a new tubulin-binding protein

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 prepared 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

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 polyacrylamide 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

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

706


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 corresponding 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

707


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 supernatant 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

708


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.

7oY


References

1. 2. 3.

4. 5. 6. 7.

Asnes, C.F., and Wilson, L. (1979) Anal. Biochem. 98, 64-73. Kumar, N., and Flavin, M. (1981) J. Biol. Chem. 256, 7678-7686. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Hamel, E., and Lin, C.M. (1981) Arch. Biochem. Biophys. 209, 29-40. Eipper, B. (1974) J. Biol. Chem. 249, 1407-1416. Raybin, D., and Flavin, M. (1977) Biochem. 16, 2189-2194. Kim, H., Binder, K.L., and Rosenbaum, J.L. (1979) J. Cell Biol. 80, 266-276.

8. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 9. Margolis, R.L., and Wilson, L. (1978) Cell 13, l-8. 10. Margolis, R.L., and Rauch, C.T. (1981) Biochem. 20, 4451-4458. 11. Nath, J., and Flavin, M. (1980) J. Neurochem. 35, 693-706.

710

a new tubulin-binding protein

Documents