Vol. 166, No. 3, 1990 February 14, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1176-1182
LYSOSOMAL ACID PHOSPHATASE IS TRANSPORTED VIA ENDOSOMES TO LYSOSOMES
Yoshitaka
Department
Tanaka, Shinji Yano, Masaru Himeno, of Physiological Sciences,Kyushu
Kazutaka Okada. Toyoko and Keitaro Katol
Chemistry, University,
Ishikawa,
Faculty of Pharmaceutical Fukuoka 812, Japan
Received December 4, 1989 Involvement of endosomes in transport of newly synthesized acid phosphatase to lysosomes was investigated using the Golgi fraction (GF1+2), enriched in endosomes. The Golgi fraction (GF1+2) was prepared from the livers of rats given [3sSlmethionine and asialofetuin conjugated-horseradish peroxidase (HRP). Newly synthesized acid phosphatase in the endosomes containing internalized asialofetuin-HRP was measured as a loss of the detectable labeled enzyme after 3,3'diaminobenzidine (DAB) and HzOz reaction, due to formation of insoluble polymers which reduce protein antigenicity. With this procedure, acid phosphatase was all but undetectable in the Golgi fraction. Thus, newly synthesized acid phosphatase is apparently transported to lysosomes by endosomes. Q1990Academic Press. Inc. Acid
phosphatase
lysosomes soluble
as
3).
lysosomes 6-P)
by
the
domain
Unlike
through
the
(4-6).
APase
manner
reported
synthesized
APase
is
evidence in
rat
liver
then
is
converted of
of
which the
transported
are
to to
moieties
translocated
into
mannose-g-phosphate to lysosomes
(l-
(Manvia
Man-g-
(3,7). of
the
Golgi
accumulation fractions,
of obtained
newly using
1 To whom correspondence should be addressed. Abbreviations: APase, acid phosphatase; HRP, horse radish DAB, 3,3'-diaminobenzidine; peroxidase: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Man-6-P. mannose-6ASGPR, asialoglycoprotein receptor; MPR, mannose-6phosphate; TfR, transferrin receptor. phosphate receptor; 0006-291X/90 $1.50 Copyright All rights
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
a
COOH-terminal
of carbohydrate
enzymes
mediation
and transported
proteolysis
and processing
P receptor-independent We
synthesized
enzyme,
limited
many lysosomal
receptor
is
a membrane-bound
form
transmembrane
(APase)
1176
Vol.
166, No. 3, 1990
subcellular
BIOCHEMICAL
fractionation
labeling
sites endosomes,
APase but
the
are
with
Golgi
region
liver
Golgi
fractions,
that
(GF2), at
of
time
internalized
fractions in All
(9).
We
of have
delivered
Golgi
endocytic these
involvement
staining
to
bodies transmost
and
cisternae
network
in
the
Golgi
intermediate
based
on
particular
the elements,
internalization,
high
were present of
structures
in
led the
in
that
endosomes
that
of
rat
findings
concentrations rat
liver
internalized
Golgi
ligand
was
galactosyltransferase
to the
transport
evidence via
the
devoid
observations
lysosomes
intracellular
in
majority
now obtained
the
using
reported
after
endosomes
the
tubulovesicular
endosomal
the
contrast,
that
in
pulse-
Kay and co-workers
[ 12BI]insulin
and that
present
is
contain
an early
In
with
multivesicular
and the (8).
RESEARCH COMMUNICATIONS
conjunction
(7).
lysosomes,
apparatus
Golgi
in
we observed
no strong
trans
fraction
*
In
microscopy
of
of
techniques
experiments
immunoelectron
AND BIOPHYSICAL
idea
of
of APase newly after
to
a
possible
lysosomes.
synthesized passage
APase
through
the
complex.
MATERIALS
AND METHODS
Materials: L-[35Slmethionine (1000 Ci/mole) was purchased Amersham. EN3HANCE was obtained from New England Nuclear Co. (type VI) was purchased from the Sigma Chemical Company. Preparation HRP conjugate al. (10).
of --
the asialofetuin-HRF! was prepared according
conjugate: The asialofetuinto the method of Ishikawa
Antibodies: The major soluble form of APase (C-APase purified from rat liver lysosomal contents and antibodies the enzyme were raised in a goat, as described (11).
from HRP
et
I) was against
Electron microscopy: Under anesthesia with Nembutal, rats were given asialofetuin-HRP (50 fig/100 g body weight) intravenously, and 10 min later the livers were fixed by perfusion through the portal vein with 0.1 M cacodylate buffer (pH 7.4) containing 2% glutaraldehyde. The tissue was additionally fixed by immersing in the same fixative at 4-C for 2 h, washed with ice cold 0.1 M cacodylate buffer (pH 7.4). The f!O - 30 Drn sections were cut with a cryostat microtome. The sections were incubated in DAB solution (lmg/mll in 50 mM Tris-HCI buffer (pH 7.0) for 20 min at room temperature and then with HZOZ (final concentration of 1177
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.01%) for 30 min. The sections were postfixed with 1% O-O4 at 4-C for 30 min. dehydrated through a graded series of ethanol, and embedded in Epon. Ultrathin sections were stained with lead citrate and examined under a JEM 1200 EX electron microscopy.
Preparation of used. g) were [35Slmethionine intervals were g body weight) animals were asialofetuin-HRP, interface of of Howell and et al. (13).
subcellular fractions: Male Wistar rats (200 - 250 Under anesthesia with ether, rats given were (500 /1Ci/lOO g body weight) and at definite further injected with asialofetuin-HRP (50 ug/lOO at 10 min before the end of pulse-labeling. The decapitated 10 min after the injection of the livers removed and GF1+2 (floating at the 0.25/0.86 M sucrose) was isolated by the procedure Palade (12), who modified the method of Ehrenreich
DAB treatment and APase detection: GF1+2 was incubated with DAB in the presence or absence of HzOz as described by Courtory et (14). The reaction mixtures were brought to 1% SDS/0.5% 2al Triton X-100/0.15 M NaC1/2 mM EDTA/lO mM Tris-HCl (pH 7.5) and 10 ,ug/ml of protease inhibitors (leupeptin, chymostatin, pepstatin A and antipain) were added. After centrifugation, the resulting supernatants were preincubated with nonimmun IgG-Sepharose 4B for 1 h at 4-C. After centrifugation, the supernatants were incubated with anti-APase IgG-Sepharose 4B 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 NaC1/2 mM EDTA/O.l% BSA/lO mM Tris-HCl (PH 7.5) (buffer A), and five times with buffer A containing 2 M KC1 and then twice with 0.1% SDS/O.S% Triton X-100/0.5 M NaCl/lO mM Tris-HCl (pH 7.5). The sedimented beads were treated for 3 min at 1OO'C with 3% SDS/5% 2-mercaptoethanol/2 mM EDTA/SO mM Tris-HCl (pH 6.8)/10% glycerol and then centrifuged. The supernatants were (10% gel) (15). Radioactive bands were analyzed by SDS-PAGE detected by fluorography using EN=HANCE on Kodak XAR-5 film.
RESULTS AND DISCUSSION
In
the
injection
Golgi
of
fraction
[ 1Z61]-asialofetuin.
was 150 - 170-fold injection mainly in
of found
Fig.
injection To during performed.
in
Thus,
the
is
predominantly
the
biosynthetic
of
not
ligand
DAB
in in
the
Golgi
GF1+2 at
or not
APase
passes
the
following
injected
from with
pulse-labeling. 1178
the
after
product
was
complex
as
shown
min
after
the
10
endosomal
elements. through
endosomes was
experiment
livers
of
asialofetuin-HRP, GF1+2
the
radioactivity
reaction
from
isolated
the
after
At 10 min
derived
pathway,
was
and end
but
of
homogenate. the
whether
GF1+2
the
that
endosomes
1.
10 min
isolated
enrichment
asialofetuin-HRP,
[36Slmethionine before
over
determine the
(GF1+2)
thus
given
rats at prepared
10
min was
Vol.
166,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
in rat Fig. 1 Intracellular distribution of asialofetuin-HRP hepatocytes. At 10 min after the intravenous administration of asialofetuin-HRP hepatocytes were fixed and processed to rats, using HRP-DAB labeling as outlined in "Materials and Methods". The HRP reaction products were seen in endosomes, including small vesicles close to the bile canaliculus (BC). Asialofetuin-HRP was not evident in the Golgi stacks (G). Ly. lysosome; N, nucleus. Bar = 1 pm, x 22,000.
incubated
with
DAB in
HzOs-catalyzed the
oxidation
potential the
APase
encounters
When
GFl+Z
(16).
would
isolated
[36Slmethionine
at
of
reduction
in
isolated
at
the
proteins
by trapping newly
asialofetuin-HRP,
30 or 60 min
after
the
administration
(Fig.
with
DAB
form, 2,
with lanes
DAB and HZ02
at
60
min 1179
them
synthesized
DAB-HzOz
(1)
and
if
HRP-
diminishes
the
immunoprecipitablee 30
The
after
a 67-kDa
with
H202.
be observed
in
GF1+2
of
vesicles
containing
incubated
chains
incubation
the
Therefore,
was
immunoprecipitated oligosaccharide
DAB within
endosomes
APase
or absence
intravesicular
DAB polymer
in
presence of
to extract
within
reduction
the
alone, complex
1 and 3).
in
resulted
APase
molecules
after
the
a reaction. of
APase type In
was
N-linked contrast,
a considerable from
GF1+2
injection
of
Vol.
166, No. 3, 1990
BIOCHEMICAL
wz
-
AND BIOPHYSICAL
-
f
RESEARCH COMMUNICATIONS
+
Fig. 2 Distribution of the newly synthesized APase in GF1+2 isolated at 10 min after the injection of asialofetuin-HRP to rats. [J5S]methionine (500 bCi/lOO g body weight) was administrated intravenously to rats at the times indicated, and asialofetuin-HRP (50 fig/100 g body weight) was injected at 10 min before end of pulse-labeling. At 10 min after the injection of asialofetuin-HRP, livers were excised and fractionated as described in "Materials and Methods". GF1+2 was then incubated with DAB in the presence (lanes , 2 and 2) or absence (lanes 1 and 3) of HnOz. APase was immunoprecipitated from each sampleand analyzed on SDS-PAGE followed by fluorography.
["5Slmethionine APase 30
(lanes
observed
min
after
APase
immediately
intensity
of
to
mean
that
endosomes
to
is
transport
supported
rapidly
transport lysosomes from by our
is
most
useful
the
Golgi
the
procedure separate
APase
from newly and
complex in
increase
the
can be interpreted
observations developed from
from
APase
as compared
to endosomes.
endosomes 1180
At
synthesized
gradually,
complex
endosomes.
that
Golgi
at 60 min
immunocytochemical
to
indicates
newly
synthesized
labeled
Additionally,
proceeded the
from
of
through
observed
of
The DAB density-shift (14)
passed
band
newly
derived
reaction
DAB-H202
APase
the
disappearance
to endosomes.
the
Thus,
is apparently the
the
synthesized moved
GFl+Z
postinjection,
GF1+2
rapid
in
2 and 4).
This
to
the
notion
(8).
by Courtory
et -2
other
cellular
al
Vol.
166,
No.
3, 1990
organelles
as
density
of
DAB. In
addition,
the
BIOCHEMICAL
there
is
endosomes
is
the
proteins
distinct
receptors,
pathway
(17.18).
evidence
of
increase
formed
or
used
Despite
the
endocytic
TfR
pathway, of
there
proteins.
evidence
that
newly
synthesized
APase
only of
in
Thus, endocytic
lysosomal
it
is
likely
processes
membrane
proteins
but
of in
the
been
advancing
to
no
obtained
present
in
role
of
direct
intracellular
in
and a ligand
the
endocytic
has
also
study
functionally
on the
endosomes
This
to
data
that
of
(16).
We
were
buoyant
renders
groups
endosomes
synthesized
endosomes.
other
and MPR,
newly
endocytosis
the
vesicles
processes
of
receptor-mediated
within
by
transport
by
in
polymerization
accumulating
involvement
the
COMMUNICATIONS
insoluble
sorting
such as ASGPR,
the
RESEARCH
detergent
being
movement
in
specific
DAB polymer
currently
intracellular
endosomes
a
BIOPHYSICAL
due to HRP-HZOz-catalyzed
intravesicular
procedure
AND
herein
internalized in
are
the
same
involved
biosynthetic
not pathway
lysosomes.
Acknowledgments: We wish to thank M. Ohara for helpful This study was supported in part by a Grant-in-Aid Ministry of Education, Science and Culture of Japan.
comments. from the
REFERENCES 1. Himeno, M., Koutoku, H., Ishikawa, T., and Kato, K. (1989) J. Biochem. 105, 449 - 456. 2. Himeno, M., Fujita, H., Noguchi, Y., Kono, A., and Kato, K. (1989) Biochem. Biophys. Res. Commun. 162, 1044 - 1053. Gottschalk, S., 3. Waheed, A., I-Iille, A., Krentler, C., Pohlmann, R., Braulke, T., Hauser, H., Geuze, H., and von Figura, K. (1988) EMBO J. 7, 2351 - 2358. 4. Gabel, C.A., Goldberg, D.E., and Kornfeld, S. (1982) J. Cell Biol. 95. 536 - 578. 5. Fisher, H.D., Creek, K.E., and S.Ly, W.S. (1982) J. Biol. Chem. 257, 9938 - 9981, 6. Natowicz, M., Hallett, D.W., Frier, C., Chi, M., Schlesinger, P.H., and Baenziger, J.U. (1983) J. Cell Biol. 96. 925 934. 7. Himeno, M., Tanaka, Y., and Kato, K. (1988) Cell Struct. Funct. 13, 578. 8. Yokota. S., Himeno, M., and Kato, K. (1989) Cell Struct. Funct. 14, 163 - 171. 9. Kay, D.G., Khan, M.N., Posner, B.I., and Bergeron, J.J.M. (1984) Biochem. Biophys. Res. Commun. 123, 1144 - 1148. 1181
Vol.
10. 11. 12.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Ishikawa, E., Hashida, S., Kohno, T., Kotani, T., and Ohtaki, S. (1986) in Monoclonal Antibodies; Hybridoma Techniques (Schook, L.B., ed.) pp. 113 -137. Marcel Dekker, New York. Himeno, M., Koutoku, H., Tsuji, H., and Kato, K. (1988) J. Biochem. 104, 773 - 776. Howell, K.E.. and Palade, G.E. (1982) J. Cell Biol. 92, 822 - 832.
13. 14. 15. 16.
and Ehrenreich, J.H., Bergeron, J.J.M., Seikevitz, P., Palade. G.E. (1973) J. Cell Biol. 59, 45 - 72. Courtoy, P.J., Quintart, J., and Baudhuin, P. (1984) J. Cell Biol. 98, 870 - 876. Laemmli. U.K. (1970) Nature. 227, 680 - 685. Ajioka, R.S., and Kaplan, J. (1987) J. Cell Biol. 104, 77 85.
17. 18.
Geuze. H.J., Stoorvogel, W., Strous, G.J., Slot, J.W., Bleekemolen, J.E., and Mellman, I. (1988) J. Cell Biol. 107, 2491 - 2501. Stoorvogel, W., Geuze, H.J., Griffith, J.M., Schwartz, A.L., and Strous, G.J. (1989) J. Cell Biol. 108, 2137 - 2148.
1182
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1176-1182
LYSOSOMAL ACID PHOSPHATASE IS TRANSPORTED VIA ENDOSOMES TO LYSOSOMES
Yoshitaka
Department
Tanaka, Shinji Yano, Masaru Himeno, of Physiological Sciences,Kyushu
Kazutaka Okada. Toyoko and Keitaro Katol
Chemistry, University,
Ishikawa,
Faculty of Pharmaceutical Fukuoka 812, Japan
Received December 4, 1989 Involvement of endosomes in transport of newly synthesized acid phosphatase to lysosomes was investigated using the Golgi fraction (GF1+2), enriched in endosomes. The Golgi fraction (GF1+2) was prepared from the livers of rats given [3sSlmethionine and asialofetuin conjugated-horseradish peroxidase (HRP). Newly synthesized acid phosphatase in the endosomes containing internalized asialofetuin-HRP was measured as a loss of the detectable labeled enzyme after 3,3'diaminobenzidine (DAB) and HzOz reaction, due to formation of insoluble polymers which reduce protein antigenicity. With this procedure, acid phosphatase was all but undetectable in the Golgi fraction. Thus, newly synthesized acid phosphatase is apparently transported to lysosomes by endosomes. Q1990Academic Press. Inc. Acid
phosphatase
lysosomes soluble
as
3).
lysosomes 6-P)
by
the
domain
Unlike
through
the
(4-6).
APase
manner
reported
synthesized
APase
is
evidence in
rat
liver
then
is
converted of
of
which the
transported
are
to to
moieties
translocated
into
mannose-g-phosphate to lysosomes
(l-
(Manvia
Man-g-
(3,7). of
the
Golgi
accumulation fractions,
of obtained
newly using
1 To whom correspondence should be addressed. Abbreviations: APase, acid phosphatase; HRP, horse radish DAB, 3,3'-diaminobenzidine; peroxidase: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Man-6-P. mannose-6ASGPR, asialoglycoprotein receptor; MPR, mannose-6phosphate; TfR, transferrin receptor. phosphate receptor; 0006-291X/90 $1.50 Copyright All rights
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
a
COOH-terminal
of carbohydrate
enzymes
mediation
and transported
proteolysis
and processing
P receptor-independent We
synthesized
enzyme,
limited
many lysosomal
receptor
is
a membrane-bound
form
transmembrane
(APase)
1176
Vol.
166, No. 3, 1990
subcellular
BIOCHEMICAL
fractionation
labeling
sites endosomes,
APase but
the
are
with
Golgi
region
liver
Golgi
fractions,
that
(GF2), at
of
time
internalized
fractions in All
(9).
We
of have
delivered
Golgi
endocytic these
involvement
staining
to
bodies transmost
and
cisternae
network
in
the
Golgi
intermediate
based
on
particular
the elements,
internalization,
high
were present of
structures
in
led the
in
that
endosomes
that
of
rat
findings
concentrations rat
liver
internalized
Golgi
ligand
was
galactosyltransferase
to the
transport
evidence via
the
devoid
observations
lysosomes
intracellular
in
majority
now obtained
the
using
reported
after
endosomes
the
tubulovesicular
endosomal
the
contrast,
that
in
pulse-
Kay and co-workers
[ 12BI]insulin
and that
present
is
contain
an early
In
with
multivesicular
and the (8).
RESEARCH COMMUNICATIONS
conjunction
(7).
lysosomes,
apparatus
Golgi
in
we observed
no strong
trans
fraction
*
In
microscopy
of
of
techniques
experiments
immunoelectron
AND BIOPHYSICAL
idea
of
of APase newly after
to
a
possible
lysosomes.
synthesized passage
APase
through
the
complex.
MATERIALS
AND METHODS
Materials: L-[35Slmethionine (1000 Ci/mole) was purchased Amersham. EN3HANCE was obtained from New England Nuclear Co. (type VI) was purchased from the Sigma Chemical Company. Preparation HRP conjugate al. (10).
of --
the asialofetuin-HRF! was prepared according
conjugate: The asialofetuinto the method of Ishikawa
Antibodies: The major soluble form of APase (C-APase purified from rat liver lysosomal contents and antibodies the enzyme were raised in a goat, as described (11).
from HRP
et
I) was against
Electron microscopy: Under anesthesia with Nembutal, rats were given asialofetuin-HRP (50 fig/100 g body weight) intravenously, and 10 min later the livers were fixed by perfusion through the portal vein with 0.1 M cacodylate buffer (pH 7.4) containing 2% glutaraldehyde. The tissue was additionally fixed by immersing in the same fixative at 4-C for 2 h, washed with ice cold 0.1 M cacodylate buffer (pH 7.4). The f!O - 30 Drn sections were cut with a cryostat microtome. The sections were incubated in DAB solution (lmg/mll in 50 mM Tris-HCI buffer (pH 7.0) for 20 min at room temperature and then with HZOZ (final concentration of 1177
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.01%) for 30 min. The sections were postfixed with 1% O-O4 at 4-C for 30 min. dehydrated through a graded series of ethanol, and embedded in Epon. Ultrathin sections were stained with lead citrate and examined under a JEM 1200 EX electron microscopy.
Preparation of used. g) were [35Slmethionine intervals were g body weight) animals were asialofetuin-HRP, interface of of Howell and et al. (13).
subcellular fractions: Male Wistar rats (200 - 250 Under anesthesia with ether, rats given were (500 /1Ci/lOO g body weight) and at definite further injected with asialofetuin-HRP (50 ug/lOO at 10 min before the end of pulse-labeling. The decapitated 10 min after the injection of the livers removed and GF1+2 (floating at the 0.25/0.86 M sucrose) was isolated by the procedure Palade (12), who modified the method of Ehrenreich
DAB treatment and APase detection: GF1+2 was incubated with DAB in the presence or absence of HzOz as described by Courtory et (14). The reaction mixtures were brought to 1% SDS/0.5% 2al Triton X-100/0.15 M NaC1/2 mM EDTA/lO mM Tris-HCl (pH 7.5) and 10 ,ug/ml of protease inhibitors (leupeptin, chymostatin, pepstatin A and antipain) were added. After centrifugation, the resulting supernatants were preincubated with nonimmun IgG-Sepharose 4B for 1 h at 4-C. After centrifugation, the supernatants were incubated with anti-APase IgG-Sepharose 4B 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 NaC1/2 mM EDTA/O.l% BSA/lO mM Tris-HCl (PH 7.5) (buffer A), and five times with buffer A containing 2 M KC1 and then twice with 0.1% SDS/O.S% Triton X-100/0.5 M NaCl/lO mM Tris-HCl (pH 7.5). The sedimented beads were treated for 3 min at 1OO'C with 3% SDS/5% 2-mercaptoethanol/2 mM EDTA/SO mM Tris-HCl (pH 6.8)/10% glycerol and then centrifuged. The supernatants were (10% gel) (15). Radioactive bands were analyzed by SDS-PAGE detected by fluorography using EN=HANCE on Kodak XAR-5 film.
RESULTS AND DISCUSSION
In
the
injection
Golgi
of
fraction
[ 1Z61]-asialofetuin.
was 150 - 170-fold injection mainly in
of found
Fig.
injection To during performed.
in
Thus,
the
is
predominantly
the
biosynthetic
of
not
ligand
DAB
in in
the
Golgi
GF1+2 at
or not
APase
passes
the
following
injected
from with
pulse-labeling. 1178
the
after
product
was
complex
as
shown
min
after
the
10
endosomal
elements. through
endosomes was
experiment
livers
of
asialofetuin-HRP, GF1+2
the
radioactivity
reaction
from
isolated
the
after
At 10 min
derived
pathway,
was
and end
but
of
homogenate. the
whether
GF1+2
the
that
endosomes
1.
10 min
isolated
enrichment
asialofetuin-HRP,
[36Slmethionine before
over
determine the
(GF1+2)
thus
given
rats at prepared
10
min was
Vol.
166,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
in rat Fig. 1 Intracellular distribution of asialofetuin-HRP hepatocytes. At 10 min after the intravenous administration of asialofetuin-HRP hepatocytes were fixed and processed to rats, using HRP-DAB labeling as outlined in "Materials and Methods". The HRP reaction products were seen in endosomes, including small vesicles close to the bile canaliculus (BC). Asialofetuin-HRP was not evident in the Golgi stacks (G). Ly. lysosome; N, nucleus. Bar = 1 pm, x 22,000.
incubated
with
DAB in
HzOs-catalyzed the
oxidation
potential the
APase
encounters
When
GFl+Z
(16).
would
isolated
[36Slmethionine
at
of
reduction
in
isolated
at
the
proteins
by trapping newly
asialofetuin-HRP,
30 or 60 min
after
the
administration
(Fig.
with
DAB
form, 2,
with lanes
DAB and HZ02
at
60
min 1179
them
synthesized
DAB-HzOz
(1)
and
if
HRP-
diminishes
the
immunoprecipitablee 30
The
after
a 67-kDa
with
H202.
be observed
in
GF1+2
of
vesicles
containing
incubated
chains
incubation
the
Therefore,
was
immunoprecipitated oligosaccharide
DAB within
endosomes
APase
or absence
intravesicular
DAB polymer
in
presence of
to extract
within
reduction
the
alone, complex
1 and 3).
in
resulted
APase
molecules
after
the
a reaction. of
APase type In
was
N-linked contrast,
a considerable from
GF1+2
injection
of
Vol.
166, No. 3, 1990
BIOCHEMICAL
wz
-
AND BIOPHYSICAL
-
f
RESEARCH COMMUNICATIONS
+
Fig. 2 Distribution of the newly synthesized APase in GF1+2 isolated at 10 min after the injection of asialofetuin-HRP to rats. [J5S]methionine (500 bCi/lOO g body weight) was administrated intravenously to rats at the times indicated, and asialofetuin-HRP (50 fig/100 g body weight) was injected at 10 min before end of pulse-labeling. At 10 min after the injection of asialofetuin-HRP, livers were excised and fractionated as described in "Materials and Methods". GF1+2 was then incubated with DAB in the presence (lanes , 2 and 2) or absence (lanes 1 and 3) of HnOz. APase was immunoprecipitated from each sampleand analyzed on SDS-PAGE followed by fluorography.
["5Slmethionine APase 30
(lanes
observed
min
after
APase
immediately
intensity
of
to
mean
that
endosomes
to
is
transport
supported
rapidly
transport lysosomes from by our
is
most
useful
the
Golgi
the
procedure separate
APase
from newly and
complex in
increase
the
can be interpreted
observations developed from
from
APase
as compared
to endosomes.
endosomes 1180
At
synthesized
gradually,
complex
endosomes.
that
Golgi
at 60 min
immunocytochemical
to
indicates
newly
synthesized
labeled
Additionally,
proceeded the
from
of
through
observed
of
The DAB density-shift (14)
passed
band
newly
derived
reaction
DAB-H202
APase
the
disappearance
to endosomes.
the
Thus,
is apparently the
the
synthesized moved
GFl+Z
postinjection,
GF1+2
rapid
in
2 and 4).
This
to
the
notion
(8).
by Courtory
et -2
other
cellular
al
Vol.
166,
No.
3, 1990
organelles
as
density
of
DAB. In
addition,
the
BIOCHEMICAL
there
is
endosomes
is
the
proteins
distinct
receptors,
pathway
(17.18).
evidence
of
increase
formed
or
used
Despite
the
endocytic
TfR
pathway, of
there
proteins.
evidence
that
newly
synthesized
APase
only of
in
Thus, endocytic
lysosomal
it
is
likely
processes
membrane
proteins
but
of in
the
been
advancing
to
no
obtained
present
in
role
of
direct
intracellular
in
and a ligand
the
endocytic
has
also
study
functionally
on the
endosomes
This
to
data
that
of
(16).
We
were
buoyant
renders
groups
endosomes
synthesized
endosomes.
other
and MPR,
newly
endocytosis
the
vesicles
processes
of
receptor-mediated
within
by
transport
by
in
polymerization
accumulating
involvement
the
COMMUNICATIONS
insoluble
sorting
such as ASGPR,
the
RESEARCH
detergent
being
movement
in
specific
DAB polymer
currently
intracellular
endosomes
a
BIOPHYSICAL
due to HRP-HZOz-catalyzed
intravesicular
procedure
AND
herein
internalized in
are
the
same
involved
biosynthetic
not pathway
lysosomes.
Acknowledgments: We wish to thank M. Ohara for helpful This study was supported in part by a Grant-in-Aid Ministry of Education, Science and Culture of Japan.
comments. from the
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