Tubulin assembly induced by cobalt and zinc

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 95, No. 4, 1980

Pages ]703-]709

August 29, 1980

TUBULIN ASSEMBLY INDUCED BY COBALT AND ZINC Kathryn M. Haskins, Randall R. Zombola, James M. Boling, Young C. Lee andRichard H. Himes Department of Biochemistry University of Kansas Lawrence, Kansas 66045 Received

July

14,1980

SUMMARY Both Co(ll) and Zn(ll) stimulate the assembly of bovine brain 6S tubulin in the absence of organic solvents, under conditions where Mg(ll) is ineffective. The products of the assembly reaction depend on the cation/tubulin ratio. At lower ratios microtubules are formed, but as the cation concentration is increased, broad sheets of protofilaments are the predominate products. The assembly reaction in Co(ll) is inhibited by high ionic strength, GDP, colchicine, and Ca(ll). Unlike other inhibitors, which will cause depolymerization of Co(ll)-induced microtubules, Ca(ll) added to Co(ll)-induced microtubules causes the formation of broad sheets of protofilaments.

INTRODUCTION Divalent

metal

self-assembly. Mn(ll)

ions play

Microtubule

an

important

(MT) formation

(2, 3) and is inhibited by Ca(ll)

but

sheet

(5,

6),

broad

sheets

(4).

in tubulin

(7).

(i) and

Other cations have been shown

For example, in the presence of

of protofilaments

formation has also been reported

role

is stimulated by Mg(ll)

to produce aberrant polymerization products. Zn(ll)

undefined

are formed.

Co(ll)-induced

It is known that the tubulin

dimer contains one tightly bound Mg(ll) (8-I0)which can be replaced by Mn(ll) (2).

The

question

arises

whether

the

actions of Co(ll)

and Zn(ll)

are a

result of replacement of the bound Mg(ll), of binding to other sites on the protein,

or

designed

to answer this question we found that both Co(ll)

stimulate

of

acting

via

metal-GTP

complexes.

In the course of studies

the formation of MTs as well as sheets.

and Zn(ll)

can

MTs are favored at low

cation/tubulin ratios and the assembly process occurs with pure 6S tubulin in the absence of other stimulatory agents.

0006-291X/80/161703-07501,00/0 1703

Copyright © 1980 by Academic Press, Inc. All rights o f reproduction in any form reserved.

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MATERIALS AND METHODS Bovine brain tnbulin was isolated through two cycles of the polymerization-depolymerization procedure (II). Tubulin 6S dimer was purified from this preparation using phosphocellulose chromatography as described previously (9), except that the phosphocellulose was placed on top of a Sephadex G-75 column (1.2 x 25 cm). Elution was performed with 20 mM PIPES, pH 6.9. The Sephadex column is included to remove DMSO, excess GTP and other buffer components which are eluted from the phosphocellulose. Unless indicated otherwise, assembly reactions were done at 37°C in 0.i M Pipes, 0.5 mM GTP, pH 7.1. In some cases 10% DMSO was included. The reaction was followed by measuring the absorbance at 350 nm as a function of time. Samples for electron microscopy were negatively stained with 2% uranyl acetate. Protein concentrations were determined by the Bradford method (12).

RESULTS Stimulation of assembly by Co(II).

Upon

the

addition

of

increasing

concentrations of CoC12 to 6S tubulin, an increase in turbidity occurred (Fig. 1).

Using

identical

concentrations

absorbance under these conditions.

of

Mg(II),

there

was

no

increase

in

Examination of negatively stained samples

showed that at the lower Co(II) concentrations, MTs were present (Fig. 2A). By

increasing

predominate

the

Co(II)/

assembly

tubulin

product.

ratio,

These

sheets

protofilament were

similar

sheets to

became

the

Zn-sheets

and

0.8"

0.5 mM

0.6 E ¢:: 0

rnM t.~ 0 . 4 z ~n

al

O2

0.0MINUTES

Fig. I. Assembly of tubulin with cobalt. Tubulin (1.5 mg/ml) was assembled under the conditions described in "Materials and Methods" and at varying concentrations of CoC12.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 2. Electron micrographs of 6S tubulin assembled in the presence o f cobalt or zinc. Magnification: A, B and D = 140,000; C = 62,100. A. MTs formed in the presence of 0.15 mM COC12. B. Sheets formed in the presence of 0.5 mM CoCI 2. C. Effect of Ca(II) on Co(ll)-induced MTs. CaCI 2 (2.0 mM final concentration) was added after tubulin was assembled in the presence of 0.2 mM Co(ll). D. MTs formed in the presence of 25 NM ZnCI 2.

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

appeared either as large flat sheets or, as in Fig. 2B, a large sheet folded over itself. of

Co(ll)

That a significant amount of assembly occurred in the presence

was

demonstrated

by

centrifuging

a sample after polymerization.

Using 0.2 mM Co(ll), a concentration which produces MTs, approximately equai amounts of protein were found in the pellet and supernatant; in 0.5 ram Co(ll), which produces nearly all sheets,

4-5 times more protein was in the pellet

than in the supernatant. Co(ll)-induced

assembly was inhibited by colchicine,

temperature, GDP, and high ionic strength.

vincristine,

cold

Moreover, MTs and sheets preformed

in the presence of Co(ll), were depolymerized by these agents.

Ca(ll) also

prevented assembly but had an unusual effect when added to MTs formed in the presence

of Co(ll).

After the addition of Ca(ll)

the absorbance

initially

decreased but then increased dramatically to a value higher than that before Ca(ll) was added (Fig. 3).

The increase was apparently due to the formation

of sheet structures (Fig. 2C). Effect of pH and DMSO on Co(ll)-induced MT formation. above were done at pH 7.1.

The

studies

described

Previously (13) we demonstrated that changing the

pH of the assembly medium in the region from 6.0 to 7.2 had a profound effect on

the

sheets

nature were

similar

of the DMSO-induced

favored

effect

is

at

the

apparent

lower

assembly product of 6S tubulin; pH

complex

and MTs at the higher pH values.

in Co(ll)-induced

assembly.

A

At pH 7.1 in the

presence of 0.15 mM Co(ll) only MTs were observed; at pH 6.5, both sheets and MTs were seen. The

presence

of

10% DMSO

greatly

stimulates

the

Co(ll) and also affects the structure of the product.

rate of assembly in

In 10% DMSO and 0.25 mM

Co(ll) numerous broad sheets as well as MTs were formed; in the absence of DMSO,

MTs

formation occurred

were at in

the

main

concentrations its

absence.

product. (25 pM) DMSO,

In

addition,

of

Co(ll)

therefore,

10%

where

extends

DMSO no the

stimulated

apparent

1706

assembly

effective

concentration range for both MT and sheet formation to lower limits.

MT

Co(ll)

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.,I

Ca (~)

1

0.3" E c

0

o

Z

,<

0.2"

o

0.1

0.0

I

i

,

,

;2

is

20

&

MINUTES Fig. 3. Effect of Ca(ll) on Co(ll)-induced assembly. Assembly was induced by 0.2 mM Co(ll). At the arrow, Ca(ll) was added to yield a final concentration of 2.0 mM.

Assembly in the presence of Zn(II).

Zn(II)-induced assembly of tubulin into

broad sheets has been reported by others (3, 4) and the Zn-sheets have been used for structural studies of the tubulin dimer in individual protofilaments (14-17).

However,

formed (Fig. 2D).

we

have found that under some conditions, MTs are also

The cation/tubulin ratio over which MTs are formed is very

narrow in the case of Zn(II).

At a tubulin concentration of 14 ~M, MTs were

found at 25-40 gM ZnCI2, but sheets were the only products observed at higher concentrations. DISCUSSION Zn(II), Co(II), and Mg(II) all stimulate the in vitro assembly of tubulin in the

absence

of

associated proteins or other assembly promoting agents.

However, the Mg(II)-induced assembly requires a cation concentration of I0 n~M and a protein concentration of greater than 2.5 mg/ml (18).

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In contrast, much

Vol. 95, No. 4, 1980

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

lower concentrations of Zn(II) and Co(ll) are effective and at lower protein concentrations.

Thus

the

rate

and possibly the K

eq

of the polymerization

reaction is higher in the presence of these cations than in the presence of Mg(II). which

Unlike Mg(II), these two cations also cause the production of sheets contain

a large number of protofilaments.

The cation/tnbnlin

ratios

which cause sheets to be formed are different

for the two cations.

causes

in fact, MTs can be observed

sheet production at fairly low ratios;

only over a very narrow range of Zn(II) concentration.

Zn(II)

Co(II) stimulates MT

formation over a broader concentration range. Gaskin (3) has shown that Zn(II) causes sheet formation when assembly is stimulated formation.

by

taxol

She

has

in

the

also

absence

shown

of

that

GTP.

sheets

Taxol are

alone

produced

stimulates when

Zn(II)

MT is

included in an assembly reaction promoted by Cr(III)OTP, which stimulates MT assembly in the absence of added GTP. that Zn(II)

These results rule out the possibility

acts in sheet production by way of a ZnGTP complex and supports

the idea that direct binding to tubulin is involved. and

Co(II)

showed Sheet

that

tubulin

these

formation

replacement Zn(II)

to

of

had previously

cations

stimulated the

and Co(II)

displace by

been

indicated

in

tightly bound Mn(II)

Zn(II)

firmly bound

Direct binding of Zn(II)

and

Mg(II)

Co(If)

could

by these cations

to other sites on the protein.

experiments

which

from tubulin be

the

result

(2). of

or of binding by

In addition, although the

data is fairly conclusive that sheet formation is caused by direct binding of Zn(II)

and

Co(II)

to

tubulin,

it

is

still

possible

that

MT

stimulated by these two cations does occur via a metal-GTP complex.

formation Binding

studies currently underway in our laboratory should distinguish between these possibilities.

ACKNOWLEDGEMENTS This research was supported by NIH research grant NS 11360 and American Cancer Society research grant CH-98 to R.H.H., by a postdoctoral training fellowship from the Kansas Cancer Society R.R.Z. and by a NICHD research service predoctoral award (HD 02528) to K.M.H.

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REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18.

Lee, J. C., and Timasheff, S. N. (1975) Biochemistry 14, 5183-5187. Buttlaire, D. H., Czuba, B. A., Stevens, T. H., Lee, Y. C. and Himes, R. H. (1980) J. Biol. Chem. 255, 2164-2168. Gaskin, F. (1980) Federation Proceed. 39, 2163. Weisenberg, R. C. (1972) Science 177, 1104-1105. Larsson, H., Wallin, M., and EdstrSm, A. (1976) Exp. Cell Res. i00, 104-110. Gaskin, F. and Kress, Y. (1977) J. Biol. Chem. 252, 6918-6924. Wallin, M., Larsson, H., and EdstrSm, A. (1977) Exp. Cell Res. 107, 219-225. Olmsted, J. B., and Borisy, G. G. (1975) Biochemistry 14, 2996-3005. Himes, R. H., Burton, P. R.,and Gaito, J. M. (1977) J. Biol. Chem. 252, 6222-6228. Williams, R. C., Jr., and Detrich, H. W., III (1979) Biochemistry 18, 2499-2503. Lee, Y. C., Samson, F. E., Jr., Houston, L. L., and Himes, R. H. (1974) J. Neurobiol. 5, 317-330. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Burton, P. R., and Himes, R. H. (1978) J. Cell Biol. 77, 120-133. Crepeau, R. H., McEwen, B., Dykes, G., and Edelstein, S. J. (1977) J. Mol. Biol. 116, 301-315. Baker, T. S.,and Amos, L. A. (1978) J. Mol. Biol. 123, 89-106. Tam, L. K., Creapeau, R. H., and Edelstein, S. J. (1979) J. Mol. Biol. 130, 473-492. Tsuprum, V. L., and Surgucheva, I. G. (1979) Mol. Biol. 13, 626-631. Herzog, W., and Weber, K. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1860-1864.

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