Proteolytic origin of a modified form of RNA polymerase I from Artemia salina larvae

Vol. 78, No. 4, 1977

BIOCHEMJCAL AND BIOPHYSJCAL RESEARCH COMMUNICATIONS

PROTEOLYTIC ORIGIN OF A MODIFIED FORM OF RNA POLYMERASE I FROM Artemia Carmen Osuna,

Jaime

Universidad

September

LARVAE

Renart

de Enzimologia

Instituto

Received

salina

and Jesik

de1 CSIC, Aut&oma,

SebastiZn

Facultad

de Medicina,

Madrid-34,

Spain

16. 1977

SUMMARY Extracts from Artemia saZina larvae contain a form of RNA polymerase (Ia) not present in the embryos. The appearance of this RNA polymerase during the larval development is correlated with a decrease in the levels of RNA polymerase I. The modification of RNA polymerase I to Ia was obtained in vitro by incubation of enzyme I with either a larvae extract or protease B, one of the multiple proteases induced during the larval development. The results indicate that the modified form of RNA polymerase present in the larvae is produced in vitro by proteolysis of enzyme I during the extraction and solubilization of the RNA polymerases. INTRODUCTION The embryos stage

from Artemia

and after

time

(1).

dehydration

The cysts

ation

of development

which

hatched

in a short

In a series merases levels

rise

it

interval

of studies

in both

dormant

of RNA polymerase is produced

in vitro

after

control

(nauplii)

that

of

reiniti-

of nauplii

subsequent

culture

the

there (3).

the larval

timing

during exist

enables of specific

three

the Artemia RNA poly-

The investigation

development

(4,5)

and the In this

in the nauplii

by proteolysis

periods

after

The isolation

of transcription

(5).

long

(2).

embryos

hatching

present

larvae

at the gastrulae

for

to investigate

of the polymerases

shortly

state

and their

previously during

development

at 30°C.

regulation

and developing

new form of RNA polymerase

MATERIAL

of time,

as their

of the RNA polymerases levels

swimming hours

on the

their

in a dormant

populations

has been reported

in the relative

opment

remain

16-18

synchronous as well

can stop

to free

in about

events

development

they

give

one to have highly developmental

salina

has shown

appearance

paper

during

of the

we report

the

of RNA polymerase

changes

of a new form

early

that larval

the devel-

I.

AND METHODS

Chemicals and Buffers. Nucleotides, phenylmethylsulfonylfluoride, and soybean trypsin inhibitor type I-S, were obtained from Sigma Chemical Co. 3H-UTP from the Radiochemical Centre, Amersham. Calf thymus DNA from Worthington.Biochemical Abbreviations:

Copyrig& AN rights

Q I977

PMSF, phenylmethylsulfonylfluoride;

by Acndemic Press. inc. ik my form reserr,erf.

of reproduction

STI,

soybean

trypsin

inhibitor.

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL ?ESEAK’-I

~“.‘~.?l?l,“\‘r~,4TI~~l~

GmbH, and ?EAE-Sephadex ~42:) from co. a-Amanitin from Boehringer Mannheim, Pharmacia. 50 mM Tris-HCl, 0.2 mM EDTA, 5 mM mercaptoetil;~nol Buffer B contains: and 20% glycerol, pH 7.5. Buffer C is buffer B plus 0.075 ?1 arm>onium sulfate. Buffer D is buffer B plus 0.05 M ammonium sulfate. Buffer H contains: 50 mM Tris-HCl. 0.2 mM EDTA. 5 mM mercaptoethanol and 0.6 M ammonium sulfate, pH 7.5. Organism-and growth conditions. A&ania saZina cysts were obtained from San Francisco Bav Brand Inc.. Division of Metaframe Co.. Menlo Park, Cal. 94025, USA. Treatment of the dry embryos and growth conditions were as described elsewhere (3). Nauplii hatched between 16-18 hours were isolated and grown synchronously at 30°C. Samples were taken at intervals and the anima1.s were collected by filtration on a cloth and washed with distilled water. The samples were kept at -70°C until use. Preparation of the extracts and enzymatic assays. The preparation of the extracts and the solubilization of the RNA polymerases were as described elsewhere (3) using buffer H. The RNA polymerase assays were carried out according to Renart and SebastiSn (3) using native calf thymus DNA as template. One unit of RNA polymerase activity was defined as the amount of enzyme that catalyzes the incorporation of 1 pmole of UTP into trichloroacetic acid insoluble material in 10 min under the assay conditions. Proteases B and C were isolated from 35 hour old nauplii by chromatography on DEAE-cellulose as described by Osuna et al. (6). RESULTS AND DISCUSSION Figure merases

la shows

which

pattern

are

four

peak of activity polymerase levels III

peaks

is eluted Ia.

Ia which

decrease

increase

during

this

II

Several

between

experiments controlled

with

icant

of nauplii amount

observed

after

Several I,

including

the

incubation

mechanisms a proteolytic

out

after insensitive

new peak was called increas'e

to test

undetectable between

I or Ia.

Therefore,

this

levels. I and the

action.

a nauplii

1391

multiple

extract

was studied

does not

contain

the RNA polymerase

by

I is that the a signifIa

of the RNA polymerase

in the modification

In fact,

I.

possibility. with

was made by modification

can be involved

The

the two enzymes,

of the incubation

incubation

I.

form of RNA pvlymerase

I was incubated

in the

of RNA

of RNA polymerase

Figure 2 shows that RNA polymerase Ia form. Control experiments showed

utilized

of RNA polymerase

a few hours

of RNA polymerase

reaching

RNA polyan elution

obtained

of the RNA polymerase

and the product

extract

This

a relationship

carried

chromatography on DFAE-Sephadex. converted to the RNA polymerase amount

I.

be a modified

RNA polymerase

with

within

are

and the levels

period

Ia could

three

A new a-amanitin

a decrease

constant

Ia suggest

conditions

lb).

RNA polymerase

the decrease

have been

purified

However,

activity

c, and d) shows a relative

remain

RNA polymerase

Partially

(3).

(Fig.

developmental

of RNA polymerase

instance,

under

1 (b,

is correlated

The correlation for

extracts

contain

on DEAE-Sephadex

polymerases

before

Figure

of RNA polymerase

of A. .saZina

of RNA polymerase

of the larvae

RNA polymerase

nauplii

by chromatography

to the embryo

hatching,

chromatography

newborn

separated

identical

after

that

proteases

of RNA polymerase are

induced

I.

Vol. 78, No. 4, 1977

BIOCHEMICAL

lo

C .L

20

i

3-

la

I

II

m

30

40

30

IO

x E

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

IO

20

I 3-

u

30

IO

T24

I

40

I

m

50

II

-f27

E” s. 2 < z

2-

Iz

It

2-

‘1b 4 1 ii-

jw 0 IO

I 30

20

-o-

40

Jo

IO

Fraclion

20

30

40

50

number

Fig.

1. Separation of RNA polymerases isolated from naupli at different times of development by DEAE-Sephadex chromatography. Extracts were prepared from newborn nauplii (Tlg) and 3, 6, and extracts from 3 g nauplii 9 hour old larvae (T21, 24, 27). The soluble (ww) were subjected to chromatography on 30 ml columns of DEAE-Sephadex A25 equilibrated with buffer C. The extracts were diluted with buffer B to get a final concentration of 0.075 M ammonium sulfate. After the application of the sample the column was washed with 30 ml buffer C and eluted with 120 ml linear gradient from 0.075 M to 0.6 M ammonium sulfate in buffer B. Fractions of 3 ml were collected and 50 ~1 were assayed for RNA polymerase activity in the presence (0) and absence (0) of 10 rig/ml of a-amanitin. The specific activity of the UTP in the assay was 230 CPM/pmole and the incubation time of the reaction was 30 min.

during

the early

proteases

extracts.

experiment is

unable

protease

with of the

chromatography.

of RNA polymerase and the effect to inhibit B, while

(6,7) the

The effect

by incubation

by DEAE-Sephadex modification

development

B and C correlates

in the nauplii was tested

larval

the in

time

I to Ia. vitro

of appearance

of proteases

isolated Protease

of several

ST1 inhibits

and the timing

of

of RNA polymerase

Fig.

modification the modification.

Ia

B and C on RNA polymerase

proteases with the polymerase B is able to produce in Vitro

inhibitors

1392

of induction

3 shows

the results

of proteolytic

I

followed the

of this enzymes.

of the RNA polymerase The pattern of the

PMSF

I by effect

of

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL

Fraction

RESEARCH COMMUNICATIONS

number

Fig.

2. In by a nauplii extract. About 1000 units of isolated RNA polymerase I (DEAE-Sephadex fraction) were incubated with 0.25 ml of-an-extract from nauplii (T24) during 60 min at 4°C. An identical amount of RNA polymerase I was kept as a control at 4°C. The treated and untreated samples of RNA polymerase I were subjected to chromatography on two 3 ml columns of DEAE-Sephadex equilibrated with buffer D. After application of the sample the column was washed with 5 ml buffer D and eluted with a 20 ml linear gradient from 0.05 M to 0.25 M ammonium sulfate. Fractions of 1 ml were collected and assayed for RNA polymerase activity. The figure shows the fractions collected after the application of the gradient. Top: elution of the RNA polymerase I control. Bottom: elution of the polymerase incubated with the nauplii extract.

the

two protease

indicated

that

These proteolytic that

protease

the presence

produced

during

nauplii

To test

Fig.

4 shows the results

only

the modification Ia.

Therefore,

previous

B on RNA polymerase the this

I.

larval

out

The results

of this

experiment.

RNA polymerase

1393

of the

Ia is

produced

levels

suggest of RNA

of RNA poly-

and absence

The presence

also

and solubilization

two extractions

I but also

by a

may be an artefact

the extraction

in the presence

of the RNA polymerase

produced

development

possibility

carried

which

to PMSF (6).

Ia is

Ia and the decrease early

results

resistant

RNA polymerase

of enzyme I during were

with

by ST1 but

that

of RNA polymerase

polymerase.

polymerase

inhibited

of protease

I observed

from

in agreement

indicate

by proteolysis

of this

is B is

experiments action

polymerase

merase

inhibitors

of STI.

of ST1 prevents the appearance in vitro

during

not

of RNA the

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

Fraction

b

number

Fig.

3. In vitro modification of RNA polymerase I by protease B and of proteolytic inhibitors. 600 units of RNA polymerase I (DEAE-Sephadex fraction) were ed with 0.3 units of protease B (6) for 6 min at 4°C in the absence the presence of 50 ug/ml of ST1 and 1 mM PMSF. After the incubation, samples were subjected to chromatography on 3 ml DEAE-Sephadex A25 as described in the legend of Fig. 2. a) RNA polymerase I control. polymerase I incubated with protease B. c) RNA polymerase I incubated protease B plus STI. d) RNA polymerase I incubated with protease B PMSF.

preparation

of the extracts.

and the decrease increase

ment

the levels

in

The kinetics

of enzyme I observed of protease

of the appearance in Fig,

B that

during

incubatand in the columns b) RNA with plus

of the form

1 can be correlated

occurs

effect

this

stage

with

la the

of develop-

(6). Fig.

4 also

shows that

the presence

of ST1 produces

indicating that RNA polymerase III activity, this enzyme may also be an artefact produced protease. The existence merases

raises

potential

role

of proteases

several

theoretical

of the proteases

with

the observed in vitro

a differential and practical

in the in &vo

1394

a recovery

of the

disappearance

of

by a STI-sensitive activity

problems, regulation

on the RNA polyincluding of the RNA

the poly-

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ra

1

II

m -ST1

h

L

1 40

20

60

+ ST1

20

40

Fraction

60

number

Fig.

4. Effect of soybean trypsin inhibitor on the extraction of the RNA if_olymerases from nauplii. 10 gr (ww) of nauplii (T27) were divided in two parts. 5 g were homogenized with buffer H and 5 gr were homogenized with the same buffer containing 100 lig/ml of STI. The proteolytic inhibitor was also present at this concentration in all buffers used for the dilution of the last The extracts were subjected to extract prior to the chromatography. chromatography on two columns as described in the legend of Fig. 1. Top: chromatography of the extract prepared in the absence of the STI. Bottom: chromatography of the extract prepared in the presence of STI.

merases

and their

role

RNA polymerases. systems in

as well

the levels

enzymes

and during

problems

as during

bacterial

are particularly

systems sporulation

including

(8),

when changes

of new proteolytic

on RNA polymerases yeast

in the

in developing

transitions,

and appearance

of proteases

heterogeneity

important

and growth

proteases

The effect

biological

of an artefactual

physiological

of preexisting

can occur.

ed in several (9)

These

in the creation

has been

L7rosoph~IZa

report-

maZanognster

(10).

ACKNOWLEDGEMENTS We thank Elvira Dominguez and Francisca de Luchi for their technical assistance. C.O. holds a fellowship from P.F.P.I., Spain. This work was supported in part by a grant from Fundaci6n Juan March and Fondo National ra el Desarrollo de la Investigaci6n CientFfica.

1395

pa-

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Dutrieu, J. (1960) Arch. 2001. Exp. Gen. 99, l-134. Hentschel, C.C. and Tata, J.R. (1976), Trends in Biochem. Sci. 1, 97-100. Renart, J. and Sebastihn, J. (1976) Cell Different. 5, 97-107. Birndorf, H.C., D'Alessio, I. and Bagshaw, L.C. (1975) Develop. Biol. 45, 34-43. Abstr. SebastiZn, J., Renart, J. and Osuna, C. (1975) 10th FEBS Meeting Paris, p. 302. M.A. and Sebastign,J. Osuna, C., Olalla, A., Sillero, A., Gunther Sillero, (1977) Develop. Biol.in press. SebastiCn, J., Osuna, C., Olalla, A., Renart, J., Cruces, J. and Sillero, M.A.G. (1976) J. Cell Biol. 70, 340a. Dezelle, S., Wyers, F., Sentenac, A. and Fromageot, P. (1976) Eur. J. Biochem. 65, 543-552. K.F. (1976) FEBS Letters 71, 205-208. Greenleaf, A.L,, Haars, R. and Bautz, Linn, T.G., Greenleaf, A.L., Shorenstein, R.G. and Losick, R. (1973) Proc. Nat. Acad. Sci. USA 70, 1865-1869.

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