Vol. 64, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VECTORIAL DISCHARGE OF NASCENT POLYPEPTIDES

ATTACHED TO CHLOROPLAST

THYLAKOID MEMBRANES Maurice

M. Margulies,

Smithsonian

Received

April

SUMMARY.

H. Lee Tiffany

and Allan

Institution Radiation Biology 12441 Parklawn Drive Rockville, Maryland 20852

Michaels

Laboratory

2,1975

Chloroplast

thylakoids with attached ribosomes were isolated from They were allowed to incorporate labeled amino acids into polypeptides. Labeled membranes were recovered from the reaction mixture, and a portion was treated with puromycin. The amount of labeled polypeptides released to the medium, and to the membranes by puromycin was determined by comparing radioactivity in soluble protein before, and after untreated, and puromycin-treated membranes were solubilized with the detergent Nonidet P-40. About 20% of the radioactive protein associated with the membranes was in nascent chains which were terminated by puromycin. Essentially all of terminated nascent chains remained with the membranes, and thus, were vectorially released. The results support the hypothesis that polypeptides which are synthesized by thylakoid-bound ribosomes are being incorporated into the membranes as they are synthesized. Chlamydomonas

reinhardti.

INTRODUCTION, ribosomes

Ultrastructural

are associated

suggestive

(Margulies, incorporates

chloroplast that

the

ribosomes

amino

Nascent

.

were

Proteins

incorporated synthesized

in another

ribosomes

(6-8).

proteins

might

into

This

(1,3).

which into

were the

by vectorial directional

release into

the

Over

50% of

are polyribosomes

and completed

are

important

These

membranes

release

(1,3).

from

The thylakoid-ribosome

synthesized

in one subcellular

be incorporated

membranes

nascent

chains

to thylakoids proteins

localized

acids

isolated

isolated

to them

unpublished).

polypeptide

ribosomes thylakoid

A.,

in arrangements

have been

attached

of the

some chloroplast

thylakoids

Thylakoids

ribosomes

and Michaels,

labeled (5)

(l-4).

ribosomes

M.M.

have shown that

the chloroplast

with

reinhardti

the thylakoid-bound

vitro

with

of polyribosomes

Chlamydomonas

in

studies

of polypeptides

structure

to the

attachment

provides

of

led us to postulate

by chloroplast

compartment

also

polypeptides

results

as they

complex

were

membrane-bound

synthesized

(1).

can be transferred from membrane-bound a mechanism

of membranes

(9).

by which Vectorial

or

Vol. 64, No. 2, 1975

release with

BIOCHEMICAL

on termination nascent

of nascent

polypeptides

reticulum

(8),

and for

cytoplasm

side

of the

to determine attached

whether

vectorial

with

to the

puromycin

ribosomes

polypeptides

mitochondrial

to thylakoid-bound

thylakoid

chains

attached nascent

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(i.e.

of the

attached

envelope

might

on the

Our experiments release

play

endoplasmic

to ribosomes

(6).

directional)

ribosomes

has been demonstrated

a role

were

of nascent

designed

polypeptides

in the biosynthesis

of

membranes.

MATERIALS AND METHODS, Chloroplast membranes were isolated from chloramphenicoltreated cells of C. reinhardti, as previously described (5). Chloramphenicol was added to cultures to preserve the ribosome-chloroplast membrane association during harvesting and breaking of cells (3). Amino acid incorporation was carried out with 80 &i of tritiated amino acids per ml of reaction mixture, but otherwise as described previously (5). The tritiated,amino acid mixture was obtained from New England Nuclear, Boston, Mass, (Cat. No. ZSO), and had an average specific radioactivity of 9.95 nCi/pmole. Samples were counted on filter paper discs (10) at an efficiency of 6.1%, giving a calculated specific radioactivity of 1,340 cpm/pmole amino acid. Amino acid incorporation was allowed to proceed for 2 h, and the reaction mixture was cooled to O*C. The membranes were recovered, and washed once with buffer (25 mM KCl, 25 mM MgCl 25 mM Tris-HCl, 1 mM dithiothreitol, pH 7.5) by centrifugation, The was is’ed membranes were stored at -8O’C. Eighty-five percent or more of the radioactive protein in the reaction mixture was recovered in the washed membranes. Membrane samples, containing 0.1 mg of chlorophyll, and about 10,000 cpm in 0.5 ml of buffer, were incubated for 1 hr at 0°C with, or without 1 mM puromycin, and with, or without 0.05 ml of Nonidet P-40 (S)/mg membrane chlorophyll, In the experiment presented, Nonidet was added at the start of the 1 h incubation at 0°C. However, Nonidet can be added at the end of the 1 h incubation at 0°C (i.e. after treatment with puromycin) with the same result (see Discussion). The treated membranes were then analyzed by centrifugation on sucrose density gradients. These were prepared from 0.31 and 1.4 M sucrose solutions in buffer, as already described (ll), but with these exceptions: 11.5 ml of gradient was prepared instead of 12.0 ml; 0.5 ml of a cushion of 2 M sucrose in buffer was layered under the gradient; 0.5 ml of buffer was layered on top of the gradient; The total the 0.5 ml sample was layered between this overlay, and the gradient. volume in the centrifuge tubes was 13.0 ml. The gradients were centrifuged for 1 h at 40,000 rpm, at 0 to 2’C in a Spinco SW 40 rotor, They were scanned at 254 nm, divided into 0.306 ml samples, and two-O.1 ml samples were taken from each fraction for determination of radioactive protein. Material that pelleted to the bottom of the tube was resuspended, and radioactivity determined, Gradient fractions l-4 contained soluble protein, 5-11 ribosomes (subunits and monomeric ribosomes), 12-35 polyribosomes, and 36-43 membranes. 80 S cytoplasm monoribosomes in 15,000 x g homogenate supernatants sedimented to fraction 11 when analyzed on replicate gradients. RESULTS, of the ment, columns

Essentially

gradients whether

when labeled or not

A and B).

membranes.

no radioactivity

the

membranes

sample

Ninety-five

When membranes

were

treated

in the

analyzed

had been treated percent

were

was found

with

of radioactivity with

736

detergent,

soluble

without puromycin

protein

region

detergent

treat-

(Table

was recovered

I, with

20% of radioactivity

the

BIOCHEMICAL

Vol. 64, No. 2, 1975

AND BIOPHYSICAL

Table Vectorial

lradient

Fraction

Release

Treatment Without

I

of Nascent

Polypeptides

of membranes

detergent

With

A puromycin

detergent

With puromycin

Without puromycin

With puromycin

(A)

@I

(Cl

PI

protein

With detergent

Without detergent

Without puromycin

percent Soluble

RESEARCH COMMUNICATIONS

(B - Al

CD - Cl

cpm

1.0

2.1

20.6

42.5

+

1.1

+

21.9

iibosomes

2.3

1.6

15.0

19.3

-

0.7

+

4.3

To1 yribosomes

2.2

0.5

42.7

25.4

-

1.7

-

17.3

94.5

95.8

21.6

12.9

+

1.3

-

8.7

dembranes

cpm recovered Total

7,320

was found portion were

with

soluble

treated

with

proteins

treated

with portion

puromycin The increase puromycin polyribosome

both

puromycin,

(Table

I,

detergent,

I,

column

with,

was discharged

or without protein

percent to the

in radioactive was accompanied

in the

by a decrease I,

puromycin

vesicles soluble

(9,190

that

However,

737

C and D).

on)

that if

of 21% between (D minus

membranes was in membranes

C) indicates

as a result protein

of the

that action

discharged

( (D-C)

- (B-A)/(D-C)

fraction

produced

from 43 to 25% of radioactivity

columns

put

42% of radioactivity

was discharged

membrane

indicating (5).

of the radioactive

protein

(Table

C),

The difference

D).

gradient

7,820

proteins

and detergent,

column

Ninety-five

fraction

(Table

was in completed

of radioactive

of puromycin,

7,270

protein

of radioactivity

soluble

that

7,170

from

by ),

by in the

Vol. 64, No. 2, 1975

DISCUSSION. tion

Over

BIOCHEMICAL

85% of label

by chloroplast

Some of the

solubilized

it

is

molecular

weight,

radioactive

However,

a portion

appears

polyribosome-like (5,

activity

associated

of the

completed

attached,

bands

I in this

radioactive

into

(i.e.

they

become incorporated

where

they

are not

tional

release

argues

for

protein were

this

treated

when puromycin

was added

discharge

microsomes

(S),

puromycin, during

considered

with

thylakoid

as spaces

of microsomes

acid

associated

is

that

having

without

the

become

through

Our observation thylakoid

I:

a stage of direc-

polyribosomes

patterns

of radioactive

1) when isolated

treated

with

radio-

protein

going

similar

of

the precursor

nascent

incorporation

enclosed

are released

membranes

detergent, instead

or 2)

of after

puromycin

experiments

with

mitochondria

by the membranes. somes are

derived), Only

(8).

However,

in the

(6).

Microsomes,

in our

by membranes.

released

the

used

has been demonstrated

Neither (6) has it are

unlike

chloroplast

in our been

experiments,

demonstrated

in the membranes, the endoplasmic thylakoids

phycobilin-containing

(3)

Some of the nascent space

can all

be

polypeptides protein-

or in the whether

the peptides

spaces (from

have no documented chloroplasts

and

upon nascent

or in the reticulum

with

mitochondria,

preparations

to the membrane-enclosed

with

are vectorially

polypeptides

mitochondria

fragments

termination

function.

still

with

and then

is

suggest

latter

(3,s).

in Table

amino

of nascent

and also

chloroplast

which

presented

it

(5).

of membranes.

Vectorial

the

1 h with

the

associated

high

by detergent-treatment

We have obtained

to those

for

because

while

membrane)

membrane

discrete

particles

the membrane

(5) (

electrophoresis

results

proteins,

of label

interpretation.

distribution

recovery

to the

by puromycin

These

the membrane into

attached

protein,

polyribosome-like membrane

and incorporated

gel

incorpora

membranes

and gives

are released

work),

with

acid

to be in completed

on acrylamide

which

these

remains

appears

to be in nascent

with

end of 2 h amino

by detergents,

particles

and Table

at the

C. reinhardti

to the membranes

because

membranes

from

bound

proteins,

with

in peptides

membranes

label

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

enclosed

which

micro-

secretory

of cryptophyte

algae

BIOCHEMICAL

Vol. 64, No. 2, 1975

has the

space

within

in cryptophytes, Our experiments rihosomes

for

the

the

only

spaces

is not suggests enclosed

attached

show that

discharged

lumen

thylakoids

to the

ribosomes

on thylakoid

Whether into

the thylakoids

been reported

to contain

to thylakoids

could

a portion

membranes protein known. that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

of nascent

vectorially

remains

with

However,

the

vectorial

function

the membrane

release

of documented occurs

produced

to the itself

(12).

Thus,

in secretion,

polypeptides

discharged

lack

protein

by

thylakoids.

or is

discharged

secretory

function

to the membranes,

and not

by them.

ACKNOWLEDGEMENTS. The work reported here is published with the approval the Secretary of the Smithsonian Institution, It was supported in part Smithsonian Research Foundation (Grant No. 450126).

of by the

REFERENCES. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Chua, N,, Blobel, G., Siekevitz, P. and Palade, G.E. (1973) Proc. Natl. Acad. Sci. U.S., 70: 1554-1558. Falk, H. (1969) T. Cell Biol. 42: 582-587. Margulies, M.M. and Michaels, A. 7974) J. Cell Biol. 60: 65-77. Philippovich, I.I., Bezmertnya, I.N. and Oparin, A.I. (1973) Exp. Cell Res. 79: 159-168. Michazs, A. , and Margulies, M.M. (In press) Biochim. Biophys. Acta. Kellems, R.E., Allison, V.F. and Butow, R.A. (1974) J. Biol. Chem. 249: 3297-3303. Redman, C.M., Siekevitz, P. and Palade, G.E. (1966) J. Biol. Chem. 241: 1150-1158. Redman, C.M. and Sabatini, D.D. (1966) Proc. Natl. Acad. Sci. U.S. 56: 608-615. shlinger, P.J. and Schimke, R.T. (1972) J. Biol. Chem. 247: 1257-1264. Mans, R.J. and Novelli, G.D. (1961) Arch. Biochem. BiophK 94: 48-53. Honeycutt, R.C. and Margulies, M.M. (1972) Biochim. Biophys. rta 281: 399-40s. Gantt, E., Edwards, M.R. and Provasoli, L. (1971) J. Cell Biol. 48: 280-290.

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