71

Biochimica et Biophysica A cta, 585 ( 1979) 71--84 © Elsevier/North-Holland Biomedical Press

BBA 28904

I N T R A C E L L U L A R L O C A L I Z A T I O N AND D E G R A D A T I O N OF A S I A L O F E T U I N IN ISOLATED R A T HEPATOCYTES

H. TOLLESHAUG, T. BERG, W. FROLICH and K.R. NORUM

Institute for Nutrition Research, School of Medicine, University of Oslo, Blindern, Oslo 3 (Norway) (Received September 29th, 1978)

Key words: Asialofetuin degradation; IntraceUular distribution: Protein transport; (Rat hepatocy re)

Summary Analysis b y isopycnic and differential centrifuging o f the intracellular distribution of radioactivity following uptake of 12SI-labelled asialofetuin by isolated rat hepatocytes showed that during incubations up to 1 h, most o f the radioactivity was associated with structures which had a subcellular distribution pattern different from both the lysosomes and the plasma membrane. The latter two organelles were followed b y means of enzyme markers. Ca 2÷ is necessary for the binding of asialofetuin to the plasma membrane, and it was also possible to differentiate between asialofetuin b o u n d to the plasma membrane and that contained in intracellular structures b y removing Ca 2÷ from the medium (by EGTA). Such experiments showed that asialofetuin became rapidly internalized. Practically all the labelled protein was located intracellularly in cells that had been incubated with asialofetuin for more that 30 min. When incubations were carried o u t for more that 1 h a peak appeared in the radioactivity distribution in the same place as the peak of activity of lysosomal marker enzymes. However, degradation of asialofetuin takes place in the lysosomes and this starts before the labelled protein can be found in the lysosomal fractions. Our data suggest that the rate-determining step in the cellular handling of asialofetuin is the transport of endocytized protein from the endocytic vesicles to the lysosomes.

Abbreviation: EGTA, ethyleneglycol bis(~-aminoethyl ether)-N, NP-tetraacetic acid.

72 Introduction The discovery by Ashwell and Morell [ 1 ] that many serum glycoproteins are bound to and taken up by liver cells in vivo when the penultimate galactose units of their carbohydrate moieties have been exposed by removal of the terminal sialic acid residues, presented an unusually promising opportunity to study receptor-mediated uptake and intracellular degradation of proteins. The present work, which is a continuation of our studies on the uptake and degradation of asialofetuin in isolated rat liver cells [2], presents an effort to elucidate the intracellular fate of proteins taken into hepatocytes by adsorptive endocytosis. Some of the findings have been presented in a preliminary report [3]. We have obtained cells from the liver by perfusing it with a solution of collagenase, and have incubated these cells with '2SI-labelled asialofetuin. We have previously shown [2] that the labelled protein is taken up exclusively by liver parenchymal cells (not by Kupffer cells) by a process which may be described as semi-saturable. Cumulative degradation, measured as acid-soluble radioactivity, was linear for 40--60 min, after a lag period of 10--15 min. Initially the radioactivity was found mostly in the microsomal fraction, but l h after exposure of the cells to labelled protein, the distribution pattern by differential centrifugation resembled that of the lysosomal enzyme N-acetyl-~-glucosaminidase. An advantage of the use of isolated liver cells for the study of uptake and degradation of protein, as compared to studies on the whole animal or the perfused liver, is that tight control of the concentration of and the time of exposure to the labelled protein becomes possible. The intracellular distribution may be studied in great detail by taking samples of the cell suspension over an extended period of time. In the present study, we have taken advantage of these possibilities. We have also compared the results obtained by labelling asialofetuin with different radioactive isotopes. Methods and Materials The procedures for isolation of rat liver cells, incubation of cells, and homogenization have all been described earlier [2,4]. Only an outline will be given here. Liver cells were prepared by a modification [2,4] of the collagenase perfusion method of Berry and Friend [5]. Non-parenchymal liver cells were removed by differential centrifugation [4]. The parenchymal cells were suspended in incubation medium [2] and incubated in Erlenmeyer flasks in a shaking water bath at 37°C. The cell density was 6--8.106 cells/ml. Cell-associated radioactivity was determined in cells separated from the medium by centrifugation through dibutyl phthalate [2,4]. Degradation of asialofetuin was followed by measuring radioactivity which remained soluble after having treated aliquots of the cell suspension with 2% phosphotungstic acid in 1 N HC1 [ 2,6 ]. Subcellular fractionation. Differential centrifugation was performed as described previously [2]. For isopycnic centrifuging a 'post-nuclear fraction'

73 or 'cytoplasmic extract' was used: following homogenization of the hepatocytes [2], the nuclear fraction was centrifuged down. This was washed once with 0.25 M sucrose, and a 4 ml aliquot of the combined supernatants ('postnuclear fraction') was layered on t o p of a sucrose gradient with a volume of 34 ml. The sucrose concentration varied linearly from 25% to 53% (w/w). The solution was buffered with 10 -3 mol/1 Tris-HC1, pH 7.2. The gradients were centrifuged for 4 h in a Beckman SW-27 rotor at 25 000 rev./min. 2-ml fractions were removed from the t o p of the tube by lifting the gradient with 58% sucrose entering at the b o t t o m of the centrifuge tube. The densities of the fractions were determined by way of their refractive indices. For determination of the 'r-radiation from ~:sI, the entire fraction was placed in the well of a "r-counter. For determination of fl-radiation, a 1 ml aliquot was diluted with 5 ml of water and mixed with 6 ml of 'insta-gel' (Packard Instrument Co., Downers Grove, IL 60515, U.S.A.). Desialylation o f fetuin. Native fetuin (Type II, Sigma Chemical Co.) was dissolved in 0.1 M phosphate buffer, pH 7.3, at a concentration of 10 mg/ml. 10 munits/ml of neuraminidase (sialidase, EC 3.2.1.18) from Clostridium perfringens (Type VI, Sigma Chemical Co.) was added and the solution was left for 24--48 h at room temperature. (Units of neuraminidase activity were standard units as determined by the supplier, without regard to deviation from optimal conditions of pH, temperature, or substrate.) Neuraminidase will destroy the activity of the asialoglycoprotein receptor on liver cell membranes [7]. However, the final concentration of neuraminidase in the cell suspension was less than 1 ~unit/ml, while the concentration required for a measurable effect on the uptake of asialofetuin is a b o u t 0.1 munit/ml (Tolleshaug, H., Unpublished observations). Accordingly, no effort was made to remove neuraminidase. For labelling with 3H or ~4C, fetuin was desialylated by mild acid hydrolysis [8]. Labelling of asialofetuin with radioactive isotopes. Iodination with ~2sI was carried o u t by the lactoperoxidase method [9] as previously described [2]. Asialofetuin was labelled with [~4C]methyllysine by allowing 5 mg of protein to react with [~4C]formaldehyde (1.9 MBq, 1.7 TBq/mol) in 1 ml of sodium borate buffer, pH 9.3. The resulting Schiff base was reduced by the addition of 0.5 mg sodium borohydride in 0.5 ml of distilled water [10]. The C-6 h y d r o x y m e t h y l group of the galactose residues of asialofetuin was labelled with 3H according to the procedure of Morell et al. [11]. The hydroxymethyl group was oxidized to the aldehyde by means of galactose oxidase (Sigma Chemical Co.) and the aldehyde was subsequently reduced, regenerating the original h y d r o x y l group, with sodium boro[3H]hydride. Determinations o f enzyme activities. 5'-Nucleotidase (EC 3.1.3.5) was determined according to the m e t h o d of E1-Aaser and Reid [12], glucose-6-phosphatase (EC 3.1.3.9) was determined according to Morr~ [13], and acid phosphatase (EC 3.1.3.2) according to Barrett [14] with glycero 2-phosphate as substrate. In all of these assays, liberated inorganic phosphate was determined spectrophotometrically b y means of the molybdate/ascorbic acid reagent of Ames [15]. Methods described b y Barrett [14] were e m p l o y e d to determine N-acetyl-~glucosaminidase (EC 3.2.1.30) and cathepsin D (EC 3.4.23.5). Barrett's method

74 for the determination of cathepsin B [16] was slightly modified, in that we used Fast Red B Salt (the stabilized diazonium salt of 2-methoxy-4-nitroaniline) at a concentration of 0.5 mg/ml instead of Fast Garnet B GBC salt in the coupling reagent. The colour was read at 540 nm. This substitution gave lower blank values and marginally higher sensitivity. Protein was determined by the method of Lowry et al. [ 17]. Results

Release of acid-soluble radioactivity after uptake of 12SI-labelled asialofetuin In experiments concerning the subcellular distribution of 12SI-labelled asialofetuin, the cells were usually incubated with asialo-fetuin in concentrations below 30 nmol/1 before they were homogenized and fractionated. At such concentrations most of the labelled protein was removed from the medium b y the cells within 10 min. After a lag period of 10--15 min the a m o u n t of acidsoluble radioactivity in the medium increased linearly. The amount of acidsoluble radioactivity in the cell pellet was of the order of 2% of the total acidsoluble radioactivity in the cell suspension. Using a Sephadex column to separate iodide from iodotyrosines by the m e t h o d described b y LaBadie and coworkers [6], we found that more than 98% of the radioactivity was released to the medium as ~2sI-. In a homogenate of washed cells only traces of m o n o i o d o t y r o sine were found along with I-.

Isopycnic centrifuging of a cytoplasmic extract prepared from hepatocytes incubated with 12SI-labelledasialofetuin H e p a t o c y t e s were incubated with ~2SI-labelled asialofetuin for 10 min, extracellular protein was then removed by washing, and the cell suspension reincubated at 37°C. Alternatively, the cells were incubated with an amount of asialofetuin (20 nmol/1) that was taken up to an extent of 85--90% within 15 min, with no washing. These procedures gave closely similar results. Samples were removed from the incubator at intervals of up to 3 h. The cells were washed 3 times and homogenized [2] in ice-cold 0.25 M sucrose. Since the isotonic sucrose solution did not contain Ca 2+ most of the asialofetuin b o u n d to the receptors on the plasma membrane was released during the washing procedure [7,18]. After homogenization, the nuclear fraction was removed, and the post-nuclear supernatant was fractionated b y isopycnic centrifuging. The percentage of radioactivity in the nuclear fraction increased from a b o u t 5 to a b o u t 15% after 3 h, which presumably reflected increasing size o f the particles containing asialofetuin [ 2]. For cells that had been incubated for less than a b o u t 1 h most of the radioactivity was found in a band in the upper part of the gradient. The peak o f the radioactivity was initially at a density of a b o u t 1.14 (Fig. 1, Table I) and was subsequently found at progressively higher densities. The change in density was small but clearly measurable. The correlation coefficient for the shift of the position o f the peak with time was 0.97. The density distribution o f the plasma membrane marker enzyme 5'-nucleotidase resembled that of the radioactivity (Fig. 1) up to a b o u t 1 h of incubation. In contrast to the radioactivity, however, the peak o f the enzyme activity was relatively unaffected by the duration

75

%

EL1

125[

10 5

0

5

10

5

%

125]

lO 5

0

5

10

%

15 lZ5[ 130 rain

10 ' ~ 5

o

I;

1'5

20 - 5'nucl, ose •/. rain) r'L 1 15

¢..,~ density

1.25 91ml 1.20 1.15

10

1.10 0 a/o

5

10

15

5

10

15

1.05

-

I0

5 0

1

fraction number Fig. 1. D i s t r i b u t i o n s o f r a d i o a c t i v i t y a n d o f m a r k e r e n z y m e s in s u c r o s e g r a d i e n t s . Cells w e r e e x p o s e d t o 3 0 n m o l / 1 of 12 S I-labelled a s i a l o f e t u i n , e x t r a c e l l n i a r a s i a l o f e t u i n w a s r e m o v e d b y w a s h i n g a f t e r 1 0 r a i n , a n d s a m p l e s o f t h e cell s u s p e n s i o n w e r e r e m o v e d f o r h o m o g e n i z a t i o n at t h e t i m e s i n d i c a t e d . T h e p o s i t i o n s of t h e p e a k s o f t h e m a r k e r e n z y m e s 5P-nucleotidasc ( p l a s m a m e m b r a n e ) a n d N - a c e t y l - f l - g l u c o s a m i n i d a s e ( l y s o s o m e s ) a t d i f f e r e n t t i m e s o f i n c u b a t i o n are listed in T a b l e I. T h e a c t i v i t y o f t h e c o n s t i t u e n t s in t h e f r a c t i o n s is g i v e n as p e r c e n t o f t o t a l r e c o v e r e d a c t i v i t y in t h e g r a d i e n t . T h e r e c o v e r y v a l u e s o f t h e m e a s u r e d c o n s t i t u e n t s in t h e g r a d i e n t s in p e r c e n t o f t h a t l a y e r e d o n t o p initially w e r e 9 3 . 6 % a n d 1 0 9 . 1 % f o r N-acetyl-floglucosaminidase a n d 5 t - n u c l e o t i d a s e , r e s p e c t i v e l y . T h e r e c o v e r y o f r a d i o a c t i v i t y v a r i e d b e t w e e n 9 6 . 2 a n d 9 7 . 5 % . T h e c y t o p l a s m i c e x t r a c t (see t e x t ) l a y e r e d o n t o p o f t h e g r a d i e n t initially c o n t a i n e d in this p a r t i c u l a r e x p e r i m e n t , in p e r c e n t o f t h e a m o u n t in t h e h o m o g e n a t e , 9 5 . 0 % o f t h e N-acetyl-floglucosa m i n i d a s e , 9 7 . 2 % o f t h e 5~-nucleotidase a n d b e t w e e n 9 9 % (initially) a n d 9 5 % ( a f t e r 1 3 0 m i n ) o f t h e radioactivity.

76 TABLE

I

POSITIONS OF THE PEAKS OF RADIOACTIVITY AND OF MARKER ENZYME A C T I V I T I E S IN SUCROSE GRADIENTS AT DIFFERENT TIMES AFTER EXPOSURE TO 12SI-LABELLED ASIALOFETUIN Cells were incubated with 20 nmol/1 labelled asialofetuin for 10 rain. Extracellular asialofetuin was removed by washing and the cells were reineubated. Samples were taken at the times indicated, homogenized, and fractionated by isopyenic centzifugation. Densities in g/ml of the peak fractions are given. Time (min)

Radioactivity

5'-Nucleotidase

N - A c e t yl-fl-glucosaminidase

10 30 70 100

1.136 1.143 1.148 1.147 1.206 * 1.153 1.203 * 1.165 1.204 *

1.155 1.152 1.158 1.160

1.199 1.202 1.195

130 190

1.206 1.163 1.203 1.163 1.204

* Minor peak.

of the incubation. The peak of activity of glucose-6-phosphatase (marker for the endoplasmic reticulum) was also found in the same region of the gradient as 5'-nucleotidase and the radioactivity (not shown). After a b o u t 1 h of incubation at 37°C, a new peak of radioactivity was found at the position of the lysosomal marker enzyme N-acetyl-fl-giucosaminidase at d = 1.20 g/ml (Fig. 1). This peak increased in size relative to the radioactivity peak at lower density during continued incubation. After 3 h, the peaks were o f the same size. At this time, the total cell-associated radioactivity had decreased to a b o u t 30% of the original amount. Other lysosomal enzymes (acid phosphatase, cathepsin D, cathepsin B) showed the same distribution in the gradient as N-acetyl-fl-glucosaminidase. In order to test the effect of different concentrations of asialofetuin on the intracellular distribution, two aliquots of the same cell suspension were each made 4 nmolar in ~2SI-labelled asialofetuin, and to one of them was also added enough cold asialo-fetuin to make the total concentration 50 nmol/1. Samples were removed from both suspensions at 45 and 90 min. The distribution Curves of radioactivity were identical in samples from both cell suspensions after 45 min o f incubation as well as after 90 min (not shown). In order to be able to follow the intracellular transport of asialofetuin b y means of isopycnic or differential centrifugation it is essential to differentiate between asialofetuin b o u n d to the plasma membrane and that contained in endocytotic vesicles (phagosomes). We have shown above that during the first hour after the uptake of asialofetuin, most of the cell-associated radioactivity is outside the lysosomes. These experiments do not, however, tell h o w the labelled protein is distributed between the plasma membrane and the endocytic vesicles inside the cell. The fact that the distribution patterns of radioactivity and the plasma membrane marker enzyme 5'-nucleotidase are quite similar, does n o t necessarily mean that the radioactive protein is b o u n d to the plasma membrane, as phagosomes containing asialofetuin are formed from the plasma membrane and may also contain 5'-nucleotidase. In the present

77 T A B L E II A M O U N T OF M E M B R A N E - B O U N D A S I A L O F E T U I N A cell s u s p e n s i o n c o n t a i n i n g 8 • 1 0 6 c e l l s / m l w a s i n c u b a t e d w i t h 20 n M l a b e l l e d a s a l o f e t u i n f o r 30 r a i n , e x t r a c e l l u l a r r a d i o a c t i v i t y was r e m o v e d b y w a s h i n g , a n d t h e cells w e r e r e i n c u b a t e d at 37°C. T h e a m o u n t o f m e m b r a n e - a s s o c i a t e d asialo-fetuin w a s d e t e r m i n e d as t h e d i f f e r e n c e b e t w e e n ceil-associated r a d i o a c t i v ity in i d e n t i c a l s a m p l e s w h i c h h a d / h a d n o t b e e n t r e a t e d w i t h E G T A . All figures are p e r c e n t o f t h e radioa c t i v i t y in t h e cell s u s p e n s i o n a t t h e s t a r t o f t h e r e i n c u b a t i o n . V a l u e s given are a v e r a g e s o f d u p l i c a t e samples. T h e s t a n d a r d e r r o r in t h e d e t e r m i n a t i o n o f cell-associated r a d i o a c t i v i t y is a p p r o x i m a t e l y 2%. Time

Total cell-associated activity

Membrane-bound radioactivity

S u m o f t o t a l cell-associated radioactivity and phosphot u n g s t i c acid-soluble radioactivity

Start of reincubation After 40 min of reincubation

98.3

8.5

100

67.8

--1.5

97.2

study, we have tried to determine the portion of asialofetuin b o u n d to the plasma membrane b y measuring h o w much of the cell-associated labelled protein could be released b y adding a 50% molar excess over Ca 2÷ of EGTA. It has been shown that the binding of asialo-fetuin to the receptors depends on Ca 2. [1,7] and that asialofetuin b o u n d to the outside of the cells is released when Ca 2÷ is removed with EGTA [1]. Internalized asialofetuin, on the other hand, remains cell associated. Table II shows that a b o u t 9% of the radioactivity associated with cells that had been incubated with 20 nmol/1 of asialofetuin for 30 min at 37°C could be released b y means of EGTA. If the cells were washed and reincubated in asialofetuin-free medium, the portion of releasable asialofetuin decreased rapidly, and after 40 min practically no radioactivity could be released b y means of EGTA (Table II). We have also tried to determine the position in sucrose gradients of asialofetuin b o u n d to receptors on the plasma membrane. This was done b y fractionating cells that had been incubated with asialofetuin at 10°C for 30 min. At this temperature almost all the cell-associated radioactivity was releasable with EGTA and therefore probably b o u n d to the plasma membrane. Fig. 2 shows the subcellular distribution of labelled protein after centrifugation in sucrose gradients of homogenates from cells that had been incubated with 20 nmol/1 of asialofetuin at 10°C for 30 rain. In these experiments the isotonic sucrose solution used in the homogenization step was supplemented with 2 mM CaC12 in order to minimize dissociation of asialofetuin from the receptor. The data obtained (Fig. 2) show that the density distribution of radioactivity coincides almost perfectly with that of 5'-nucleotidase and is slightly different from the distribution of radioactivity for cells that were incubated with labelled protein at 37°C for 10 min and then reincubated for 30 rain in fresh medium (Fig. 1). We have previously studied the distribution of radioactivity in fractions prepared by differential centrifugation of homogenates from cells exposed to labelled asialofetuin [2]. These experiments showed that the radioactivity was found in progressively 'heavier' fractions with time of incubation after the cells

78

./"

./

124

o/

,.,.,.-:7\ 1.20

.

lO

1.16 1 E3

1.12

1.08 I

1

I

I

1

5

10 Fraction number

15

I0

2

Fig. 2. D i s t r i b u t i o n s o f r a d i o a c t i v i t y (~), 5 r - n u c l e o t i d a s e (~) a n d a c i d p h o s p h a t a s e (o) in a s u c r o s e g r a d i e n t a f t e r c e n t r i f u g i n g a p o s t - n u c l e a r f r a c t i o n p r e p a x e d f r o m cells t h a t h a d b e e n i n c u b a t e d a t 1 0 ° C f o r 3 0 rain w i t h 2 0 n m o l / l 1 2 S i . l a b e l l e d a s i a l o f e t u i n . T h e a c t i v i t y o f t h e c o m p o n e n t s in t h e f r a c t i o n s is g i v e n as p e r c e n t o f t o t a l r e c o v e r e d a c t i v i t y in t h e g r a d i e n t . T h e o r d i n a t e t o t h e r i g h t represents d e n s i t y o f the f r a c t i o n s in g / c m 2 "(o). R e c o v e r y v a l u e s o f t h e m e a s u r e d c o n s t i t u e n t s : a c i d p h o s p h a t a ~ e , 9 8 . 2 % ; 5P-nucleoti d a s e , 1 1 0 . 3 % ; r a d i o a c t i v i t y , 9 8 . 9 % . T h e p o s t - n u c l e a r f r a c t i o n c o n t a i n e d in percent o f the h o m o g e n a t e values 96.1% of the acid pbosphatase, 92.7% of the nucleotidase and 88.2% of the radioactivity.

had been exposed to the labelled protein. After 60--90 min the distribution of radioactivity resembled that of lysosomal enzymes [2]. We have in the present study repeated these experiments and have in addition measured the plasma membrane marker enzyme 5'-nucleotidase in the fractions. The results are shown in Fig. 3. It can be seen that the relative specific activity of 5'-nucleotidase showed a peak in the particulate (microsomal) fraction. This distribution was relatively unaffected by the duration of the incubation. The radioactivity was, as earlier [2], found in progressively heavier fractions with time of incubation (not shown). Thus, the cell-associated radioactivity shows a distribution pattern after differential centrifugation which early after the uptake of the labelled protein is smaller to that of 5'-nucleotidase (not shown). With time of incubation the two patterns become quite dissimilar (see Fig. 3).

Degradation in vitro by lysosomes previously loaded with 12SI-labelled asialofetuin Hepatocytes were incubated with 12SI-labelled asialofetuin (30 nmol/1) for 10 min. Extracellular protein was removed by washing with fresh medium, and the incubation was continued for 2 h. A lysosome-rich fraction, comprising the usual 'mitochondrial' and 'light mitochondrial' fractions, was obtained by differential centrifugation [2]. This fraction was homogenized in a solution containing sucrose (0.25 M), hydroxyethylpiperazine (20 mmol/1, pH 7.2) and MgC12 (1 mmol/1) and subsequently incubated at 37°C. During the incubation there was a small but measurable increase in acid-soluble radioactivity. This

79

RADIOACTIVITY

M

N

P

S

I

>

"G ,j

L

2

p-AOA

U~

3

5'NUCLEOTIDASE

2

1 E' ij I

0

l

I

I

I I

I

0

I

I

"/.

I

I

I0 0

Fig. 3. I n t r a c e l l u l a r d i s t r i b u t i o n p a t t e r n s o f r a d i o a c t i v i t y a n d o f t h e l y s o s o m a l a m r k e r e n z y m e N - a c e t y l ~ - g l u c o s a m i n i d a s e a n d t h e p l a s m a m e m b r a n e e n z y m e 5 S - n u c l e o t i d a s e . A s a m p l e o f t h e cell s u s p e n s i o n w a s t a k e n 9 0 m i n a f t e r t h e a d d i t i o n o f 1 2 S I - l a b e l l e d a s i a l o f e t u i n t o t h e cells. T h e h o m o g e n i z e d cells w e r e f r a c t i o n a t e d i n t o a n u c l e a r f r a c t i o n (N), a h e a v y m i t o c h o n d r i a l f r a c t i o n (M), a l i g h t m i t o c h o n d r i a l f r a c t i o n ( L ) , a m i c r o s o m a l f r a c t i o n (P) a n d a f i n a l s u p e r n a t a n t (S). T h e a b s c i s s a r e p r e s e n t s r e l a t i v e p r o t e i n content (cumulatively from left to right). The ordinate represents relative specific activity (percentage of total recovered activity divided by percentage of total protein in each fraction). The recovery values of t h e m e a s u r e d c o n s t i t u e n t s as p e r c e n t o f h o m o g e n a t e values: r a d i o a c t i v i t y , 9 8 . 9 % ; N - a c e t y l - ~ - g l u c o s a m i n i dase, 93.8%; 5Lnucleotidase, 111.5%; protein, 97.5%.

increase, which a m o u n t e d to a b o u t 8% of the total initial acid-precipitable radioactivity, could be largely abolished if the lysosome-rich fraction was incubated in hypotonic medium (the control medium diluted with one part of distilled water) or in medium containing 0.1% (v/v) of the detergent Triton X-100 (Fig. 4). Practically no increase in acid-soluble radioactivity could be demonstrated if the soluble or the particulate (microsomal) fractions were incubated under the conditions used for the lysosome-rich fraction.

Uptake of asialofetuin labelled in different parts of the molecule In order to compare the kinetics of degradation of asialofetuin molecules labelled b y different means, it is necessary to establish that the kinetics o f uptake are similar. Fig. 5 shows that this is indeed the case for labelling with 12sI in the tyrosine residues, with [ 14C] methyl groups on the amino groups of lysine residues, and with 3H in the galactose residues o f the carbohydrate moiety.

80

°/° I

I

6

O

/x_ O

0

0

15

30

/.L5 rain 6LO

Fig. 4. D e g r a d a t i o n of 1251_labelled a s i a l o f e t u i n b y l y s o s o m e s in v i t r o . L y s o s o m e s w e r e isolated f r o m cells w h i c h h a d b e e n a l l o w e d to t a k e u p labelled a s i a l o f e t u i n a n d i n c u b a t e d in a n i s o t o n i c m e d i u m (o), a h y p o t o n i c m e d i u m ( e ) or in i s o t o n i c m e d i u m c o n t a i n i n g 0.1% o f the d e t e r g e n t T r i t o n X - I O 0 ( \ ) . T h e o r d i n a t e r e p r e s e n t s increase in acid-soluble r a d i o a c t i v i t y .

100

/ :

o

.,~/I

80

/';;," /,,x" /,'/

-o

/,':/

,.Bd// 20

,/;" 10

20 time (rain

30

40

Fig. 5. U p t a k e o f a s i a l o f e t u i n labelled in t h e p e p t i d e c h a i n w i t h 1251 in i o d o t y r o s i n e ( a ) o r w i t h 14 C in m e t h y U y s i n e (±). a n d w i t h 3 H in the g a l a c t o s e r e s i d u e of t h e c a r b o h y d r a t e m o i e t y (o). T h e initial c o n c e n t r a t i o n o f a s i a l o f e t n i n w a s 30 n m o l f l in all flasks, a n d t h e cell c o n c e n t r a t i o n w a s 107 ceHs/ml. T h e u p t a k e of t h e labelled p r o t e h l is e x p r e s s e d as t h e s u m o f cell-associated r a d i o a c t i v i t y a n d t h e i n c r e a s e in acids o l u b l e r a d i o a c t i v i t y in t h e m e d i u m .

81 When the uptake was expressed as the sum of cell-associated radioactivity plus the increase in acid-soluble radioactivity, the uptake curves were almost identical (Fig. 5).

Degradation of asialofetuin labelled with 3H in the galaetose residues Degradation of the carbohydrate side chain to acid-soluble small molecules starts immediately, and the level of intracellular acid-soluble radioactivity is high throughout the incubation. After a lag of some 40 min, acid-soluble radioactivity is also found in the medium (Fig. 6). In order to study the intracellular localization of degradation products, the post-nuclear supernatant from cells that had been exposed to [3H]galactoselabelled asialo-fetuin for 15 rain was fractionated by isopycnic centrifuging. More than 80% of the radioactivity was found in the upper half of the gradient. A b o u t half o f the radioactivity entered the gradient, b u t there was no separate peak at d = 1.14 g/ml. There was no accumulation of radioactivity at d = 1.20 g/ml; however, a small but reproducible increase in the relative amount of radioactivity in the lower part of the gradient was observed 60--90 min after the exposure to [3H]galactose-labelled asialofetuin (data not shown).

Degradation of asialofetuin labelled with [14C]methyllysine The increase in acid-soluble radioactivity in a cell suspension incubated with asialofetuin labelled with [14C]methyllysine showed a lag period lasting for a b o u t 10 min, followed by a steady rise (Fig. 7). The change in acid-soluble radioactivity resembled that seen for 12SI-labelled asialofetuin, b u t a direct

80 %

^J°

joS

60 50 /.0

15 I]o

25

10

20

0

L-o--o 0



J

/.0

-

i

80

I

min

120

o

/

y

0

0

10

20 rain 30

Fig. 6. U p t a k e and d e g r a d a t i o n o f a s i a l o f e t u i n labelled w i t h 3 H in t h e g a l a c t o s e residue o f t h e c a r b o h y d r a t e side c h a i n . T h e i n i t i a l c o n c e n t r a t i o n o f a s i a l o f e t u i n w a s 4 2 n m o l / 1 . T h e diagram s h o w s t h e c h a n g e w i t h t i m e o f a c i d - p r e c i p i t a b l e r a d i o a c t i v i t y in t h e cells (o), a c i d - s o l u b l e r a d i o a c t i v i t y in the cells (~), a n d a c i d - s o l u b l e r a d i o a c t i v i t y in t h e s u p e r n a t a n t ( e ) . Fig. 7. U p t a k e (o) and d e g r a d a t i o n (e) o f [ 1 4 C ] m e t h y l l y s i n e . l a b e l l e d a s i a l o f e t u i n . T h e l e f t - h a n d o r d i n a t e a p p l i e s t o u p t a k e , t h e r i g h t - h a n d t o d e g r a d a t i o n , m e a s u r e d as the increase o f a c i d - s o l u b l e r a d i o a c t i v i t y . The initial c o n c e n t r a t i o n o f a s i a l o f e t n i n w a s 1 3 nmol/1.

82 comparison of the degree of degradation, measured as increase in the percentage of acid-soluble radioactivity is not possible because N(6)-methyllysine is incompletely soluble in phosphotungstic acid. Discussion We have in the present s t u d y used cell fractionation by means of isopycnic and differential centrifugation with the object of getting information a b o u t the intracellular transport o f asialo-fetuin i n rat hepatocytes. Fractionation in density gradients indicated that the bulk of the cell-associated asialofetuin was outside the lysosomes (or at least in a cell organelle different from that containing the bulk of the acid hydrolases) until a b o u t 60 min after the uptake of the labelled protein had started. Degradation of asialofetuin starts already 10--15 min after the start of its uptake b y the cells, and we have presented data elsewhere [2,3 ] indicating that this early degradation at least partly takes place in the lysosomes. We therefore believe that the early degradation is so effective that the labelled protein is n o t detectable in the lysosome-rich fractions. If one assumes that the degradation of asialofetuin follows the usual heterophagic pathway, then the cell-associated, non-lysosomal asialofetuin can conceivably be b o u n d to the plasma membrane and/or be contained in endocytic vacuoles (phagosomes). The fact that the binding of asialoglycoproteins to their specific receptors is dependent on Ca:+ [1,7], and that the protein may be released from the receptor b y removing free Ca :+ with EGTA can be used to determine the fraction of cell-associated asialofetuin which is bound to the plasma membrane. This m e t h o d showed that the cell-associated labelled asialofetuin became rapidly internalized at 37°C, and practically all the labelled protein was contained in intracellular structures in cells that had been preincubated with asialofetuin (for instance for 10 min) and then reincubated w i t h o u t the labelled protein in the medium for more than 30 min. We believe that these intracellular, non-lysosomal structures containing labelled asialofetuin are phagosomes. That the radioacticity had left the plasma membrane during reincubation in asialofetuin-free medium was also reflected in the density distribution curves for radioactivity and the plasma membrane marker enzyme 5'-nucleotidase. With time of incubation these t w o curves became slightly but distinctly different. Labelled asialo-fetuin b o u n d to the plasma membrane at low temperature and releasable when Ca 2÷ was chelated with EGTA had a density distribution closely similar to 5'-nucleotidase. The dissimilarity between the subcellular distribution of radioactivity (non-lysosomal) and 5'-nucleotidase was seen particularly clearly in subcellular fractions prepared with differential centrifugation o f homogenates from cells that had been incubated with asialofetuin for more than a b o u t 30 min. We have found earlier that the rate of uptake of asialofetuin b y the cells exceeds the rate of degradation at all concentrations of asialofetuin. Consequently, asialofetuin accumulates in the cells until the extracellular supply is exhausted. The reason for this accumulation is not, however, that the lysosomal degradation as such does n o t keep pace with the uptake [2,3]. The data obtained in the present study indicate that asialofetuin accumulates in endo-

83 cytic vesicles and that the pathway from phagosomes to lysosomes is rate limiting for the cellular degradation of asialofetuin (at least for the first hour after the uptake has started). Phagosomes evidently start to fuse with lysosomes early after their formation at the plasma membrane, as indicated by the release of acid-soluble radioactivity already 10--15 min after the start of the uptake. The capacity of this pathway from endocytic vesicles to lysosomes seems, however, to be rather limited as noted previously [ 2,3]. The rate of transport of material from phagosomes to lysosomes is either directly dependent on the ease with which the membranes of the two organelles fuse with each other, or the intracellular movements which make contact between the two organelles possible. The movement of these organeUes may depend on intact microfilaments or microtubuli, and that such movements may be a rate-determining step in heterophagy is indicated by our recent finding that colchicine and cytochalasin B inhibit the degradation of asialo-fetion and asialo-orosomucoid in rat hepatocytes (Kolset, S.O. and Tolleshaug, H., unpublished data). By incubating cells for 2 h, we may 'load' the lysosomes with asialofetuin, and, by isolating the lysosomes by differential centrifuging and incubating them in vitro, we may show that degradation of labelled asialofetuin actually takes place in the lysosomes. Furthermore, this degradation is inhibited by the detergent Triton X-100 and by a hypotonic incubation medium. Therefore degradation is dependent on intact lysosomes. The accumulation of labelled protein in the lysosome-rich fractions observed after prolonged incubation (more than 1 h) may perhaps be a consequence of suboptimal physical conditions for proteolysis inside the lysosomes. When we exposed cells to asialofetuin labelled in the galactase residue of the carbohydrate side chain, no 'lysosomal' peak was observed in sucrose gradients after 90 min of incubation. This result is in agreement with the data of Aronson and de Duve [19] on the degradation of asialofetuin. They concluded that the galactose residue is split off first. In living cells, [3H]galactose may be reutilized as such, or it may be converted to other hexoses without loss of the label in the C-6 hydroxymethyl group. This may explain the unusual lag period of 40 min before release of acid-soluble radioactive material to the medium. In a previous study [2], we have established that binding of asialofetuin to isolated hepatocytes and internalization of the bound protein are relatively rapid processes. In this study, we have tried to form a picture of the intracellular events that follow binding of the protein. We believe that the protein enters the cytoplasm as a part of an entity which is derived froin the plasma membrane and subject to intracellular processing. The existence of a lag period of some 15 min before the onset of degradation indicates that the protein has to be transported through the cytoplasm to the lysosomes. When asialofetuin has entered the lysosomes, degradation is, however, rapid.

Acknowledgements This investigation was supported by the Norwegian Research Council for Science and the Humanities. The authors wish to express their gratitude to Ms. Turid Ose and Ms. Kari Holte for expert technical assistance.

84 References 1 Ashwell, G. and Morell, A.G. (1974) Adv. Enzymol. 41, 99--128 2 Toneshaug, H., Berg, T., Nilsson, M. and Norum, K.R. (1977) Biochim. Biophys. A c t a 499, 73--84 3 Tolleshaug, H., Ose, T., Berg, T., Wandel, M. and Norum, K.R. (1978) in Kupffer Cells and Other Liver Sinusoidal Cells (Wisse, E. and Knobk, D.L., eds.), pp. 333--341, Elsevier/North-Holland Binmedical Press, A m s t e r d a m 4 Nilsson, M. and Berg, T. (1977) Biochim. Binphys. Acta 4 9 7 , 1 7 1 - - 1 8 2 5 Berry, M. and Friend, D.S. (1969) J. CeB Biol. 43, 506--520 6 LaBadie, J.H., Chapman, K.P. and Aronson, N.N. (1975) Biochem. J. 152, 271--279 7 Pricer, W.E. and Ashwell, G. (1971) J. Biol. Chem, 246, 4 8 2 5 - - 4 8 3 3 8 Tlippy, H. and Gottschalk, A. (1972) in G l y c o p r o t e i n s (Gottschalk, A., ed.), 2rid edn., p. 440, Elsevier, A m s t e r d a m 9 Frantz, W.L. and Turkington, R.W. (1972) Endocrinolo gy 91, 1 5 4 5 - - 1 5 5 2 10 Means, G.E. and Feeny, R.E. (1968) Biochemistry 7, 2192--2201 11 Morell, A.G., van den Hamer, C.J.A., Scheinberg, I.H. and Ashwell, G. (1965) J. Biol. Chem. 241, 3 745--3749 12 EI-Aser, A.A. and Reid, E. (1969) Histoehem. J. 1 , 4 1 7 - - 4 3 7 13 Morr6, D.J. (1971) Methods Enzymol. 22, 138 14 Barrett, A.J. (1972) in Lysosomes, a Laboratory H a n d b o o k (Dingle, J.T., ed.), pp. 111--125, NorthHolland Publ. Co., A m s t e r d a m 15 Ames, B.N. (1966) Methods Enzymol. 8, 115--116 16 Barrett, A.J. (1972) Anal. Biochem. 47, 280--293 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 18 Van Lenten, L. and Ashwell, G. (1972) J. Biol. Chem. 247, 4633---4640 19 Aronson, N.N. and de Duve, C. (1968) J. Biol. Chem. 243, 4 5 6 4 - - 4 5 7 3

Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes.

71 Biochimica et Biophysica A cta, 585 ( 1979) 71--84 © Elsevier/North-Holland Biomedical Press BBA 28904 I N T R A C E L L U L A R L O C A L I Z A...
807KB Sizes 0 Downloads 0 Views