Tyrosination state of free tubulin subunits and tubulin disassembled from microtubules of rat brain tissue

Vol. 89, No. 3, 1979 August

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

13, 1979

Pages

TYROSINATION

STATE OF FREE TUBULIN SUBUNITS AND TUBULIN DISASSEMBLED FROM MICROTUBULES OF RAT BRAIN TISSUE Julio

Laboratory

A. Rodriguez

of Molecular

Received

893-899

June

Biology,

and Gary G. Borisy

University

of

Wisconsin,

Madison,

WI 53706

25,1979

Abstract: The content of endogenous COOH-terminal tyrosine in unpolymerized tubulin subunits from the brain extracts of new-born rats was twice as high as that of tubulin isolated from disassembled native microtubules. It was also observed that the proportion of free sites in tubulin from in vitro incorporation of tyrosine at the COOH-terminus in unpolymerized proteinwas about one-half of that obtained for the protein from disassembled microtubules. In the extracts of young adult rats the value for the endogenous COOH-terminal tyrosine content in unpolymerized subunits decreased and almost equaled the value for the polymerized tubulin which remained virtually constant. Cytoplasmic of

tubulin

an aromatic

c1 chain

of

residue,

a proportion

by an ATP-dependent (10).

has been found

Because

of

mainly

tyrosine,

into

of

molecules

(1,3,11).

unique

tyrosine

has been considered

function

of

far

the

that

presence

tubulin.

observation sination

of

relative

distribution

Conditions the

the

not

configuration

determined dure

by the

to stabilize

unpolymerized ditions

rule

play

out

of tubulin

between that

of the

COOH-terminal

nature

of

native

microtubules

the

Our purpose the

state

of the

specific

ct tubulin

for

for

the

is

not

that

portion

brain

the

tyroof

the

states.

discriminated

by

be ultimately

We have

tissue

was to isolate COOH-terminus

could

end.

This

mechanism

are

so

by the

and monomer

which

at that

cell

or

indicated

end (2,9).

control

molecules

of

affected

in the

polymer

tubulin

catalyzed

activity

have

COOH-terminal

the

from

is

role

in the

residue

The reaction

of evidence

possibility

end of the

of incorporation

--in vitro at the

addition

COOH-terminal

reaction

pieces

participate

be such

the

a regulatory

several

the

a postsynthetic

be highly

the

could

could

subunits.

aind analyze

to

of tyrosine

CL subunit

--in vivo

features,

of polymerization

absence

does

seems to

However,

reaction

or the

the

enzyme which its

to undergo

used a proce-

and to separate

tubulin by assaying

from

both

them from con-

tyrosine

after

0006-291X/79/150893-07$01.00/0

893

Copyright All rights

@ 1979 by Academic Press, Inc. of reproduction in any form reserved.

Vol.

89,

No.

3, 1979

release

with

though

tubulin

carboxypeptidase from

sine,there

were

availability

of

ences the

as well

Materials

the

the

sites the

AND

A.

for

--in

vitro

of

amino

acid

tubulin

COOH-terminal

here

contained

tyrosine,

indicate

observed

showed in such

that

lower

that the

altyro-

that

quantitative

a fashion the

COMMUNICATIONS

COOH-terminal

We have also

tyrosination of

RESEARCH

reported

of assembly

differences.

two forms

content

BIOPHYSICAL

The data

two states

quantitative

for

endogenous

incorporate

BIOCHEMICAL

the differ-

the

higher

capacity

to

--in vitro.

and Methods

Native microtubules and unpolymerized subunits were prepared from brains of male albino rats (Holtzman Co., Madison, WI) according to the procedure of Pipeleers et al. (1977) with minor changes. The tissue (5 g) was homogenized with 10 vol of a stabilizing solution containing 50% glycerol (Sigma), 5% dimethyl sulfoxide (Fisher), 0.5 mM GTP (Aldrich), 0.5 mM MgC12, 0.5 mM EGTA, 10 mM sodium phosphate buffer, pH 7.2 and 0.1 mM CuClp. The CuCl*, which did not affect the recovery of microtubules, was included to prevent the action of an endogenous carboxypeptidase activity reported in brain (4). The homogenization was performed in a 50-ml teflon-in-glass homogenizer with a difference in diameter between pestle and tube of 0.23-0.25 mm, at 850 rpm for 5 up-and-down strokes. The homogenate was centrifuged at 1000 x g for 10 min to remove large debris, and then the supernatant was centrifuged at 105,000 x g for 45 min. The resulting pellet and supernatant contained the stabilized microtubules (polymer fraction) and unpolymerized subunits (monomer fraction), respectively. The supernatant fraction was removed and the pellet was resuspended in an equal volume of a solution containing 0.1 W Pipes, pH 6.94, 0.1 mM MgClz and 0.1 ti GTP. All operations up to this point were carried out at room temperature (23'C). The polymer fraction was made 0.25 M in KCl, kept in ice for 30 min to depolymerize the microtubules, centrifuged at 105,000 x g for 30 min at 3°C and the supernatant containing the subunits from the disassembled microtubules was removed. The monomer fraction was applied to a Sephadex G-50 column which was equilibrated and eluted at 4" with the same solution as The voided above to change the sample to the same buffer as the pellet. fraction was eluted and made 0.25 II in KC1 to correspond to the treatment of the pellet. Tubulin was isolated from both polymer and monomer fractions by ionexchange chromatography on DEAE-Sephadex and analyzed for its content of COOH-terminal tyrosine by the fluorimetric technique as described previously (11). Anlaytical SOS-electrophoresis of the tubulin fraction showed a degree of purity for the protein in the range of 95 to 99%. was measured in the Maximal capacity for incorporation of '"C-tyrosine two fractions as obtained just before the ion-exchange chromatography step. The conditions forthe assay were as described previously (5) except for the addition of partially purified porcine tubulin-tyrosine ligase to insure Ligase was purified from pig brain tissue approximaximal incorporation. mately 15-fold according to the procedure of Raybin and Flavin (1977). Samples taken described by Gorisy

for electron microscopy et al. (1974).

894

were

negatively

stained

as

Vol. 89, No. 3, 1979

Protein with

(1951) Results

the

conditions

tion.

Electron

native

microtubules.

of the

microscopy

after

addition the

fraction

(see

Materials

This

content

of

of

Lowry

Animals

et al.

the

amount

the

calculated

value

terminal

of

tyrosine

tubulin tyrosine,

the

to tubulin both

is

polymer

of

of

tyrosinated

the

the

protein

same relaitive

difference

rats

the

from young

shown for

the

absolute adult

protein

amino 1.

was found

for

values

were

that

895

content

rats. to

by of

studied.

tyro-

Although

COOH-terminal

In one-week-old tubulin

obtained

proportion

rats

the

was 52% and

microtubules

lower. the

in

of COOH-

ratio

material

1.

comparable

The molar

contained

indicated

adult

released

fractions

slightly

in Table

acid

age points

the

of

(1975).

three

disassembled

amount

was recovered

is

its

of non-assembled

from

was 95-

endogenous

and young

for

were observed.

molecules

rats

Fig.

monomer

is compared

et al.

the

the

by carboxypeptidase

two fractions

DEAE-tubulin

of

by DEAE-chroma-

used and the

new-born

by Arce

andmonomer

purified

release

fraction

in the

is shown in

depolymerized

of the

of age were

of

but

of purification

60% of the

reported

number

and from

degree

both

a large

was isolated

after

each

in

differences

proportion

although

data

analysis

quantitative

one for

from

recovered

A digestion

from

assayed

centrifuga-

cold

determinations

by spectrofluorimetry

carboxypeptidase bound

direct

microtubules

from

in the

and the

to preserve

and unpolymerized

showed

microtubules

1, 7 and 60 days

of tubulin

The result

fraction

Tubulin

used approximately

of pelletable

The total

for

recovered

conditions

form

M KCl.

(1977)

by differential

disassembled

tyrosine of

separated

native

was used

(DEAE-tubulin)

Under

slowly

et al.

Microtubules

polymer

and Methods)

of COOH-terminal

protein

tissue. were

the

depolymerized

material

A digestion.

brain

of 0.25

from

protein

method

by Pipeleers

tissue

These

tography

99%.

of rat

in extracts

rapidly

described

microtubules

subunits

sine

RESEARCH COMMUNICATIONS

and Discussion

native

the

AND BIOPHYSICAL

concentration was determined by the bovine serum albumin as standard.

We used the

BIOCHEMICAL

was 26%. from

Measurements of

The

one-day-old in the

tyrosinated

Vol.

89,

No.

3, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE Recovery

Tubulin chromatography measured in was determined

of

from monomer as described the dialyzed by analytical

tubulin

RESEARCH

COMMUNICATIONS

I

from

rat

brain

tissue

and

polymer fractions was isolated by ion-exchange previously (11). Protein concentration was eluates from the DEAE-columns. The degree of purity SDS-electrophoresis (see Materials and Methods)

cl

MONOMER POLYMER

FRACTION FRACTION

60

I

AGE

(days)

Histogram representing the relative content of COOH-terminal Figure 1. tyrosine in tubulin from the monomer and polymer fractions of rat brain. Tubulin from the fractions prepared as described under Material and Methods was partially digested with carboxypeptidase A and the tyrosine released was assayed fluorimetrically as described elsewhere (11). Molar ratios were calculated assuming a molecular weight for tubulin dimer of 110,000 daltons.

free

tubulin

tubulin

which

Several of

the

had decreased remained

almost

attempts

COOH-terminus

to

the

level

corresponding

unchanged.

have been made to correlate of

a

tubulin

to microtubular

and the

896

capacity

the

state

of the

of

tyrosination

protein

to

poly-

Vol.

89,

No.

merize

3, 1979

in vitro.

without the

BIOCHEMICAL

Tubulin

COOH-terminal

conditions

to the

conditions

two states not

assembly

be equlivalent of of

sination that

of

of

noteworthy

that

of

age of

chick

few days

animals

the

crude

after

desalting

of pellet

porcine

tubulin-tyrosine

pattern sites that ligase

for for

the

the

ligase

tion

was negligible

with

pelleted

complementary

equal.

previously

It

is

tubulin

for

changes

cyto-

in the

in the

brain

rat

of

the

amino

materials

the

two fractions

for from

the

Partially

to

a

unassembled

In the

of incorporation

of

14X-tyrosine

microtubules. relationship

shown)

indicating

The results between

the

897

the

tubulin

polymer.

of amount

Fig.

of

no ligase experiment

of endogenous

and

in order

to

of the The kinetic of available

was only

in the

this

2.

proportion

absence

that

the

purified

The time-course

but

capacity

in

was used

shown in

the

(not

(10))

acid. is

its

and resuspension

respectively. (according

for

was performed

centrifugation

in both

same for

protein level

double

of unassembled

compared

The assay

differential

incorporation the

the

of tyrosination

seems to occur

was also

in vitro.

and supernate,

reaction

was the

direct

of tyro-

is

almost

of maximal

report

of assembly

incorporation

incorporation

are

reported

The period

in that

levels

of tyrosination

to that

(11).

stages

14C-tyrosine

maximal

analogous

discussed

to incorporate

insure

state

level

brain

the

rats

from

the

developing

adult

might

out

isolated that

that

which

birth.

from

material

in the

is

but

in young

tubulin

the

of

microtubules,

change

brain

after

in extracts

two fractions

embryo

Tubu'lin

tubulin

the

now carried

in tubulin indicate

between

factors

We have

Our findings

applicable

of tubulin

of different

experiments.

or

Nevertheless,

be directly

The distribution

on a number

with

(2,9).

may not

of tyrosination

native

the

chick

the

extent

from

of the

plasmic

the

unassembled

tubulin

the

may depend

polymerization.

of tubulin

with

in vivo.

COMMUNICATIONS

same extent

conditions

--in vitro

in the --in vitro

determinations two states.

experiments

RESEARCH

to the

in --in vit‘ro

obtaining

of

BIOPHYSICAL

seems to polymerize

tyrosine

of the

AND

one-half

of

exogenous

porcine

microtubular

frac-

was associated indicated tyrosine

a at the

Vol. 89, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

I

I-

O

20

I

I

I

40

60

80

TIME In vitro were tion

corporation state

of

of young

position of the

number

acid.

between

of

It also tubulin

sites

available

confirms

the

of

the

for

--in vitro

difference

two fractions

in-

in the

from

the

brains

animals.

of

development,

of

assembly

shown in this tubulin but

of tyrosination

GM 25062

No. F05-TW0246-02

report

does

a selection

Acknowledgements: Grant

and the

amino

tyrosination

The data

extent

(min)

incorporation of 14C-tyrosine into tubulin of the (0) fractions from 7-day-old rat brain. Conditions in Materials and Methods. Tubulin content in each fracby electrophoresis on SDS-acrylamide gels.

as described was estimated

COOH-terminal

RESEARCH COMMUNICATIONS

occurs

of the This

(to

not

distribute

cell

COOH-terminal

and a P.H.S.

that, at

in the

work was supported

G.G.B.) (to

demonstrate

at least

random which

end of by National

International

between is

the

in early the

stages

two states

correlated

with

the

c1 subunit. Institutes Research

of

Health

Fellowship

J.A.R.).

References 1.

Arce, C.A., J. Biochem.

Rodriguez, J.A., 59, 145-149.

Barra,

898

H.S.,

and Caputto,

R. (1975)

Eur.

Vol. 89, No. :3, 1979

2. 3. 4. 5. 6. 7. 8. 9. :1":

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Arce, C.A., Hallak, M.E., Rodriguez, J.A., Barra, H.S., and Caputto, R. (1978) J. Neurochem. 31, 205-210. Argaraiia, C-E., Arce, C.A., Barra, H.S., and Caputto, R. (1977) Arch. Biochem. Biophys. 180, 264-268. Argaraiia, C.E., Barra, H.S. and Caputto, R. (1978) Molec. Cel Biochem. 19, 17-21. Barra, H.S., Rodriguez, J.A., Arce, C.A. and Caputto, R. (1973) J. Neurochem. 20, 97-108. Borisy, G.G., Olmsted, J.B., Marcum, J. M. and Allen, C. (1974) Fed. Proc. 33, 167-174. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Pipeleers, D.G., Pipeleers-Marichal, M.A., Sherline, P. and Kipnis, D.M. (1977) J. Cell Biol. 74, 341-350. Raybin, II. and Flavin, M. (1977a) J. Cell Biol. 73, 492-504. Raygin, 0. and Flavin, M. (1977b) Biochemistry 16, 2189-2194. Rodriguez, J.A. and Borisy, G.G. (1978) Biochem. Biophys. Res. Commun. 83, 579-586.

899