler et al.,
1996
; Knop et al., 1997
). Based on molecular weight estimates and densitometry of Coomassie stained gels, we
estimate that Drosophila
TuSC is a heterotetrameric complex containing one Dgrip84, one Dgrip91, and two
molecules of
-tubulin. This stoichiometry is identical to
the proposed composition of the S. cerevisiae 6 S complex
(Sc
TuSC). Immunoprecipitation experiments with tagged
proteins indicate that Sc
TuSC contains one Spc97p, one
Spc98p, and two or more molecules of
-tubulin (Knop et
al., 1997
; Knop and Schiebel, 1997
). Together, these results support the hypothesis that the organization of
-tubulin into
TuSC is likely to be conserved among all
organisms where
-tubulin is found. One question of fundamental importance that needs to be addressed in the future is how the two
-tubulin molecules within
TuSC are
arranged. Are they arranged in a head to tail dimer as
would be suggested by the model proposing that
TuRC is a protofilament of
-tubulin (Erickson and Stoffler,
1996
), or are they arranged in a side-by-side configuration
(Zheng et al., 1995
)?
In metazoa, TuSC is further assembled into
TuRC.
Based on our hydrodynamic analysis of Drosophila complexes, we estimate that
TuRC contains approximately
four to six
TuSCs. An exact determination awaits a more
accurate appraisal of the molecular weight of
TuRC, currently being attempted using scanning transmission electron microscopy. It will also be interesting to identify the structural correlate of
TuSC within
TuRC. When viewed
by cryo-electron microscopy,
TuRC has a modular structure with ~13 structural repeats organized in a radial symmetric pattern. Based on our current estimates, we speculate that one
TuSC might correspond to two of the radial
symmetric structural repeats visible by cryo-electron microscopy.
Nucleation Activity of TuSC and
TuRC
An important issue with respect to the in vivo roles of
TuSC and
TuRC is their relative MT nucleating activity.
The fact that S. cerevisiae does not appear to contain a
TuRC-like complex raises the question of whether Sc
TuSC has nucleating activity or whether it must assemble
into a larger structure at the spindle pole body to become
active. Conversely, in metazoa it is possible that
TuRC is
a storage form for
-tubulin and it could be
TuSC that
nucleates MTs at centrosomes (Knop and Schiebel, 1997
).
To begin to address this question, we compared the nucleating activity of peptide-eluted complexes (in which ~75%
of
-tubulin is
TuRC) to isolated
TuSC at similar concentrations of
-tubulin. Using both solution and coverslip
nucleation assays, we found that both preparations had
nucleating activity. However, whereas the solution nucleating activity of the peptide-eluted complexes was robust, typically 80-100-fold above the level of spontaneous nucleation, under similar conditions the level of nucleation
for isolated
TuSC was only two- to threefold above background. Thus, per mole of
-tubulin
TuRC is ~25 times
more active than
TuSC in promoting nucleation. Combining these data with our stoichiometry measurements, we estimate that per mole of complex
TuRC is ~150
times more active than
TuSC, suggesting that organization of
TuSC into
TuRC facilitates MT nucleation activity.
We emphasize that the nature of the nucleating activity
of TuSC is still unclear.
TuSC may have intrinsic nucleating activity or it may need to assemble into larger
TuRC-like complexes in order to nucleate MTs. In Xenopus extracts, high-salt dissociated
TuRC components can
be reassembled by desalting. This reassembly is blocked
by depleting Xgrip109, suggesting that intact
TuSC is required for assembly of
-tubulin into a
TuRC-like structure (Martin et al., 1998
). Here we separate
TuSC from
the remaining components of
TuRC and do not find any
evidence for assembly of larger structures after desalting.
This occurrence suggests that
TuSC is required but not
sufficient for assembly of a
TuRC-like structure. We cannot exclude the possibility that
TuSC, like
-tubulin
dimer, assembles into larger complexes when the temperature is raised during nucleation assays. Because the level
of activity of
TuSC is very low compared to that of
TuRC, we also cannot rule out the possibility that the nucleating activity of
TuSC depends on trace levels of other
TuRC components that contaminate our
TuSC preps.
Expression and purification of the
TuSC will be necessary to further characterize
TuSC activity.
Nucleotide Binding Properties of -tubulin
Cross-linking experiments showed that -tubulin in both
TuSC and
TuRC can bind guanine nucleotide. To investigate how
-tubulin compares to other members of the tubulin family in its nucleotide binding properties, we compared the nucleotide bound to
-tubulin in
TuSC to that
bound to
-tubulin dimer after desalting from buffers
containing GDP or GTP. We found, like
-tubulin (Weisenberg et al., 1976
),
-tubulin in
TuSC binds guanine nucleotide exchangeably. However, in contrast to
-tubulin,
which has about a threefold higher affinity for GTP than
GDP (Zeeberg and Caplow, 1979
),
-tubulin in
TuSC
strongly prefers binding GDP to GTP. Our results suggest that the affinity of
-tubulin for GTP is much lower than
that of
-tubulin, based on nucleotide recovery after desalting under similar conditions (Zeeberg and Caplow,
1979
). Determination of the absolute affinities of
-tubulin
for GDP and GTP will be important to know whether the
affinities of
- and
-tubulin for GDP are similar. If they
are, this will suggest that the strong preference of
-tubulin for GDP is primarily because of a reduction in its affinity for GTP relative to
-tubulin. These experiments will require a reliable supply of
TuSC, currently limited by
antibody availability for immunoisolation and by lack of
an expressed source. A structural comparison of the nucleotide binding pocket of
-tubulin to those of
- and
-tubulin should also be revealing. Development of procedures
to prepare more concentrated and highly purified
TuRC
should allow a comparison of nucleotide binding by
-tubulin in
TuRC to that in the
TuSC. If the nucleotide binding properties of
-tubulin in
TuRC are similar
to those of
-tubulin in
TuSC, it will suggest that GTP hydrolysis by
-tubulin may not be important for its function
in vivo.
![]() |
Footnotes |
---|
Received for publication 9 September 1998 and in revised form 11 January 1999.
Address correspondence to Karen Oegema, European Molecular Biology
Laboratory, Cell Biology Program, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel.: 49-6221-387-337. Fax: 49-6221-387-512-306. E-mail:
Karen.Oegema{at}EMBL-Heidelberg.DE or Yixian Zheng, Department of
Embryology, Carnegie Institute of Washington, Baltimore, MD 21210. Tel.: 410-554-1232. Fax: 410-243-6311. E-mail: zheng{at}mail1.ciwemb.edu
We dedicate this work to Christine Mirzayan. The authors thank Mike
Sepanski and Mei Lie Wong for the negative stain EM. We also thank Arshad Desai for much help during the course of this work and Arshad Desai and Doug Kellogg for critical reading of the manuscript.
This work was supported by grants from the National Institutes of Health to T.J. Mitchison and Y. Zheng (RO1-GM56312-01). Y. Zheng was also supported by the Pew Scholar's Award and C. Wiese by a postdoctoral fellowship from the American Cancer Society.
![]() |
Abbreviations used in this paper |
---|
Dgrip, Drosophila gamma ring protein;
TuRC,
-tubulin ring complex;
TuSC,
-tubulin small complex;
MT, microtubule;
PCM, pericentriolar material;
PEG, polyethylene glycol;
Sc
TuSC, Saccharomyces cerevisiae 6 S complex.
![]() |
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