Transport of acid phosphatase to lysosomes does not involve passage through the cell surface

Vol. 170, No. :3, 1990 August

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16, 1990

Transport

of Acid Phosphatase Passage through

1067-1073

to Lysosomes Does Not Involve the Cell Surface

Yoshitaka Tanaka, Shinji Yano, Koji Furunol, Toyoko Ishikawa, Masaru Himeno, and Keitaro Katoz Division of Physiological Chemistry, Faculty of Pharmaceutical Fukuoka 8 12, Japan Received

J'une 21,

Sciences, Kyushu University,

1990

SUMMARY: In foregoing studies, we reported that LGPl07, a major lysosomal membrane glycoprotein in the rat liver, distributes in and circulates continuously throughout the endocytic membrane system (endosomes, IySOSOmeS and plasma membrane), in hepatocytes (1,2). In the present study we examined whether acid phosphatase (APase), an enzyme that is transported to lysosomes as a transmembrane protein, passes through the cell surface during intracellular transport, because transport of newly synthesized APase to lysosomes involves the passage of endosomes containing a ligand which is internalized via receptors on the cell surface and is finally dispatched to lysosomes for degradation (3). When localization of APase in rat hepatocytes was investigated by immunoelectron microscopy, APase was found to be localized in lysosomes and endosomes, but not in coated pits on the cell surface, which are positive for LGPlO7, and from which antibodies for LGP107 are internalized. Further, unlike LGP107, newly synthesized APase was not detected in plasma membranes isolated from livers of rats given [~Slmethionine, and when cuttured hepatocytes were exposed to 1251-labeled anti APase IgG at 37°C there was no transfer of the antibody to lysosomes even after 24 h incubation. Therefore, these results indicate that intracellular movement of APase does not involve cell surface passage in rat hepatocytes, and clearly differs from the recent report that human APase is transported to lysosomes via the cell surface in BHK cells transfected with its cDNA (4) . 0 1990 Academic mess, 1°C.

The biogenesis components. segregation

of lysosomes

involves the selective

transport

of soluble

and membrane

In particular, the formation and maintenance of lysosomes is apparently regulated by or by selective sorting mechanisms of lysosomal membrane proteins from other cellular

components. Transport of lysosomal membrane glycoproteins to lysosomes is independent phosphate-receptors

system (5). which is responsible

for the targeting

of the mannose 6-

of soluble

lysosomal

hydrolases to lysosomes, their final destination (6). It is considered that newly synthesized lysosomal membrane glycoproteins are delivered to lysosomes from the most trans side of Golgi, referred to as trans Golgi network, where they are sorted from proteins destined to be transported surface (7).

On the other hand,

lysosomal membrane glycoproteins

‘Present address: Faculty of Pharmaceutical 729-02, Japan. zTo whom correspondence

to the cell

such as LEPlOO in chicken

Sciences, Fukuyama University, Fukuyama, Hiroshima

should be addressed.

Abbrevtattu APase, acid phosphatase; LGP107, 107kDa lysosomal membrane glycoprotein; IgG, lmmunoglobulin G; HRP, horseradish peroxidase; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0006-291X/90 1067

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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fibroblasts (8,9) and LGP107 in rat hepatocytes (1,2) resides in lysosomes, endosomes and the cell surface and are recycled between the cell surface and lysosomes, via endosomes. Acid phosphatase

(APase) is also transported as a transmembrane

protein to lysosomes in a

mannose 6-phosphate

receptors-independent

manner (10,l I). After delivery to lysosomes, APase

undergoes

of the COOH-terminal

membrane anchoring

processing

domain, resulting in a soluble

form (1 O-1 3). We found that in rat hepatocytes, newly synthesized APase is delivered from the Golgi complex to lysosomes via endosomes,

which contain a ligand internalized

by receptor-mediated

endocytosis (3). Other investigators noted that transport of human lysosomal APase to lysosomes includes the passage through the cell surface in BHK cells transfected with its cDNA (4). We have now addressed the question of whether or not the intracellular pathway of APase involves passage through the cell surface in rat hepatocytes, by making a comparison with finding’s with LGPI 07, which constitutively recycles between the cell surface and lysosomes via endosomes in rat hepatocytes (1,2). We obtained evidence that APase does not pass through the cell surface during intracellular transport.

EXPERIMENTAL

PROCEDURES

Cell Culture: Hepatocytes were isolated from male Wistar rats (200 g), using the collagenaseperfusion procedure originally described by Seglen (14). Antibodies: Antibodies against APase were raised in a goat and those against LGPI 07 were raised in rabbits, respectively, as described (1516). Preparation of labeled Proteins: The HRP-Fab’ conjugate was prepared according to the procedure outlined by lshikawa et a/. (17). lodination of IgG (0.5 mg) was carried out with Nalzsl (0.5 mCi) (Amersham, U.K.) and Enzymobead Radioiodination Reagent (Bio-Rads Lab., Cali., U.S.A.). The antibody’s speclii radioactivity was -800 cpm per ng protein. Immunoperoxidase E/e&on Microscopy: Hepatocytes were fixed first at 4°C with a solution of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 10 min, and subsequently with another solution containing 4% paraformaldehyde, 0.01 M sodium periodate, and 0.075 M L-lvsine in 0.375 M sodium DhOSDhate buffer IDH 6.5) for 4 h (18). The cells were treated for 3 h in cold 7% sucrose, 10% rabbii nonimmune serum,‘and O.O25%‘saponin in 0.1 M sodium phosphate buffer (pH 7.4). then they were exposed to HRP-anti APase Fab’ or HRP-anti LGP107 Fab’ for 12 h. After several rinses with a cold 0.1 M sodium phosphate buffer (pH 7.4), the cells were fixed for 30 min in cold 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The fixed cells were incubated in 0.5 mM 3,3’-diaminobenzidine tetrahydrochloride and 0.005% Hz02 in 0.05 M Tris-HCI buffer (pH 7.5). The cells were then postfixed for 1 h with 1% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.4) and embedded in epoxy resin after dehydration treatment. The sections were examined with and without uranyl stain, using a JEM-1200EX electron Microscope. Preparation of S&cellular fractionation and Immunoprecipitation: Male Wistar rats (200-250 g) were used. Plasma membrane fractions were prepared from livers of ether anesthesized rats killed at definite intervals following intravenous administration of [35S]methionine (250 f&i/l00 g body weight) (Amersham, U.K.), by the procedure of Hubbard et a/. (19). The plasma membrane fractions were brought to 1% Triion X-l 0010.5% deoxycholatel0.15 M NaCV2 mM EDT&10 mM Tris-HCI (pH 7.5) and 10 ug/ml of protease inhibitors (leupeptin, chymostatin, pepstatin A and antipain) were added. After centrifugation, the resulting supematants were preincubated with nonimmune IgGSepharose 48 for 1 h at 4°C. After centrffugation, the supernatants were incubated with anti APase IgG-Sepharose 46 or anti-LGP107 IgG-Sepharose 48 for 16 h at 4°C. The beads were sedimented, and washed five times with 1% Triton X-100/0.5% deoxycholate/0.15 M NaCV2 mM EDTA/O.l% BSAJIO mM Tris-HCI (pH 7.5) (buffer A), and five times with buffer A containing 2 M KCI and then twice with 0.1% SDS/O.S% Triton X-100/0.5 M NaCVlO mM Tris-HCI (pH 8.5). The sedimented beads were treated for 3 min at 100°C with 3% SDS/5% P-mercaptoethanoM mM EDTA/50 mM Tris-HCl (pH 6.8)/l 0% glycerol and then centrifuged. The supematants were analyzed by SDS-PAGE (10% gel) (20). Radioactive bands were detected by fluorography using EN3HANCE on Kodak XAR-5 film. Assay of ‘%/gG Degradation: 1251-lgG in 0.55 ml of medium was added to cell monolayers on 35 mm tissue culture dishes. Following incubation at 37°C the medium was removed and the degradation of lsl-lgG was determined in a gamma counter by measuring radioactivity in the medium, soluble in 7% trichloroacetfc acid.

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RESULTS Localization

of LGP107

One day-cultured cytochemical

and

APase

hepatocytes

in Cultured

Hepatocytes

on collagen-coated

plastic dishes were fixed and subjected to

staining for LGP107 (Fig. 1) and for APase (Fig. 2), at the ultrastructural

immunoperoxidase were observed morphological

techniques.

As shown in panel a of Fig. 1, immunoreactive

in coated pits in the ventral membrane

products for LGP107

facing the substratum,

features noted in hepatocytes cultured on plastic dishes.

level, using

that is the same

In addition, LGP107 was

localized in small vesicles at peripheral structures lying close to the ventral membrane (Fig. 1 b) and in relatively

larger vesicles (-700

complex in the deeper

nm) containing

numerous

regions (Fig. lc), presumably

inclusion

representing

vesicles close to the Golgi early and late endosomes,

respectively. Various types of lysosomes were also positive (Fig. 1 d). Thus, LGP107 distributes in all compartments observations

involved in endocytic

vacuolar systems,

finding’s consistent

with our previous

(1).

On the other hand, immunoreactive staining for APase was rare in the coated pits (Fig. 2a) and in vesicular

structures close to the ventral membrane.

The labeling was predominantly

endosomes close to the Golgi complex and in lysosomes (Fig. 26 and c, respectively). suggested

two possible explanations;

visible in

These results

one is that newly synthesized APase is not transported to the

cell surface, and the other is that APase does not recycle between the cell surface and lysosomes, as was noted with

LGP107

(1,2).

For elucidation,

we designed

two independent

experiments

as

follows. Newly

synthesized

APase

is not transported

to the cell surface

We reported that part of the newly synthesized APase is delivered to lysosomes even 30 min after synthesis (11). To examine whether the newly synthesized APase is transferred to the cell surface prior to delivery to lysosomes, plasma membranes were isolated from rat livers at 30 and 60 min after the administration immunoprecipitated

of [35S]methionine,

after which APase and LGP107 were separately

from the detergent-extracted

plasma membranes and analyzed by SDS-PAGE,

followed by fluorography.

As shown in Fig. 3. LGP107 was detected as a 107kDa form at 30 and 60

min after the injection of [35S]methionine

(lanes, 1 and 2). However, no immunoprecipitable

APase

band was found at either time (lanes, 3 and 4). Therefore, transport of newly synthesized APase to lysosomes does not involve passage through the cell surface. APase

does

not recycle

between

the cell surface and Iysosomes

To further investigate whether APase recycles between the cell surface and lysosomes, cells were continuously

the

exposed, separately, to either 1z51-labeled anti LGP107 IgG or 1251-labeled

anti APase PgG for up to 24 h, and transport of the antibody to lysosomes measuring the amount of degradation

was determined

of Q5l-IgG released into the medium (TCA-soluble

by

radioactive

products). As shown in Fig. 4, the degradation of 1251-anti LGP107 IgG increased linearly up to 24 h, amounting to about 8% of the administered amount afler 24 h of incubation. However, under these conditions, there was no significant increase in the degradation of lzsl-anti APase IgG, which was at the same level as that of nonimmune IgG. 1069

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EieL, lmmunochytochemical localization of LGP107 in cultured rat hepatocytes. Cuttured hepatocytes were fiied and labeled for LGP107 by immunoperoxidase cytochemistry. a shows a horizonal thin section of the ventral membrane of a hepatocyte rich in coated pits. Reaction products are seen in coated pits but not along the celluhr membrane. In b, the labeling is also visible in peripherally localized endosomes (arrows) below the surface of the ventral membrane and the reactive endosomes become larger in size in the deeper regions (arrowhead). c and d show immunoreactiie structures located in the internal portion of hepatocytes. (c) Reaction products are seen in inclusion vesicles as well as on the surrounding membranes in multiiesicular vacuoles close to the Golgi apparatus (G). (o) Reaction products are seen on the amorphous materials in the matrix as well as along the limiting membrane in those secondary lysosomes (L). Bar, 1 pm. (a) - (c), x25,000; (d), x12,000.

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u lmmunocytochemtcal localization of APase in cultured rat hepatocytes. Cultured hepatocytes were fixed and labeled for APase by immunoperoxidase cytochemistry. a. b and c show portions corresponding to a, c and d of Fig. 1, respectively. (a) lmmunoreactiin staining of APase is rare in coated pits on the cell surface. (b) The labeling is found in inclusion vesicles close to the Golgi apparatus (G). (c) Reaction products are seen on the amorphous materials in the matrix as well as along the limiting membrane in those secondary lysosomes (L). Bars, 1 urn. (a) and (b), x25000; (c), xl 2.000.

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LGP107 2

3

4

03

04

Time

(h)

E@& Appearance of biosynthetically labeled APase and LGP107 in plasma membranes. [WSjMethionine (250 pCci/lOO g body weight) was administrated intravenously to rats at 30 (lanes 1 and 3) and 60 min (lanes 2 and 4), livers were excised and fractionated as described in “EXPERIMENTAL PROCEDURES”. APase (lanes 1 and 2) and LGPl07 (lanes 3 and 4) were separately immunoprecipitated from plasma membrane fractions and analyzed on SDS-PAGE followed by fluorography. E&& Kinetics of degradation of lz51-anti LGP107 IgG and lz51-anti APase IgG by hepatocytes. Cultured hepatocytes were incubated for the times indicated at 37°C independently with ‘25l-labeled IgG for LGP107 (0) or for APase (o), or ‘Bl-nonimmuna IgG (x) at the concentration of 5 wml. After acid-soluble the incubation, the medium was removed and the amount of 1251-labaled trichbroacetic materials was measured.

DISCUSSION The experiments the cell surface We passage after

reported through

synthesis,

synthesized

described

during

that

newly

the Golgi and

herein

its intracellular

were

designed

movement

synthesized

complex

(3),

is completed

APase arrival

within

to test whether

or not APase

passes

through

in rat hepatocytes. is delivered

of APase

3 h (11).

to lysosomes

at the lysosomes In the

present

via endosomes is evident

study,

after

from

transport

30 min of newly

APase to the cell surface was not detected at either 30 or 60 min after pulse-labeling

(Fig. 3). It is, therefore, evident that newly synthesized APase does not traverse the cell surface en route to lysosomes.

On the other hand, the time observed for arrival of the newly synthesized

LGP107 at the plasma membranes as shown in Fig. 3 (30 min) was the same as that for the arrival at the lysosomes (data not shown), thereby suggesting

that the newly synthesized LGP107

transported to lysosomes along two different pathways; one is that LGPl07 lysosomes from

the Golgi complex

delivered

to the

Cell

endocytic

pathway.

surface

via

through endosomes,

endosomes

and

then

1072

may be

is directly transported to

and the other is that LGP107

is transported

to lysosomes

through

is first the

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The possibility of recycling APase between the cell surface and lysosomes was examined using the same protocal as that for our previous study (1,2).

In contrast to LGP107,

APase was not

localized in the coated pits on the cell surface and the transport of antibody for APase to lysosomes was not detected even when the cells were exposed to the antibody at 37°C continuously for up to 24h. Therefore,

it is most likely that in rat hepatocytes,

APase does not constitutively

circulate

between the cell surface and lysosomes. Braun, e/a/. (4) reported that newly synthesized human lysosomal APase expressed in BHK cells transfected with the APase cDNA is transported to dense lysosomes via the cell surface. In this case, APase is transferred from the transQolgi

to the cell surface within 45 min and recycles between the

cell surface and endosomes with a half-time of 5-6 h before being transferred to dense lysosomes. Further, they noted that APase molecules on the cell surface are internalized

via clathrin-coated

Their observations differ from our results presented here and from our previous observations

pits. in that

newly synthesized APase was seen delivered to lysosomes within 3 h after synthesis (11). These differences may relate to cell lines or to the experimental

design.

APase in BHK cells was at least P-fold higher than the untransfected

The fact that the expression of cells suggests to us that the

normal APase delivery system might be perturbed because of overexpression overexpressed

of the protein, and the

APase would result in delivery to the cell surface.

We tentatively conclude that in rat hepatocytes, APase is transported from the ER through the Golgi complex to lysosomes via endosomes and without passing through the cell surface.

ACKNOWLEDGMENT:

We wish to thank M. Ohara for helpful comments.

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