Polysome interaction in vitro with smooth microsomal membranes from rat liver

Vol. 45, No. 5, 1971

BIOCHEMICAL

POLYSOME

INTERACTION

--In

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vitro

MEMBRANES

T. McArdie

K.

Shires,

Laboratory

L.

for

FROM

M.

RAT

and

H.

and

Wisconsin

Madison,

MICROSOMAL

LIVER

Research

of

SMOOTH

Narurkar

Cancer

University

WITH

C.

the

Pitot

Department

Medical

of

Pathology

School

53706

Wisconsin,

Received September 15, 1971 Summary The formation of a stable complex between polysomes and microsomal membranes isolated from smooth endoplasmic reticulum can be formed --in vitro Interaction required no substances other than those in the at 25-37’C. reactant’s preparations. Membrane binding sites shmed a distinct preference for native bound polysomes.

Polyscme

attachment

for

preparations

described

(l-4). --in

Site

vitro

reported

for

required

and

at

studies

membrane was

change

enzyme

previously microsomal

liver

of

amyloliquifaciens,

employed

with

membranes

vitro

in as

rough from

preparations The

with

rat

the of

(8-11).

membranes liver

1212

ribosomeinter-

disulfide

inter-

Uslng the been

and

microsomes

the

(3-b), have

been

that bacterial

of

a function

membranes

have

rabbit

(S-11).

those

of

sites

determination

while

rfbosomes.

independent

binding

microsomal

demonstrable

endogenous

was

Bacillus

been reticulum

was

membranes’ which

direct

the

polysomes

the

--In

have

endoplasmic

polysome

estimated on

rough

However,

27”-37°C

activity

membranes

exogenous

smooth

(5-71,

indirectly

liver

mechanism

Involved

interaction

action

ionic

from rat

Incubation

reticulocyte

binding

(4).

membranes

reticulocytes,

smooth

an

(a”-37°C)

rat from

displacement

entailed

temperature

we

in

after

on

derived

activity

only

Attachment

sites

the

direct

bindlng investigated.

method sites

on

Vol. 45, No. 5, 1971

Polysomes smooth

BIOCHEMICAL

derived

microsomal

layered Considerable

over

with

AND BIOPHYSICAL

deoxychoiate

treatment

membranes

and

post-microsomai

sucrose,

and

centrifuged

dense association

with

membranes

mg MEMBRANE

were

mixed

supernatant, as

was

RESEARCH COMMUNICATIONS

previously

observed

with incubated,

described as

shown

In Fig.

(3-4).

I.

PROTEIN

of a constant amount of poiysomes (100 pg) to different 1. The binding concentrations of flooth microsomai vesicles. Male Hoi tzman rats were fasted and iabeiied with P-orthophosphate 12-14 hrs before sacrifice. Polysomes were prepared from post-ml tochondriai supernatant incubated with 1.25% deoxychoiate, and layered over a two-step gradient of 1.3iM and 2.OM sucrose prior to centrifugation for 3 hrs at 226000 g. Smooth mlcrosomal fractions were made from an uniabelled post-mitochondriai supernatant which was adjusted to a sucrose density of 1.35M, spun 3.5 hrs over 2M sucrose at vesicles were taken from the top of the i.35M 275000 g (131, and the smooth layer were washed three times in sucrose. Fresh iy prepared smooth membranes, polysomes, and a constant amount of 226000 g post-microsomal supernatant were mixed in 0.44M sucrose, incubated at 3-37" for 30 minutes, chi I led to O’, layered over 1.8M sucrose, and spun at 408000 g for 8-10 hrs. The tubes were cut to separate the membranes and their associated polysomes, which together overrode the dense sucrose, from the non-binding poiysomes which pelleted, and the relative distribution of radioactivity between these The buffer for all sucrose solutions compartments was determined (3-4). contained SO mM trIs-hydrochloride (pH 7.1 at 25”)) 25 mM KCl, and 5 mM

Fig.

MgC12. 1213

Vol.

45,

No.

The of

extent the

of

amount

Using (SD

BIOCHEMICAL

5, 1971

interaction

of

of

material

membrane

10.02%)

membrane

of

preparation

temperature

by

range

of

equilibrium

at

At

membrane

enhance

the

extent

15 hrs

after

of

from of

the the

its

ribonuclease

Table

1

label

Stability

of .

the

1).

an

of

average

55.2%

the

smooth

membranes

was

membranes

and

or

a 30 min

or

longer

did

in

1). the

incubation If

membranes, the

capacity

not

demonstrable

(12).

with

37’-binding

37”

(Table

activity

the

binding

remained

included

polysomes

over

Apparent

hour

it

was

61.6%

mlcrosomes

of

mixture omitted

(SD

5 16.7%)

(polysome-to-

smooth

*

Membrane 0 hr

O0

an

negligible

sites

remained

Preincubation Temperature

37”

mixture.

from

at

of

was

of

ribosomal

mi crosomes

*

was

incubation

inhibitory

mixture

a function

incubation

microsomal

Rinding of

the

(Fig.

periods

isolation

in

was

inseparable

smooth

min

supernatant

incubation admixed

the

attachment.

Post-microsomal because

were

concentrations a 20

polysomes

(ug:ug),

interaction

Incubation of

5:1

of

centrifugation.

by 3-4”

after

25”.

of

speed

polysomes

occurred

incubation

included

polysomes

high

dependent.

entire

to

added

of

amount

ratio

the

Acceptance

up

a fixed

a membrane-to-polysome f

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Preincubation

1 hr

53.7%

f 2.3

47.5%

50.2%

f

59.4%** 57.6%

4.9

f

4.5 1.4

51.0%

Tlme

hr f

15 hr 5.7

--m---m

56.4%

+ 1.0

55.2%

f

6.4

Freshly prepared smooth mlcrosomal membranes were suspended in 3 mls 0.44 sucrose per g liver equivalent of membranes. After incubation in a water bath or ice bucket, the membrane suspensions were clarified at 2000 rpm for 10 min and protein determined on the supernatant (2). 3 Membranes were mixed with P-labelled polysomes in a ratio of 5:1 in the presence of post-microsomal supernatant and incubated at 37” for 30 Values least

from only 5 experiments.

two

experiments. Standard

All deviations

1214

other of

averages the mean

are are

based shown.

on

at

min.

Vol.

45,

No.

5, 1971

membrane

ratio,

association

5:l). was

earlier

Thus,

not

only

considered

(10).

a smooth

soluble since and

to

buffer,

the

in

our

COMMUNICATIONS

membrane-polysome

factor,

Moreover,

intrinsic

studies

on

membrane-associated membrane away

above

non-binding

used

these

the

agreement

with

incubation

mixtures

ribosome

binding

smooth

microsomal

binding

sites

must

be

preparations

of

this

procedure

this

assessment

from

trapped

the

stoichiometry

The are

0.13

had

an

sucrose

layer,

homogenized,

particles,

ratio

average

for

ratio earlier

rough of

not not

It

I. the

of

the

does

for

the is

of

f

failure based

--in on

Because

but it

is

vitro

1215

that

amounted of Because particles

for

correcting

quantitatively

to

set

interaction. membrane

used

and

membrane

known

layer

dense

particles.

serve

complexes many

from

dissociated

preliminary

ribosome-membrane profiles

dense

labeiled

ratio

of

A repetition

fractions

average

methods

over

ribosome-smooth

O.OOg),

the

recovered

reliable

the

37”

freed

rIbosome-membrane

microsomal

an

the

however,

of

(SD

had

the

by

recentrifuged

distinguish

ratio

0.03

(2))

of

be wholly

extent

Smooth

membranes

ribosome particles.

does

RNA-protein

average

of

more

the

assessed

radioactivity.

2-6%

may

Fig.

available

examinatton associated

level

f 0.02).

explanatlon

it of

and

the

centrifuging

pellicle,

through

associated

trapping

was

in

in

was

pelleting

removed

of

(SD

reported

attached

ultracentrifugation,

for

a component

membrane

by

only

a confidence

The

particles

originally

used

membrane

(4).

particles

the

as

the

entrapped

layer

Trapping smooth

encountered

physically

sucrose

(4).

studies

Dislodged

g-12%

were

dense

with

earlier

dense

sucrose.

the

polysomes

non-associating above

membrane which

polysomes of

in

rough

polysomes

pelllcle

association

as

of

RESEARCH

se. Eariler

to

any

membranes

activity

BIOPHYSICAL

formation

by

polysomes, an

AND

the

mediated

investigations

included

Per

BIOCHEMICAL

rough of

the

mi crosomal

about to

for

The an

electron

binding fractions,

0.2.

attain

compiexes

tentative

RNA-protein

microscopic

(14-15))

showing

clearly

vesicles

without

any

these

smooth

microsomal

studies

Vol.

45,

No.

pteparatfons Golgl

BIOCHEMICAL

5, 1971

from

rat

apparatus,

uncertain

as

whether

responsible

free

in

and

earlier

both

studies of

that

with

shcms polysomes

was

membranes

were

or

liver

tissue

(161,

the

of

type,

were

plasma

COMMUNICATIONS

membrane

reticulum or

all

and

(131,

types,

in

of

average

from

rat

of

of

rough

to

about

the

native

a mixture

of

a proportion

liver

37"

at

it

mlght

IS

be

divided the

bound

of

same

the

attaching

endoplasmlc

of

bound

radioactivity

to

and

smooth

native

bound

free

free

smooth polysomes,

determined was

that Table

If and

that

shown

(10).

polysomes.

native

groups reticulum,

had

extent

binding

free

bound

into

membranes

approximately

of

percentage

.

membranes

the

percentage

The

naturally

incubation

with

the

occurs

sites

bound

that

32 P,

to

binding

twice

membranes

I on

attached

incubated with

I ncubat Temperature

endoplasmic

a 30 minute

labelled

2

smooth

population

polysomes

both

Table

elements

RESEARCH

acceptance.

on

about

BIOPHYSICAL

contain

membrane

polysome

cytoplasm

groups

as

ribosoma

hepatlc the

well

a single

for

The

liver

AND

for

comparable

polysomes

mlcrosomal

*

Native Free Polysomes

Native Bound Polysomes

Bound : Free PO1 ysomes

(3:I) 3-4" 37"

*

0.9% f 0.8 36.1%

f 0.2

15.3% f 8.7

17.4% f 2.8

62.9%

57.7% f 0.2

f

2.9

A post-mltochondrial supernatant was made from livers of rats labelled with 32~ and fasted for 12 hrs then centrifuged immediately over a twolayer gradient described in Fig. 1. Native free polysomes were collected as pellets from the bottom of the tubes, and rough microsomes occurred banded above the dense sucrose. The rough membranes were harvested, suspended In 0.44fl sucrose-buffer, and layered over a second two-layer gradient for removal of any trapped free polysomes. After centrifugation, these polysomes were discarded. The membrane band overriding the 2M sucrose in the second gradient was collected and treated with 1.25% deoxychol ate. Native bound polysomes were pelleted from thls detergentcontaining suspension by centrifuging through a third two-step gradient. A total of 100 ug of polysomes was incubated wlth 0.5 mg of freshly prepared and washed smooth microsomal membranes In the presence of post-mlcrosomal supernatant. Incubations were carried out for 30 min at 37'. Standard deviations of the mean are shown for S-10 experiments.

1216

2

Vol.

45,

to

No.

that

5, 1971

obtained

post-ml

could

gave

membrane

steroids

for The

binding

of

that

a portion

on

rough

of

microsomai

on

temperature

the

smooth

the

formed

ribosomes

the

--in time

(8-il),

with

the

membrane

incubation at

rough at

microsomal

membranes

medium

dependence,

Whereas

during

attaching

available

as

rough

smooth

membranes

vitro.

of

but

these

smooth or

no

steroids

is

implicit

in

of

rough

paramount

over

the

release

complexes

formed

are

with (4).

ribosomes

Based

ultracentrifugating also

stable,

on

rough

polysome, unlike

might

function

the

1217

reported

as

consequently

an

generating

isolated

(201,

membranes to

complexes

even

shed with

steroid-dependent

has

(21-22). had

rat

under

depletion

membranes

failure

from

and

template rough

their

consideration

reticulum.

vitro,

conditioned

shown derived

worth

stable

from

Polio

membranes

units

quite in

in

perhaps

assembly, endoplasmic

incorporation

vitro

thus

proposed

RNA was on

the

a smooth-to-rough

messenger

is

that

where,

polysome-membrane

fractions

of

(lg),

reticulum

complex

acid

were

It

polysome

of of

translated

endoplasmic

the

reverse

Tamm

but

of

amino

the

protein

point

of

extended

coat

conversion

Suggestive

and

reticulum.

microsomal

dissociate

is

(17-18).

viral

conditions

to

is

Callgurl

evidence,

--in

membrane

cells,

the site

By

membranes

smooth

al ready

on

membranes

cei of

polysome-membrane

during

sites

incubation

smooth

endoplasmic

binding

tendency

detergent-treated

in

a feature

smooth-to-rough

findings

replicated

liver

in

the

to

HeLa

the

from

polysomes.

generated or

involved

of

the

virus-infected

the

in

of

membranes

free

the

present

parenchymal

are

initial

(4),

surface

rouqh

being

products

rlbosomes

from

COMMUNICATIONS

binding.

eukaryotic

be

of

novo

be

sequence of

pathway

to

de --

enrichment

required

for

as

might

obligatory

RESEARCH

population

the

native

evidence

already

preparation

on

for

isolation

viewed

be

on

those

affinity

sites

of

BIOPHYSICAL

polysome

sites

from

lower

membrane

whole

binding

differ

their

time

the

AND

supernatant.

poiysome

preparations

Sex

with

tochondrial The

and

BIOCHEMICAL

been The

a very

low

particles smooth

microsomal complexes

Vol.

45,

No.

which

BIOCHEMICAL

5, 1971

would

current

not

withstand

methods

cells

as

may

preclude

the

difference

This Institute, Cancer Agency Research

well

for as the

isolating

our finding

between

high

own

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

speed pieces

methods of

ultracentrifugation of

for

forming

transient

complexes,

steroid-dependent

work was supported The National Institute Society, and a fellowship to L. M. N. The present Centre, Bombay, India.

in

rough

and

(11).

endoplasmIc

reticulum

polysome-membrane and independent

part by grants from of General Medical from The International address of L. M. N.

Both

thus

the from

complexes serve

to

emphasize

sites.

the National Cancer Sciences, The American Atomic Energy is Bhabha Atomic

References

I. 2. 3. 4.

65: ;: 3.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Suss, R., Blobel , G., and Pltot, H. C., Biochem. Biophys. Res. Corn. i?J, 271 (1966). Khawaja, J. A., and Raina, A., Blochem. Biophys. Res. C~mm. 3, 512 (1970). Ragland, W. L., Shires, T. K., and Pitot, H. C., Biochem. J. 121, 271 (1971). Shires, T. K., Narurkar, L. M., and Pitot, H. C., Biochem. J. In Press. Coleman, G., Biochem. J. 114, 753 (1969). Burka, E. R., and Schickling, L. F., Biochemistry 2, 453 (1970). Aronson, A., J. Mol. Biol . , &, 505 (1960). Williams., D. J., and Rabin, B. R., FEBS Lett. 4, 103 (1969). Blyth, G. A., Freedman, R. B., and Rabin, B. R., Nature 230, 137 (1971). Roobol, A., and Rabin, 8. R., FEBS Lett. 14, 165 (1971). Sunshine, G. H., Williams, D. J., and Rabin, B. R., Nature 230, 133 (1971). Blobel , G., and Potter, V. R., Proc. Nati. Acad. Sci., U.S.A. SJ, 1238 (1966). Moyer, G. H., Murray, R. K., Khairal lah, L. H., Suss, R., and Pitot, H. C., Lab. Investig. 23-, 108 (1970). Shires, T. K., Narurkar, L. M., Fed. Proc. 3, 814 (1970). Shires, T. K., Narurkar, L. M., Ekren, T., and Pitot, H. C. J. Cell Biol. 2, 190a (1970). Blobel , G., and Potter, V. R., J. Mol. Biol. 28, 533 (1967). Dal lner, G., Siekevitz, P., and Palade, G. E., J. Cell Blol. 0, 73 (1966). Dallner, G., and Palade, G. E., J. Cell Biol. Siekevitt, P., 2, 97 (1966). Callguri, L., and Tamm, I., Virology 42, 112 (1970). Biobel , G., and Potter, V. R., J. Mol. Biol. 26, 293 (1367). Weksler, H. E., and Gelboln, H. V., J. Biol. Chem. 3, 727 (1967). Loab, J. N., Arch. Biochem. 139, 306 (1970).

1218