a new tubulin-binding protein
TRANSCRIPT
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Vol. 106, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 15, 1982 Pages 704-710
A NEW TUBULIN-BINDING PROTEIN
Nirbhay Kumar and Martin Flavin
Section on Organelle Biochemistry, Laboratory of Cell Biology National Heart, Lung, and Blood Institute, Bethesda, Maryland 20205
Received April 19, 1982
A new brain protein is described which forms an insoluble complex with tubulin, with concomitant stoichiometric hydrolysis of GTP. The complex contains a maximum of one tubulin-binding protein (MW 52,500) per two tubulin dimers. The tubulin-binding protein (TBP) does not compete with colchicine, but in the presence of microtubule-associated proteins tubulin appeared less accessible to it. Proteins such as TBP might sequester tubulin and thereby function either to inhibit indis- criminate polymerization, or to promote ordered nucleation by maintaining high local concentrations.
Little is known about the cellular regulation of microtubule assembly,
although in mammalian brain a number of proteins have been identified
which bind to tubulin with some specificity, and which may facilitate or
inhibit assembly. We report here a new protein isolated from brain
extract which binds to tubulin, with characteristics different from any
previously described.
Materials and Methods
Microtubule protein (tubulin-3xP) was prepared from freshly obtained bovine brains by three cycles of temperature-dependent assembly-disassembly as described by Asnes and Wilson (1). Pure tubulin (tubulin-PC) was pre- pared by phosphocellulose chromatography (2). The supernatant from the first warm cycle of assembly was stored at -70°C for the preparation of tubulin-binding protein (TBPI. The reassembly (RA) buffer, unless other- wise specified, was 100 I&I K MES, pH 6.7, 0.5 m&l MgC12, 1 n+l each EGTA and GTP.
For turbidimetric assay of microtubule assembly or tubulin aggregation, the tubulin sample was incubated at 32°C with the TBP fraction in RA buffer, 250,ul final volume, and the absorbance at 350 nm was recorded with a Beckman DU-8 spectrophotometer. TBP was added to the other components in ice-cold cuvettes, which were then placed in a cuvette holder maintained
Abbreviations: tubulin-3xP, microtubule protein purified by three cycles of assembly; tubulin-PC, tubulin purified by phosphocellulose chromatography; TBP, tubulin-binding protein; MAPS, microtubule-associated proteins; RA buffer, reassembly buffer; SDS, sodium dodecyl sulfate; MES, 2-(N-morpholino)ethane sulfonic acid.
0006-291X/82/110704-07$01.00/0 Copwghf 0 I982 h.~ .4cademic Press, Inc. ,411 rights of reproduction in any form reserved. 704
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vol. 106, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
at 32 + O.l"C. For quantitation, samples were transferred from the cuvettzs to centrifuge tubes, and centrifuged for 60 min at 32°C at 48,000 x g in a Sorvall SS-34 rotor. The protein in the pellets was determined by the procedure of Lowry et al. (3).
Assays have been described for GTPase (4) and for carboxypeptidase- tubulin (2), an enzyme which specifically cleaves C-terminal tyrosine from tubulin alpha-chain. Samples were analyzed in SDS and urea polyacryl- amide gel electrophoresis as described by Eipper (5). Samples were prepared, and slab gels stained, as previously described (6). Photo- graphic negatives of wet slabs were scanned with a Quick Scan R.S.D. densitometer. Procedures for electron microscopy were as previously described (2).
MAP-2 was prepared by the procedure of Kim et al. (7). Alpha- C32~l-~~~ and was obtained from Amersham, and trypsin and chymotrypsin from Worthington.
Results
The results reported here stem from the chance observation that a
turbidity appeared when large amounts of a partially purified carboxy-
peptidase-tubulin were added to tubulin. This enzyme had been purified
by ammonium sulfate precipitation, passage through a bed of DEAE-cellulose,
and gradient elution from a column of carboxymethylcellulose (CM-52) at
pH 6.3 (2). The TBP, which was also present in the final fraction, could
not be measured at the earlier steps, nor unequivocally identified in the
complex polyacrylamide gel electrophoresis patterns. To obtain TBP we
have followed the procedure exactly as described for the carboxypeptidase
(2), except that we added elution of the CM-52 with 50 ml of 50 mM KC1
in 50 mM K+MES, pH 6.35, before starting the KC1 gradient. Under
these conditions more than 95% of the protein added to the column has
been eluted before the start of the gradient.
Fig. 1 shows the gradient elution patterns for carboxypeptidase,
and protein as measured by dye binding (8). Eluates were pooled in five
fractions, A to E, and after concentration as previously described (2),
analyzed by gel electrophoresis in the presence of SDS and urea (Fig. 1).
The carboxypeptidase activity was primarily in fraction C, but fraction
D was most effective in causing aggregation of tubulin. Moreover, the
principle component in D, which represents 2/3 of its total protein, is
the TBP, since only it is found in the pelleted tubulin aggregates (see below
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Vol. 106, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 250
200
8 16 24 32 40 48
FRACTION NUMBER
!-A+B*C +-0+-E--1
2 800 co
8 z m
600 n P T
ABCDE
FIG. 1 Elution of carboxypeptidase-tubulin and TBP from a CM-52 column. -the left is shown the KC1 gradient, and the protein ( - 0 - 1 and carboxypeptidase activity ( - H- 1 profiles. Fractions were pooled as indicated. PAGE analysis of the concentrated pooled fractions is shown by photographs of stained gels on the right.
The turbidity observed on adding fraction D to tubulin-PC (1 mg/ml)
was maximal at the earliest measurable time, at either 0" or 32°C. No
ordered structures have been detected by electron microscopic examination
of negatively stained samples. The amount of tubulin that could be pellet-
ed was proportional to the TBP concentration, and gel electrophoretic scans
of pellets showed that, above 0.4 mg/ml of fraction 0, the ratio of tubulin
to TBP (i.e. the principle band in fraction D) was constant at about 5:l
by weight, giving a molar ratio of 2:l based on a TBP molecular weight
of 52,500 (as determined by SDS-gel electrophoresisl.
The aggregation of tubulin-PC was prevented, but not reversed, by
100 mM NaCl or KCl, or 5 mM ATP or GTP. EDTA in excess over the Mg2+
(added with the tubulin) had no effect. Merceptoethanol (0.2 M) or
podophyllotoxin, at up to 2OO)M, did not prevent the aggregation, and
colchicine binding was not affected by addition of an equal weight of
TBP to the tubulin solution. Since MAPS are sensitive to proteolysis
and might yield fragments capable of binding to tubulin, we carried out
partial digestions of MAP-Z with trypsin and chymotrypsin, to see if this
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vol. 106, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TIME lminutesl
FIG 2 A Hydrolysis of GTP accompanies the aggregation of tubulin by TBP ( - 0 - ). Tubulin-PC (1 mg/ml) was incubated at 37°C wi
3 h TBP
(fraction D) at 0.5 mq/ml in RA buffer containing 0.1 mM alpha-[. 2Pl-GTP. At the indicated time; 5~1 aliquots were added lo tubes containing 20~1 of 30% acetic acid, and 10&l of these mixtures were spotted on polyethylene-imine cellulose thin layer plates (Brinkman, Polygram Cell 300 PEI). The plates were developed by ascending chromatography in aqueous M KH2P04, dried and subjected to radioautography. Spots corres- ponding to GDP and GTP were cut out and counted in Aquasol. For comparison, the time course of GOP formation when MAP-2 (1 mg/ml) was added to another aliquot of tubulin-PC is also shown ( - A- 1. No measurable GDP was formed when tubulin ( - q - 1. MAP-2 ( - A - ) or TBP fraction ( - 0 - ) were incubated individually.
would yield a component corresponding to TBP. None could be detected by
gel electrophoretic analysis.
Neither tubulin-PC nor TBP had any measurable GTPase activity, but
when the two were incubated together at 37°C there was an initial burst
of GTP hydrolysis in parallel with the development of turbidity (Fig.
2). The amount of GDP formed, 7 nmol/ml, may be considered stoichiometric
with the 9 nmol/ml of tubulin, since the latter usually contains some
denatured species. For comparison, the data in Fig. 2 for tubulin-PC
+ MAP-Z illustrate the time course of GTP hydrolysis at the exchangeable
site of tubulin which accompanies microtubule assembly and subsequent
treadmilling (9).
Fig. 3 shows the time course of turbidity development when a solution
of assembly-competent tubulin-3xP was warmed in the absence (curve 1)
or presence (curve 2) of TBP fraction. TBP caused an elevation of the
baseline turbidity at zero time, and also enhanced the temperature- and
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Vol. 106, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
atm’r.e*rr 0.6 -
2
a b c d
5 10 15 minutes
FIG 3 a The chart on the left shows the time course of turbidity development when tubulin-3xP (1 mg/ml) was allowed to assemble at 37°C in the absence (curve 1) or presence (curve 2) of TBP (1 mg/ml). The reassembly buffer contained 1 mM GTP. Curves 3 and 4 show the results obtained when tubulin + TBP were incubated in the presence of 2.5 mM CaC12, or 10 uM podophyllotoxin. respectively. The photograph of stained gels on the right shows the SDS-PAGE analysis of the following samples: (a) TBP fraction; (b) tubulin-3xP; ( 1 c and (d), respectively, pellet and super- natant obtained by centrifuging the mixture of tubulin-3xP incubated together with TBP (from curve 2).
time-dependent increase, both in proportion to the TBP concentration
(not shown). The final turbidity was much less than that observed with
the same concentration of tubulin-PC. Gel electrophoretic analysis of
pellet and supernatant obtained by centrifuging the mixture of curve 2
showed the pellet (lane c) to contain tubulin, MAPS and the 52,500-dalton
band of the TBP fraction; all the other bands in the latter were found
in the supernatant (lane d). This result was the same when tubulin-PC
was used. Mixtures of MAPS and TBP fraction did not show enhanced
turbidity.
When 2.5 mM CaC12 (curve 3, Fig. 3) or 10~M podophyllotoxin (curve
4) were present in mixtures of tubulin-3xP and TBP fraction, the time-
dependent turbidity was abolished, though the baseline elevation was not.
When 25 )M podophyllotoxin was added to the mixtures of curves 1 and 2
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after '20 minutes of assembly, the turbidity decreased by about 30% in
the former, but by only 3% in the latter.
Negatively stained aliquots of the mixture of tubulin-3xP and TBP
fraction showed, when examined by electron microscopy, microtubules and
amorphous material, the latter sometimes in the form of 30-40 nm spherical
globules, presumably microprecipitates of protein. The globules decorated
the microtubules, sometimes with a suggestion of periodicity, but were
also abundant in the background. Occasional microtubules were elevated
off the grid coat surface throughout their length.
Discussion
Under assembly conditions our results appear generally consistent
with the view that, in the presence of TBP, both MAPS-induced microtubules
and TBP-tubulin aggregates are formed. The latter could adhere non-speci-
fically to microtubules and confer some stability. Once MAPS are bound
to tubulin, the latter is less accessible to TBP. Proteins such as TBP
are more likely to have a physiological role in relation to tubulin,
rather than to microtubules.
Tubulin is acidic, and TBP is probably a basic protein since it binds
tightly to carboxymethyl-cellulose at low pH. Aggregates of the two
contain about one TBP to two tubulin dimers. It seems likely that we
have isolated only a minor fraction of the total TBP in brain: the
tubulin-TBP complex is not solubilized at low temperature, so one might
expect to find the bulk of TBP in the particulate fraction of a brain
homogenate, perhaps in association with the cold-stable (10) or membrane-
bound (11) moieties of tubulin. Hydrolysis of GTP at the exchangeable
site of tubulin accompanies MAPS-induced assembly into microtubules.
The observation that an apparently stoichiometric hydrolysis also accom-
panies tubulin aggregation by TBP suggests there might be some structural
specificity in the interaction between the two.
Acknowledgements
The authors thank Terry Jones and Dr. Ernest Hamel for help with the electron microscopy and the GTPase assay, respectively.
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