Subcellular site of glycoprotein synthesis in liver

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 4, 1969

SUBCELLULARSITE OF GLYCOPROTEINSYNTHESIS IN LIVER J. Molnarl,

M . Tetas*

and H. Chao

Department of Biological Chemistry University of Illinois College of Medicine

ReceivedSeptember 19, 1969 SUMMARY

The smooth and rough m icrosomal fractions of rat and rabbit liver were shown to carry out transfer of N-acetylhexosamine, galactose and mannose to endogenous protein acceptors from correThe rough m icroeomal fraction was sponding Sugar-nUCleotideS. more active than the Smooth m icrosomal fraction for the incorpoas active ration of hexosamine and mannose, but only one-third for the incorporation of galactose. Data also are presented which suggest that among the subcellular fraction8 only the m icrosomal fractions are active in the hexosamine and mannose transfer reactions. Regarding lian

researcher8

tissues,

these

the mechanism of glycoprotein

complex proteins

tablished

agree that

and completion

hypotheses

have been suggested.

chain

is built

by a multienzyme

according

According

cisternae

complex of carbohydrate

This

is based on the original

observation8

and found

that

3H-labeled

macromolecules,

glucose

other

chains

transferases,

region

(1) who injected

side

two

of mammalian cells

in the membranes of this

Leblond

es-

to one the carbohy-

localized idea

backbone of

to the well

of the oligosaccharide

up in the Golgi

in mamma-

As to the glycosylation

SyntheSi8.

of protein

drate

the polypeptide

is assembled

pathway of protein

formation

which

(single-site

hypothesis).

of Neutra

or galactose

than glycogen,

is

and in rats,

were labeled

% ,his investigation was supported by Public Health Service Grant No. PHS ROl-HD-02591 and a Career Development Award 5-K3-Ca-11, 158. *Leave of absence from University

of Chile, 684

Santiago,

Chile.

Vol. 37, No. 4, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only

in the Golgi

this

interpretation,

and Eylar

zone of liver

Wagner and Cynkin

(3) have reported

glucosamine

observations

is initiated

(rich

fractions

were reported

According

to the other in liver

of rat

membranes (9)

out by

fragments)

but not

The purpose

the Golgi

region,

of sugars

thus

from experiments

the

However,

the

is assembled within

experiments

to endogenous acceptors

supporting

of protein8

tumor (8) during

multi-site

both the rough and smooth m icrosomal

Simi-

(4).

on the ribosomes. chain

of the present

liver.

glycosylation

of the rough and smooth endoplasmic

(which may include

conditions,

is carried

HeLa cells

and plasma cell

of the oligosaccharide

transfer

of N-acetyl-

and rabbit

by using

chain

Supporting

and Caccam, Jackson,

in Golgi

hypothesis

(S-7)

of the polypeptide

remainder

(21,

incorporation

fractions

by the rough m icrosomal

growth

that

tissues.

and mannose in endogenous proteins

the smooth m icrosomal

lar

and other

reticulum hypOtheSi8).

is

to show that

are carried

fractions

the multi-site

the

out by

under --in vitro

hypothesis

derived

in vivo. -MATERIALS AND METHODS

Smooth and rough m icrosomal Dallner

procedure

had been starved

fractions

(10) from the livers for

20 hr.

Isolation

were prepared

of rats

and rabbits

of subcellular

and measurements

of protein,

were described

previously

UDP-N-acetylhexo8amine-14C

CrC/pmole, glucosamine-14C:galactosamine-14C by incubating

Ehrlich

UDP-galacto8e-3H purchased

Ascites

Tumor with

and GDP-manno8e-14C (both

from New England Nuclear

which

fractions,

enzyme assays,

(5).

by the

RNA and radioactivity

= 3:l)

(4-5

was prepared

glucosamine-

14c (11);

10 pC/pmole> were

Corporation.

RESULTS AND DISCUSSION In order

to determine

which

of the subcelluar 685

component8

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 4, 1969

TABLE I DISTRIBUTION

OF SUGAR TRANSFERASE, AND MARKER ENZYME-ACTIVITIES AMONG SUBCELLULAR FRACTIONS OF RAT LIVER Sugar Transferase'

Fraction

hNAc3 Debris1 Heavy mitochondria Light mitochondria Microsomes 106,OOOg supernatant Results

are

Marker

t:

;3 20 40 0

k28 2

expressed

g-6-Pase

Man

in

Scc.Dh

E 4 of

8Gal.ase

24 55 16 5 0

2':

percent

Enzyme

recovered

fi 41 8 8

activity.

when the debris fraction was further separated according to Blobel and Potter (16) to nuclei and mixed membranous fraction (cell membrane; residual mitochondria and microsomes) no sugar transferase, or glucose-6-Pase activity was found in the nuclear fraction. 2 The mixtures in 0.5 ml volume contained: 5mM MgC12, 5 m M MnC12, 1mM EDTA, O.lM Na-maleate, pH 6.9, O.OlM KCl, 1% Triton X-100 (Triton caused 2-3 fold increase in the incorporation of sugars for each fraction), 3 membrane protein and 35,000 dpm of y4C (4 uC/umole) or 100 000 dpm of GDPReactions were c:rried out at 30' for 15 min and stopped by tie addition of 2 ml of 10% CC1 COOH. The precipitates were washed 5 times with 5 ml portions o % 10% CC13COOH, once with chloroform-methanol (2:l) and then with 95% ethanol; they were finally dissolved in 0.5 ml of 0.2N NaOH and counted in a liquid scintillation counting system. 3 hNAc, Scc.Dh,

could

acetylhexosamine; Man, succinic dehydrogenase;

carry

genous

measurable

acceptors,

to yield somal

out

debris, and cell

14C as well genase

for

livers heavy

mitochondria,

and glucose-6-Pase sentative tivity

for

experiment for

both

sugar of

sugars

transfer

rats

light

were

were

12;

8-galactosidase

I)

parallels

show that the 686

fractionated micro-

(succinic for

the

distribution

mannosedehydro-

lysosomes,

The results

5).

endo-

of hNAc-14C,

determined

microsames,

into

mitochondrial,

Incorporation

enzymes

(Table

reactions

and rabbits

mitochondrial,

sap fractions.

as marker

mannose; G-6-Pase, glucose-6-Pase; PGal.ase, P-galactosidase.

13;

of

a repre-

incorporation

ac-

of

glucose-6-

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 4, 1969

TABLE II EFFECT OF WASHING ON THE ENZYMEACTIVITIES M ITCCHONDRIALFRACTION Sugar Transferase

OF THE

Marker Enzyme

hNAc

Man

G-6-Pase

Scc.Dh.

PGal.ase

M itochondria

42

51

40

95

88

M icrosomes

58

50

60

5

12

Data are given

as % of recovered

activity

The combined light and heavy m itochondrial fraction of a rabbit liver (about 500 mg protein, containing about 50% of the G-6-Pase and hNAc-transferase activity of the liver fractions) was suspended in 60 m l of 0.25 M sucrose containing 1 O m MTris-HCl, pH 7.6, and centrifuged at 15,000 rpm for 10 m in (Sorvall RC 2B, SS-34 rotor). The sediment was treated with fresh sucrose two more times. The combined supernatants were centrifuged further at 30,000 rpm for 1 hr (Beckman L2 centrifuge, No. 30 rotor). phosphatase chondrial three

fractions

washings

activities devoid

The relatively

activity.

could

be reduced

of succinic

activity

6-Pase activity.

the m icrosomal

structures

the hexosamine

and mannose transfer

subcellular

explain

fractions

(14).

In subsequent

activity were also

After

washings.

fraction

These results cell

(Table

II)

60% of

suggest

that

constituents

the m icrosomes

of the other

were further

to rough and smooth m icnosomes and incorporation

with

rabbits

In I.9 experiments

as the smooth m icrosomes more active

with

the rough m icrosomes (Table

III).

than smooth m icrosomes 687

in

Thus contamination

reactions.

most of the activities

experiments

sugars were determined. periments

of the m ito-

but containing

are the principle

could

fractionated

by repeated

in a new m icrosomal

dehydrogenase

glucose

by m icrosomes

activity

58% of the hNAc and 50% of the mannose transferase

were recovered

the original

high

rats

of

and 5 ex-

showed twice

as much

Rough m icrosomes in the incorporation

Vol. 37, No. 4, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE III INCORPORATIONOF SUGARS INTO PROTEINS OF SMOOTHAND ROUGH M ICROSOMALFRACTIONS Source of Liver

Additions' UDP-N-Acetylhexosamine-14C

Smooth'

Rough

No. of EXP .

100 (60-140)

200 (113-300)

19

Rat

UDP-N-Acety hi hexosamineC

156 (130-200)

270 (255-300)

5

Rabbit

GDP-Mannose-14C

Rabbit

130

190

3

UDP-Galacto8e-3H

Rabbit

300

90

2

UDP-Galacto8e-3H+ fetuin acceptor3

Rabbit

1900

450

1

The results are expressed as dpm/mg protein in average obtained in the number of experiments indicated.

value8

The 'The reaction8 were Carrie out as described in Table I. activity of UDP-galactose- 4 H was 100,000 dpm (10 pC/pmole>. 2The incorporation of sugars by a m ixture m icrosomal fractions was additive. 'Fetuin (CalBiochem) was desialized after periodate oxidation according

of mannose- 14C . times

faster

On the other

rough fraction

for

exogenous acceptor It

more active

of galactose

when tested

the probable

synthesized

are the plasma glycoproteins

In the oligosaccharide

chains 688

basis

with

(5.6)

for

these

fractions.

carbohydrate

forms are assumed to be the major

study.

than the

from fetuin.

to consider

the most rapidly

macromolecules

sent

prepared

than by rough m icrosomes.

seen to be far

incorporation

removed

was taken up 3-4

between smooth and rough m icrosomal

the liver

complete

hand galactose-3H

was also

is of interest

difference8

and its galactose to Spiro (17).

by the Smooth m icrosome

The smooth fraction

of smooth and rough

In

containing and their

acceptor8

in-

in the pre-

of most of these

pro-

an

BIOCHEMICAL AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

Vol. 37, No. 4, 1969

teins,

the glucosamine

polypeptide

backbone than the galactose,

According

(15). studies

and mannose apparently

(5,6),

to the multi-site it

than galactose

findings

agree with

Cynkin

previously (2)

prediction

rather only

with

and mannose

The present

than that

of the

the smooth m icrosomes transferase

the reports

(3) who claim

have no glucosamine

Presently

based on --in vivo

paper and those of -in vivo disagree

to the

and fucose

or fucose).

carbohydrate

and Caccam et al.

somal fractions tivities.

(5,6)

acid,

glucosamine

acid,

from which

of this

closer

by the rough m icrosomal

(or sialic

to display

The results scribed

this

hypothesis,

would be expected

that

more efficiently

fraction

single-site

sialic

hypothesis,

would be expected

would be incorporated

lie

that

activity. studies

de-

of Wagner and

the rough m icro-

and mannose transferase

we have no explanation

for

this

ac-

discrepancy.

References 1.

M . Neutra

2.

R.R. Wagner and M .A. Cynkin, 35, 139 (1969).

3.

J.F. Caccam, J.J. Jackson and E.H. Eylar, Res. Commun. 35, 505 (1969).

Biochem.

4.

H.B. Bosmann, A. Hagopian Biophys. 128, 51 (1968).

Arch.

5.

J. Molnar, G.B. Robinson 240, 1882 (1965).

6.

G.R. Lawford (1966).

7.

J. Molnar

8.

F. Melchers and P.M. Knopf, Biol. 32, 255 (1967).

9.

E.J. Sarcione, (1964).

10. G. Dallner, 11. J. Molnar, In press.

and C.P.Leblond,

J. Cell

Biol.

Biochem.

and R.J. J.

and D. Sy, Biochemistry

Winzler, Biol.

689

Biophys.

Biochem.

J. Biol.

Chem. 5408

2, 1941 (1967).

M . Bohne and M . Leahy,

H. Chao and G. Markovic,

Res. Commun.

Chem. 241,

Cold Spring

M icrobial.

137 (1966).

Biophys.

and E.H. Eylar,

and H. Schachter,

Acta Path.

30,

Harbor

Symp. Quant.

Biochemistry

Stand. Arch.

Suppl.

3, 1973 166,

Biochem.

(1963).

Biophys.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 4, 1969

12. E.C. 13. T.J. 312 14.

H.B.

Slater

and W.D. Bonner,

Langley

and F.R.

Biochem.

Jevons,

Arch.

J. 2,

185 (1952).

Biochem.

Biophys.

(1968).

Bosmann and S.S. Martin,

15. N. Sharon, 16. G. Blobel 17. R.G. Spiro,

Science

Ann. Rev. Biochem. and V.R. J. Biol.

Potter,

35,

Science

Chem. 239,

690

3,

190 (1969).

623 (1966). 154,

1662 (1966).

567 (1964).

128,