Autolysosomal Membrane-associated Betaine Homocysteine
Methyltransferase
LIMITED DEGRADATION FRAGMENT OF A SEQUESTERED CYTOSOLIC ENZYME
MONITORING AUTOPHAGY*
Takashi
Ueno
,
Kazumi
Ishidoh
,
Reiko
Mineki§,
Isei
Tanida
,
Kimie
Murayama§,
Motoni
Kadowaki¶, and
Eiki
Kominami
From the
Department of Biochemistry and
§ Central Laboratory for Medical Sciences, Juntendo
University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8421 and the
¶ Department of Applied Biochemistry, Faculty of Agriculture,
Niigata University, Igarashi, Niigata 950-21, Japan
 |
ABSTRACT |
We compared the membrane proteins of
autolysosomes isolated from leupeptin-administered rat liver with those
of lysosomes. In addition to many polypeptides common to the two
membranes, the autolysosomal membranes were found to be more enriched
in endoplasmic reticulum lumenal proteins (protein-disulfide isomerase, calreticulin, ER60, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58-kDa protein) than lysosomal membranes. The autolysosomal membrane proteins
include three polypeptides (44, 35, and 32 kDa) whose amino-terminal
sequences have not yet been reported. Combining immunoblotting and
reverse transcriptase-polymerase chain reaction analyses, we identified
the 44-kDa peptide as the intact subunit of betaine homocysteine
methyltransferase and the 35- and 32-kDa peptides as two proteolytic
fragments. Pronase digestion of autolysosomes revealed that the 44-kDa
and 32-kDa peptides are present in the lumen, whereas the 35-kDa
peptide is not. In primary hepatocyte cultures, the starvation-induced
accumulation of the 32-kDa peptide occurs in the presence of E64d,
showing that the 32-kDa peptide is formed from the sequestered 44-kDa
peptide during autophagy. The accumulation is induced by rapamycin but
completely inhibited by wortmannin, 3-methyladenine, and bafilomycin.
Thus, detection of the 32-kDa peptide by immunoblotting can be used as
a streamlined assay for monitoring autophagy.
 |
INTRODUCTION |
Autophagy is a universal process by which cellular proteins are
degraded via a lysosomal/vacuolar system. Depending on the extracellular nutrient conditions, the rate of autophagic protein degradation fluctuates between 1-1.5% and 4-5% of total cell
proteins per hour (1, 2). There are two pathways of autophagy,
microautophagy and macroautophagy (for reviews, see Refs. 3 and 4). In microautophagy, relatively small portions of the cytoplasm are directly
enclosed by invaginating lysosomal membranes for subsequent sequestration and degradation. Degradation of bulky cell constituents by the lysosome/vacuole system occurs via macroautophagy. In the initial step of macroautophagy, various cytosolic proteins, as well as
cytoplasmic organelles such as mitochondria, endoplasmic reticulum
(ER),1 and peroxisomes, are
sequestered in the lumen of double-membraned autophagosomes.
Autophagosomes then fuse with endosomes or lysosomes to become mature,
single-membraned autolysosomes. Acidification of the lumen and
acquisition of lysosomal hydrolytic enzymes enable this specialized
membrane system to degrade sequestered cytoplasmic components.
The origin of the autophagosomal membrane is a subject of controversy.
Extensive morphological analyses by Dunn (5) indicated that the
autophagosomal membrane derives from the rough ER. However, the
post-Golgi membrane, as well as a unique de novo synthesized membrane, the phagophore, have also been proposed as sources (6, 7). In
recent morphological studies on yeast autophagy (8, 9), autophagosomal
membranes were found to have features distinct from those of other
pre-existing cell membranes. This appears to support the notion that
the membrane may have a unique origin. Understanding the molecular
organization of the autophagosomal membrane is important for
understanding the mechanism of autophagy at the membrane level, since
various key molecules involved in or necessary for the formation and
fusion of autophagosomes are likely to exist on the autophagosomal
membrane. In order to characterize the autophagosomal membrane, it is
necessary to isolate autophagosomes. However, autophagosomal maturation
proceeds so quickly that it is very difficult to isolate autophagosomes
of sufficient purity and in quantities suitable for biochemical
analyses. Therefore, we decided to take an indirect approach.
Autolysosomes isolated from leupeptin-administered rat liver have some
advantages. First, they can be easily purified by Percoll-gradient
centrifugation and obtained in quantity (10, 11). Second, effective
inhibition of lysosomal proteolysis by leupeptin keeps many of the
sequestered cytoplasmic proteins apparently active or undegraded (12,
13). As a result, it is expected that some membrane components
characteristic of autophagosomes may also be preserved on autolysosomal
membranes. In a previous study (13), we found that isolated
autolysosomal membranes possess two ER membrane proteins, cytochrome
P450 and NADPH-cytochrome P450 reductase. These results are consistent with those of Dunn (5) in showing that autophagosomes originate from
the ER. It is interesting to clarify other components in isolated
autolysosomal membranes, especially in relation to autophagosomes. In
this study, we systematically analyzed membrane proteins in isolated
autolysosomes by two dimensional gel electrophoresis and compared the
results with those of lysosomes isolated from dextran-loaded rat liver.
 |
EXPERIMENTAL PROCEDURES |
Animals--
Male Wistar rats (250-300 g) were maintained in an
environmentally controlled room (lights on 6:00 to 20:00) for at least 2 weeks before experiments. All rats were fed a standard pelleted laboratory diet and tap water ad libitum during this period.
For all experiments, the rats were starved for 12-18 h before use.
Primary Culture of Rat Hepatocytes--
Hepatocytes were
isolated from 18 h-starved male Wistar rats by a collagenase perfusion
procedure (14). The hepatocytes were seeded at a density of
105 cells/0.2 ml/cm2 and cultured in Williams E
medium supplemented with 10% fetal calf serum (Williams E/10%
FCS).
Analytical Methods and Reagents--
Protein was determined by
the BCA protein assay following the manufacturer's protocol (Pierce).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was carried out according to the method of Laemmli (15). Immunoblot
analyses were performed according to the method of Towbin et
al. (16) except that 2,4-dichloro-1-naphthol (17) or an ECL
Western blot detection kit (Amersham Pharmacia Biotech) was used as the
substrate for the horseradish peroxidase conjugate of the second
antibodies.
-Hexosamidase activity was measured as described by
Lusis et al. (18) using
4-methyl-umbelliferyl-
-D-glucosaminide as the substrate.
Antibodies against synthetic pentapeptides and decapeptides
corresponding to the amino-terminal sequences of three unidentified polypeptides in autolysosomal membranes were produced in rabbits as
described by Liu et al. (19). Antibodies were
affinity-purified on immobilized peptide-Sepharose columns. Antibody
against a synthetic peptide corresponding to a sequence in rab 7 (residues 175-191) was produced in rabbits as described by Chavrier
et al. (20). Antibody to cation-independent mannose
6-phosphate receptor (CI-M6PR) was produced in rabbits as described
(21). Commercially available antibodies were purchased from the
following sources: antibodies against rab 5A, trimeric GTP-binding
protein subunits Gs
, Gi-2
, and
Gi-3
from Santa Cruz Biochemicals; antibody to Golgi
58-kDa protein from Sigma. A monoclonal antibody against transferrin receptor was produced as described by White et al. (22) and kindly donated by Dr. Ian Trowbridge. Percoll was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Pronase E
(Streptomyces griseus protease, type XXV) and dextran
(Mr = 70,000) were obtained from Sigma.
[
-32P]GTP was obtained from NEN Life Science Products.
Leupeptin and pepstatin were purchased from Peptide Institute Inc.
(Osaka, Japan). E64c and E64d were donated by Dr. Kazuhiro Hanada
(Taisho Pharmaceutical Co., Saitama, Japan). Rapamycin and wortmannin
were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Isolation of Autolysosomal and Dextran-loaded Lysosomal
Membranes--
Autolysosomes (referred to as autophagic vacuoles in
our previous study) and dextran-loaded lysosomes (hereafter designated lysosomes) were prepared from rats according the procedure of Ueno
et al. (13). Autolysosomal and lysosomal membranes were isolated by centrifugation on a discontinuous sucrose gradient (13),
except that the final carbonate treatment of the membranes to remove
peripheral membrane proteins was omitted. As for autolysosomes, the
vast majority of proteins were recovered in the pellets after discontinuous sucrose gradient centrifugation. This sediment fraction consisted of sequestered protein aggregates denatured in the acidic milieu of the autolysosomal lumen (23) and was used for the experiments
shown in Fig. 5.
Two-dimensional Gel Electrophoresis and Protein Sequence
Analysis--
Protein separation by two-dimensional gel
electrophoresis was performed according to the method of O'Farrell
et al. (24) with slight modifications. For isoelectric
focusing, the gels were polymerized in Pyrex tubes (inner diameter, 3 mm; height, 12 cm) to give a gel height of 9.5 cm. Gels containing 4%
acrylamide, 0.22%
N,N'-methylene-bis-(acrylamide), 9.2 M urea, 2.5% Nonidet P-40, 1.5% octyl glucoside, 1.2%
Ampholine (pH 5-8), 0.4% Ampholine (pH 3.5-9.5), and 0.4% Ampholine
(pH 4-6) were prepared for analyses at acidic pH (pH 4-7). The
electrode solutions used were 20 mM NaOH (cathode) and 0.2 M phosphoric acid (anode). Gels containing 4% acrylamide,
0.22% N,N'-methylene-bis(acrylamide), 9.2 M urea, 2% Nonidet P-40, 0.5%
n-dodecyl-
-D-maltoside, 1.6% Ampholine (pH
7-9), and 0.4% Ampholine (pH 3.5-9.5) were prepared for analyses at
alkaline pH (pH 6-8). The electrode solutions used were 0.1 M NaOH (cathode) and 10 mM phosphoric acid
(anode). All samples applied to a gel contained the same amount of
protein. SDS-PAGE in the second dimension was performed using
5-15% linear gradient gels. After SDS-PAGE, the gels were fixed
in 50% methanol containing 10% acetic acid, and subsequently
silver-stained.
For amino-terminal amino acid sequence analysis, the proteins separated
by two- dimensional gel electrophoresis were electrophoretically transferred onto PVDF membranes (Immobilon PSQ, Millipore
Corp.) using 10 mM Caps (pH 11) containing 20% methanol as
the electrode buffer. The membrane was stained with Coomassie Brilliant
Blue, destained with 10% acetic acid containing 50% methanol
(destaining solution), and air-dried. Pieces containing individual
spots were cut out and soaked in destaining solution, washed several
times with 10% acetic acid, and air-dried. The amino-terminal amino
acid sequences of proteins fixed on Immobilon PSQ membranes
were determined with a protein sequencer (Hewlet Packard, model G1005A).
GTP Blot Analysis--
GTP blotting assays were carried out
according to the method of Huber et al. (25). Autolysosomal
and lysosomal membrane proteins resolved by isoelectric focusing and
subsequent SDS-PAGE in the second dimension (15% gels), as described
in the previous section, were electrophoretically transferred onto
nitrocellulose membranes (Advantec Toyo, Tokyo, Japan). The
nitrocellulose membrane sheets were incubated with
[
-32P]GTP (10 µCi) in the presence of 4 µM ATP at room temperature for 2 h, washed with 50 mM NaH2PO4 (pH 7.5) containing 10 µM MgCl2, 0.2% Tween 20, 4 µM
ATP, and 2 mM dithioerythritol, and then air-dried. The
incorporation of radioactive GTP was detected by autoradiography. Identical sheets were used for immunoblots to identify rab GTP-binding proteins after washing the sheets with 1% acetic acid to remove membrane-bound GTP.
Pronase Treatment of Autolysosomes--
Freshly isolated
autolysosomes were incubated at 0 °C in medium (1 ml) containing 5 mM Tes (pH 7.5), 0.3 M sucrose, and Pronase E
(0.2-0.8 mg/ml) in the presence or absence of 0.2% Triton X-100. The
reaction was stopped by adding an equal volume of 10% trichloroacetic acid. The pellets were collected by centrifugation at 5,000 × g for 5 min, neutralized with a minimal volume of 0.5 M Na2CO3, and vigorously shaken on
a microtube shaker for 5 min. The samples were then solubilized with
SDS-PAGE sample buffer, boiled in a water bath for 3 min, and
electrophoresed in 10% SDS-polyacrylamide gels.
RT-PCR--
Total RNA extracted from rat liver was subjected to
guanidium thiocyanate/CsCl ultracentrifugation (26). Poly(A) RNA was isolated from total RNA on oligo(dT)-cellulose according to the manufacturer's protocol.
Degenerated primers deduced from the amino acid sequences at the
protein level were synthesized: CCNATHGCNGGNAARAARGC designated as
p44-1, AAYGCNGGNGARGTNGTNATTHGG as p44-2, GCNACYTCNGCRTCNCCYTCRTC as
p32-1R, and GCNGCYTCRTTNACYTTYTGNCC as p32-2R. RT-PCR using the
primer set p44-1 and p32-1R from rat liver poly(A)+ RNA was
accomplished at an annealing temperature of 55 °C by an RT-PCR kit
(Toyobo, Tokyo, Japan). An aliquot of sample was further subjected to
nested PCR with the primer set p44-2 and p32-2R at an annealing
temperature of 57 °C. After subcloning into pCRII vector
(Invitrogen), some of the insert-positive clones were sequenced in an
Applied System 373A sequencer by the dye-primer method. The amino acid
sequence deduced from the nucleotide sequence was compared with the
amino acid sequence determined at the protein level, and two clones
were identified as the cDNA for p44. To isolate the 5'-region
further upstream of the cDNA, we carried out the RACE reaction
using nucleotide sequence primers including CCAGCTTGTCCTCACTTGCATAGA
designated as Up-1 and CTGCATGACGTTGGATCCAGCTCTG as Up-2 for 5'-RACE at
an annealing temperature of 57 °C with rat liver
poly(A)+ RNA with a MarathonTM cDNA
amplification kit (CLONTECH). After subcloning into
pCRII vector, the nucleotide sequences were determined by the
dye-primer method in an Applied 373A DNA sequencer.
 |
RESULTS |
Identification of Autolysosomal Membrane Polypeptides Separated by
Two-dimensional Gel Electrophoresis--
In order to identify as many
polypeptides present in greater quantity in autolysosomal membranes
than in lysosomal membranes as possible, we performed preparative
two-dimensional gel electrophoretic analyses at different pH ranges to
allow us to identify major membrane polypeptides directly by amino acid
sequence determination. It should also be noted that the carbonate
treatment of the membranes carried out in the previous study (13) was
omitted so as not to overlook peripheral membrane proteins. Fig.
1 shows silver-stained protein spots
separated at pH 4-7 (Fig. 1, A and B) and pH
6-8 (Fig. 1, C and D). Basically, autolysosomal
membranes and lysosomal membranes closely resemble one another,
indicating that autolysosomes from leupeptin-treated liver have reached
a substantial level of maturation. However, some spots appear to be
more enriched, or present only in autolysosomal membranes (Fig. 1,
A and C). The spots (a-u) shown by
arrows (Fig. 1, A and C) were
reproducibly found among different preparations, and because almost all
of these spots could be stained by Coomassie Brilliant Blue after electrophoretic transfer to PVDF membranes, we attempted to identify the polypeptides by protein sequence analysis.

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Fig. 1.
Polypeptide composition of autolysosomal
membranes and lysosomal membranes separated by two-dimensional gel
electrophoresis. Eighty micrograms of protein from autolysosomal
(A and C) and lysosomal (B and D)
membranes were separated by isoelectric focusing performed at either
acidic pH (A and B) or alkaline pH (C
and D). SDS-PAGE was carried out in the second dimension
using linear gradient (5-15%) SDS-polyacrylamide gels. After
electrophoresis, the gels were silver-stained. The polypeptides that
were present solely or more abundantly in autolysosomal membranes are
indicated by arrows with lowercase
letters (a-u). The positions of molecular mass
markers (in kDa) are shown on the right.
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The amino-terminal sequences of these polypeptides are summarized in
Table I. Unambiguous sequences were not
obtained for 9 of the 21 polypeptides analyzed, possibly due to blocked
amino termini (spots d, p, and t) or insufficient amounts of amino
acids detected (spots g, h, i, j, l, and n). Several major polypeptides separated by isoelectric focusing in the acidic pH range (Fig. 1A) were identified as ER lumenal proteins, including
protein-disulfide isomerase, calreticulin, ER60 protease, and BiP. In
contrast, no polypeptides were identified as being of post-Golgi
membrane origin. There are five polypeptides (spots m, o, q, r, and u) whose amino-terminal sequences have not yet been reported. No further
analysis was made of spot m, because the quantity obtained was too
small; information on the sequence beyond residue 10 is not yet
available. The remaining four components were identified as betaine
homocysteine methyltransferase and its partially degraded fragments, as
described below.
Identification of Golgi/Endosome Membrane Markers, Rab GTP-binding
Proteins, and Trimeric GTP-binding Protein Subunit
s--
As
clearly seen in the previous section, there are no major Golgi/endosome
components in amounts sufficient for identification by amino acid
sequence determination. The immunoblots shown in Fig.
2A show that autolysosomal but
not lysosomal membranes possess three Golgi/endosome markers,
transferrin receptor, CI-M6PR, and Golgi 58-kDa protein. As transferrin
receptor and CI-M6PR are early and late endosome markers, respectively,
we took notice of the rab GTP-binding proteins, because it has been
found that distinctive rab GTP-binding proteins characterize distinct
compartments of intracellular membranes (27). The GTP blots indicate
that both autolysosomal and lysosomal membranes have almost identical sets of rab GTP-binding proteins, except that rab 5A, identified by
immunoblot, is more abundant in lysosomal membranes than in autolysosomal membranes (Fig. 2B). Although no incorporation
of [32P]GTP into rab7 is seen in the GTP blots, it is
detected by immunoblot in both autolysosomal and lysosomal membranes
(Fig. 2C).

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Fig. 2.
Golgi/endosome marker proteins, rab
GTP-binding proteins, and subunits of
trimeric GTP-binding proteins in autolysosomal and lysosomal
membranes. A, 100 µg of protein from autolysosomal
(AL) and lysosomal (L) membranes were
electrophoresed on an SDS-polyacrylamide gel (7.5%). The separated
proteins were electrophoretically transferred onto Millipore membrane
filters (GV, 0.22-µm pore size) and analyzed by immunoblot using
monoclonal anti-transferrin receptor antibody (TfR),
polyclonal anti-CI-M6PR antibody (CI-M6PR), and anti-Golgi
58-kDa protein antibody (G58K). B, 80 µg of
protein from autolysosomal (AL) and lysosomal (L)
membranes were separated by isoelectric focusing at alkaline pH as
described under "Experimental Procedures" and subsequently
electrophoresed on 15% acrylamide gels. The separated proteins were
electrophoretically transferred onto a nitrocellulose membrane filter,
and the membrane filter was incubated with [ -32P]GTP.
The radioactive spots were visualized by autoradiography. For details,
see "Experimental Procedures." The membrane filter was then used
for the identification of rab 5A and rab 7 by immunoblots. The
arrowhead indicates the radioactive spot identified as rab
5A. C. Immunoblot showing rab 7 on the same membrane filter
previously used for GTP blot analysis. D, equal amounts of
protein (100 µg) from autolysosomal membranes (AL) and
lysosomal membranes (L) were electrophoresed in 10%
SDS-polyacrylamide gels. Separated proteins were transferred onto a
Millipore membrane filter (GV, 0.22-µm pore size). The membrane
filter strips were then immunoblotted using anti-Gs
antibody, an antibody recognizing both Gi-2 and
Gi-3 , and anti-Gi-3 -specific
antibody.
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The roles of trimeric GTP-binding proteins in ER-Golgi transport,
homotype membrane fusion of lysosomes, and in the maintenance of Golgi
structures have been reported recently (28-30). It has also been
reported that trimeric GTP-binding protein subunits are associated with
Golgi membranes and the trans-Golgi network (31, 32). We confirmed by
immunoblotting that the trimeric GTP-binding protein subunits,
Gs
and Gi
, are associated with isolated
autolysosomal and lysosomal membranes (Fig. 2D). Both Gi-2
and Gi-3
appear to be evenly
distributed in the two membranes, whereas Gs
seems more
abundant in autolysosomal membranes.
Identification of Three Major Polypeptides Associated with
Autolysosomal Membranes as Betaine Homocysteine
Methyltransferase--
As mentioned in the previous section, there are
four major polypeptides (spots o, q,
r, and u in Fig. 1) in autolysosomal membranes
whose amino-terminal sequences have not yet been reported. Based on the
apparent molecular sizes determined by mobility in SDS-PAGE, we
designate spot u as p44 and spot q as p35. Spot o and spot r have
identical sequences despite their apparently different pI values. We
therefore designate the two components as p32 without further
discrimination. We first attempted to determine as many amino acid
residues as possible toward the carboxyl terminus by protein
sequencing; the data are summarized in Table
II. It appears obvious that p32 must be
processed from p35, because, except for the first four amino acid
residues, Y, V, A, and E, the two polypeptides have identical
sequences. Immunoblot analyses using antibodies raised against
synthetic decapeptides corresponding to 10 amino-terminal residues
further support the possibility that both p35 and p32 derive from a
common precursor form, p44. As Fig. 3
shows, the antibody to p32 (
-p32-10R) recognized all three
polypeptides and the p35 antibody (
-p35-10R) reacted with both p35
and p44 (Fig. 3, A and B).
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Table II
Protein sequence determination of three unidentified polypeptides
associated with autolysosomal membranes
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Fig. 3.
Immunoblot analysis on BHMT and its partially
degraded fragments, p35 and p32. Equal amounts of protein (80 µg) from autolysosomal (AL) and lysosomal (L)
membranes were separated in 10% SDS-polyacrylamide gels, and the
separated proteins were electrophoretically transferred onto a
Millipore membrane filter (GV, 0.22-µm pore size). The membrane
filter strips were incubated with either -p32-10R (A),
-p35-10R (B), or -p44-10R (intact BHMT,
C) antibody followed by the horseradish peroxidase conjugate
of anti-rabbit IgG. The positions of molecular size markers (myosin
(200 kDa), -galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), and trypsin inhibitor (22 kDa)),
as well as p44 (44 kDa), p35 (35 kDa), and p32 (32 kDa), are shown on
the left.
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In order to confirm the above possibility further, we carried out
RT-PCR using degenerated primer sets deduced from the amino acid
sequences of p44 and p32 as described under "Experimental Procedures." Fragments of about 250 base pairs were amplified and
sequenced after subcloning into pCRII vector. The nucleotide sequences
were 258 base pairs in length, and the deduced amino acid sequences
contained the amino acid sequences of both p44 and p32 (Fig.
4A). To isolate a region on
the 5'-upstream side of the cDNA for the precursor protein, 5'-RACE
was carried out using the specific primers, Up-1 and Up-2. One clone
isolated from the 5'-RACE reaction when subcloned into pCRII vector
encodes the 5'-end of the precursor protein (Fig. 4B). The
nucleotide sequence of this cDNA, which includes a region that
overlaps with the first RT-PCR product, was subjected to a nucleotide
sequence homology search using the computer package, BLAST. It was
found that the nucleotide sequence of the cDNA for human betaine
homocysteine methyltransferase (BHMT) (33) shows 86% identity at the
nucleotide sequence level and 93% identity at the deduced amino acid
sequence level to that of the cDNA clone isolated in our study.
Thus, p35 and p32 have been identified as the proteolytic products of
p44, i.e. rat BHMT. (Recently, the full-length cDNA for
rat BHMT was registered in GenBank with accession number U96133.)

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Fig. 4.
Nucleotide sequences of RT-PCR products and
their deduced amino acid sequences. A, the nucleotide
sequence of a cDNA clone obtained by RT-PCR followed by nested PCR
is numbered from 1 to 232 above the nucleotide sequence. The
deduced amino acid sequence is shown under the nucleotide
sequence in single-letter code. Amino acids in
bold letters indicate identity to the amino acid
sequences determined at the protein level. Underlined
nucleotide sequences indicate the synthetic primers used in nested PCR.
Undetermined nucleotides and amino acids are represented by
X. B, nucleotide and deduced amino acid sequences
of a cDNA obtained by 5'-RACE are shown. The small
lettered nucleotide sequence with negative
numbers is the 5'-noncoding sequence. Nucleotides in the
coding region are shown in large letters and with
positive numbers. Amino acid sequences identical
to those determined at the protein level are in bold
letters. The underlined nucleotide sequence
indicates the Up-2 primer sequence. Nucleotides and amino acids
indicated as X are undetermined.
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Sequestration and Degradation of BHMT during Autophagy--
BHMT
is a major cytosolic protein accounting for nearly 1.6% of the total
cytosolic protein in the liver (33). We therefore reasoned that BHMT is
originally sequestered as a cytoplasmic component, the substrate of
autophagy, into autophagosomes, and subsequently degraded to p35 and
p32 by some steps during the maturation process from autophagosomes to
autolysosomes. To examine this possibility further, we attempted to
determine if autolysosomal BHMT and the two fragments truly exist in
the lumen. Since antibodies raised against synthetic peptides
corresponding to the 10 amino-terminal residues of p35 and p32
(
-p35-10R and
-p32-10R) also recognized p44, we first tried to
prepare peptide antibodies that do not react with p44 but more
specifically recognize p35 or p32 to avoid complications to the
experimental data. As the results of immunoblotting show (Fig.
5A, lane
2), antibody raised against the five amino-terminal residues
of p35 (
-p35-5R) was found to react only with p35. Likewise, antibody raised against the five amino-terminal residues of p32 (
-p32-5R) recognized p32 but not p35 or p44 (Fig. 5A,
lane 3). Using these newly prepared antibodies
together with an anti-p44 antibody (
-p44-10R), we next analyzed the
distribution of p44, p35, and p32 in autolysosomal subfractions. As
shown in Fig. 5B, both p44 and p32 exist in autolysosomal
membranes and the sediment fraction, which consists mainly of
sequestered cytoplasmic components, whereas p35 exists in autolysosomal
membranes but not in the sediment. Furthermore, Pronase treatment of
isolated autolysosomes confirmed that p44 and p32 are present in the
autolysosomal lumen but that p35 is associated with the outer surface
of autolysosomes (Fig. 5C). Incubation of autolysosomes with
Pronase at 0 °C for 10 min resulted in the complete digestion of p35
irrespective of the presence or absence of Triton X-100 (Fig.
5C, lanes 4-6). Under these
conditions, p44 was degraded in the presence of Triton X-100 but
resistant to digestion in its absence (Fig. 5C,
lanes 1-3). p32 could not be degraded by Pronase
regardless of the presence of added Triton X-100 (Fig. 5C,
lanes 7-9). This suggests that most
intra-autolysosomal p32 exists in denatured protein aggregates (sediment fraction) together with other sequestered proteins. From
these data, we conclude that BHMT (p44) is sequestered into autophagosomes and subsequently degraded upon maturation of the autophagosomes to autolysosomes. p32, a limit-digested fragment of p44,
accumulates as a result of the cessation of autophagic proteolysis in
the presence of leupeptin/E64c; otherwise, it would be degraded rapidly
and completely. In fact, p32 could not be detected in any subcellular
fraction, even under starvation conditions, unless leupeptin/E64c was
injected into the rat (data not shown).

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Fig. 5.
Location of p44, p35, and p32 in
autolysosomes as revealed by immunoblot analysis using specific
antibodies. A, autolysosomal protein (25 µg) was
electrophoresed in 10% SDS-polyacrylamide gels and the separated
proteins were electrophoretically transferred onto a Millipore membrane
filter (GV, 0.22-µm pore size). The membrane strip was incubated with
either an -p44-10R (lane 1), -p35-5R
(lane 2), -p32-5R (lane
3), or -p32-10R (lane 4) antibody,
followed by the horseradish peroxidase conjugate of anti-rabbit IgG.
The positions of molecular mass markers are shown on the
left, while the positions of p44, p35, and p32 are shown on
the right. B, autolysosomal membranes
(lanes 1, 3, and 5) and the
sediment fraction (lanes 2, 4, and
6), each containing 60 µg of protein, were electrophoresed
in 10% SDS-polyacrylamide gels, and the separated proteins were
electrophoretically transferred onto a Millipore membrane filter (GV,
0.22-µm pore size). The membrane strip was probed by immunoblotting
with -p44-10R (lanes 1 and 2),
-p35-5R (lanes 3 and 4), or
-p32-5R (lanes 5 and 6)
antibodies. C, freshly prepared autolysosomes were incubated
at 0 °C in medium (250 µl) containing 5 mM Tes (pH
7.5), 0.3 M sucrose, and Pronase (0.4 mg/ml) in the
presence (lanes 3, 6, and
9) or absence (lanes 2, 5,
and 8) of 0.2% Triton X-100. As a control (lanes
1, 4, and 7), autolysosomes were
incubated in medium without added Pronase or Triton X-100. The reaction
was terminated by adding an equal volume of ice cold 10%
trichloroacetic acid. The mixture was then centrifuged at 5,000 × g, and the pellets were solubilized and electrophoresed as
described under "Experimental Procedures." The separated proteins
were electrophoretically transferred onto a Millipore membrane filter
(GV, 0.22-µm pore size). The membrane strip was probed by
immunoblotting using -p44-10R (lanes 1-3),
-p35-5R (lanes 4-6), or -p32-5R
(lanes 7-9) antibodies.
|
|
p32 as an Endogenous Marker of Autolysosome
Maturation--
Detection of cellular p32 using
-p32-10R and
-p32-5R in the presence of leupeptin or E64 will thus become a
useful means for monitoring the maturation process from autophagosome
to autolysosome. We tested this hypothesis in primary cultures of rat
hepatocytes. Hepatocytes cultured in 6-cm dishes with Williams E/10%
FCS were washed and incubated with 1.18 mM
KH2PO4/23.5 mM NaHCO3
(pH 7.4) containing 118.5 mM NaCl, 4.74 mM KCl,
2.5 mM CaCl2, 1.18 mM
MgSO4, and 6 mM glucose (Krebs-Ringer
bicarbonate (KRB) buffer) to induce autophagy. At various times after
induction, hepatocytes were harvested and homogenized. The homogenate
was then subjected to SDS-PAGE and immunoblot analyses. The data shown
in Fig. 6A represent the time
course of p32 accumulation in the presence of E64d (membrane-permeable form of E64)/pepstatin (each 10 µg/ml). Using
-p32-10R, which recognizes p44, p35, and p32, the specific accumulation of p32 could be
clearly seen in the presence of cathepsin inhibitors. The amount of p32
increased for 4 h after medium exchange and then remained almost
constant until 24 h. The amount of p44 decreased slightly and
gradually during nutrient starvation. The accumulation of p32 was
firmly dependent on E64d (Fig. 6B, lane
2), because only p44 could be detected in its absence.
Essentially the same tendency was observed using
-p32-5R (Fig.
6A, lower panel). Often a lower
molecular mass component (26 kDa) was found just below p32. The 26-kDa
protein could be detected by both
-p32-10R and
-p32-5R, and was
also found in autolysosomes (Figs. 3 and 5). However, we could not
detect the 26-kDa protein as an obvious spot in two dimensional gel
electrophoresis (Fig. 1). It is also noted that p35 could not be
detected in immunoblots using
-p35-5R (data not shown). In the
experiments shown in Fig. 6B, the effects of various
inhibitors and an activator of autophagy on the accumulation of p32
were investigated. The accumulation of p32 was completely blocked by
3-methyladenine (34), wortmannin (35), and bafilomycin (36), known
inhibitors of autophagy (Fig. 6B, lanes
7-9). In contrast, rapamycin, which stimulates hepatic
autophagy by inhibiting S6 kinase activity (37, 38), induced p32
accumulation even when the hepatocytes were incubated with Williams
E/10% FCS (Fig. 6B, lanes 4-6).
Thus, the E64d-dependent accumulation of p32 in cultured
hepatocytes corresponds well to the activity of hepatocyte autophagy at
the time when E64d inhibits lysosomal cysteine proteinases under
starvation conditions.

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|
Fig. 6.
E64d-induced accumulation of a BHMT fragment
(p32) in cultured hepatocytes under starvation conditions as
demonstrated by immunoblots. A, hepatocytes cultured at
37 °C in 6-cm dishes with Williams E/10% FCS were washed twice and
incubated with 5 ml of KRB buffer containing 0.1% dimethyl sulfoxide
(control, lanes 1-5) and 10 µg/ml E64d plus 10 µg/ml pepstatin (lanes 6-10). At 2 h
(lanes 1 and 6), 4 h
(lanes 2 and 7), 8 h
(lanes 3 and 8), 12 h
(lanes 4 and 9), and 24 h
(lanes 5 and 10) after the transition,
cells were harvested and homogenized by sonication for 10 s in 0.5 ml of ice-cold 20 mM NaH2PO4 (pH
7.5) containing 0.15 M NaCl. The homogenates were
solubilized in SDS-PAGE sample buffer and electrophoresed in 10%
SDS-polyacrylamide gels. The separated proteins were
electrophoretically transferred onto a Millipore membrane filter (GV,
0.22-µm pore size). The membrane filter was incubated with either
-p32-10R (upper panel) or -p32-5R
(lower panel) antibody followed by the
horseradish peroxidase conjugate of anti-rabbit IgG. B,
hepatocytes cultured in 6-cm dishes with Williams E/10% FCS were
washed with KRB buffer and incubated at 37 °C for 4 h with
Williams E/10% FCS (lanes 4-6) or KRB buffer
(lanes 1-3 and 7-9) containing the
following reagents: 0.2% dimethyl sulfoxide (control, lanes
1 and 4); 10 µg/ml E64d (lane
2); 10 µg/ml E64d and 10 µg/ml pepstatin
(lanes 3 and 5); 10 µg/ml E64d, 10 µg/ml pepstatin, and 0.2 µM rapamycin (lane
6); 10 µg/ml E64d, 10 µg/ml pepstatin, and 0.1 µM wortmannin (lane 7); 10 µg/ml
E64d, 10 µg/ml pepstatin, 10 mM 3-methyladenine
(lane 8); 10 µg/ml E64d, 10 µg/ml pepstatin,
and 0.1 µM bafilomycin (lane 9).
After incubation, the hepatocytes were harvested, homogenized by
sonication, solubilized, and electrophoresed in 10% SDS-polyacrylamide
gels. The separated proteins were electrophoretically transferred onto
a Millipore membrane filter (GV, 0.22-µm pore size). The membrane
filter was incubated with either -p32-10R (upper
panel) or -p32-5R (lower panel)
antibody, followed by the horseradish peroxidase conjugate of
anti-rabbit IgG.
|
|
 |
DISCUSSION |
As shown by two-dimensional gel electrophoresis, we were able to
use lysosomal membranes isolated from dextran-loaded liver as a
reference to identify autolysosomal polypeptides of nonlysosomal origin. Most of these polypeptides derive from either the ER or endosome/Golgi compartment, i.e. from pre-existing
membranes. The question, then, is can these nonlysosomal polypeptides
be somehow related to the origin and maturation of autophagosomal membrane?
The identification of major autolysosomal membrane-associated
polypeptides as ER lumenal proteins (protein-disulfide isomerase, calreticulin, ER60 protease, and BiP) is apparently consistent with the
data of Dunn (5), indicating that ER lumenal content exists in the
outer limiting membranes of autophagosomes. Together with the
cytochrome P450 and NADH-cytochrome P450 reductase detected in our
previous study (13), these ER markers in the autolysosomal membrane may
be explained as surviving autophagosomal membrane components. However,
the existence of ER components on autophagosomal membranes has been a
subject of dispute. In immunoelectron microscopic studies, Masaki
et al. (39) and Yamamoto et al. (40) reported that cytochrome P450 does not exist on the surface membranes of autolysosomes and autophagosomes, but rather in the lumen as a substrate to be degraded via autophagy. The different antibodies used
and the different stages of maturation of the autophagosomal and/or
autolysosomal particles observed may partially explain the discrepancy
in the data among these laboratories. Moreover, an extreme possibility
may also be considered; a specific region of the ER membrane may be
used as a source of surface limiting membranes as reported by Dunn (5)
on the one hand, and bulk ER membranes may be sequestered in
autophagosomes on the other. In order to obtain a firmer conclusion
about this problem, a thorough morphological examination using
antibodies raised against as many ER components as possible should be
performed on autophagosomes/autolysosomes at various stages of autophagy.
There were no major polypeptides of endosomal/Golgi origin in amounts
sufficient for amino acid sequence analysis. Thus, we could not obtain
evidence that autolysosomal membranes are structurally related to the
Golgi/endosome compartment. The existence of endosomal markers, such as
transferrin receptor, CI-M6PR, and several rab GTP-binding proteins,
should rather be evaluated with respect to the convergence of
autophagic and endocytic pathways as reported by Gordon and Seglen (41)
and Dunn (42). Our data are consistent with a more recent report by
Berg et al. (43), showing that amphisomes (prelysosomal
autophagic/endosomal vacuoles) possess both early and late endosome markers.
The association of transferrin receptor may reflect the possibility
that the receptor is also brought in from recycling endosomes that have
been found to be distinct from sorting early endosomes (44). In
contrast, rab 5, another early endosome marker, is more enriched in
lysosomal membranes than in autolysosomal membranes. It is possible
that dextran-loaded lysosomes, the source of lysosomal membranes,
include a small portion of dextran-loaded early and late endosomes,
which may explain for the more association of rab 5 with lysosomal membranes.
The roles of trimeric GTP-binding proteins in the membrane fusion of
lysosomes, the assembly of the Golgi apparatus, and the autophagic
protein degradation of certain cell lines have recently drawn attention
(28, 30, 31, 45). To accomplish these functions, it is necessary for
the GTP-binding proteins to become associated with target membranes. We
confirmed that Gi-2
, Gi-3
, and
Gs
are associated with both autolysosomal and lysosomal
membranes. Although the exact functions of these molecules must be
clarified by further investigations, it is of potential interest that
Gs
is more abundant in the autolysosomal membranes.
Three polypeptides associated with autolysosomal membranes have been
newly identified by RT-PCR as BHMT and its partially degraded
fragments. BHMT catalyzes a reaction essential for the catabolism of
betaine and homocysteine in mammalian liver, thus potentially affecting
the regulation of methionine metabolism. BHMT is a major cytosolic
enzyme consisting of six identical subunits (33). One intact subunit
corresponds to p44 in this study. The two fragments, p35 and p32, lack
the amino-terminal 87 and 91 residues, respectively, of p44. Since all
the analyses of autolysosomal BHMT have been done by SDS-PAGE and
immunoblots, we do not know whether these intact and partially degraded
subunits exist as disassembled monomers or in polymeric forms. The
Pronase digestion experiment and a close examination of the
distribution of p44, p35, and p32 in the autolysosomal subfractions
(Fig. 5) clearly demonstrated that p44 and p32 are present in the lumen
of autolysosomes, showing that p44 (BHMT) is sequestered as a substrate
for autophagy in autophagosomes and that p32 accumulates as a
degradation product of p44 in the presence of leupeptin/E64c. In
contrast, p35 is associated with the outer surface of the autolysosomal
membrane (Fig. 5), which strongly suggests that the cleavage of p44 to yield p35 occurs in the cytosol. It has been reported previously (46)
that the partial degradation of BHMT to produce low molecular mass
forms occurs during purification and that this degradation can be
inhibited by dimethylglycine and homocysteine, the product and
substrate, respectively, of the BHMT reaction. In accordance with this
observation, we performed immunoblotting to confirm that the addition
of dimethylglycine and homocysteine (each 2 mM) to the
homogenization buffer markedly reduces the p35 level in liver
homogenates (data not shown). Furthermore, we could hardly detect p35
in primary cultured hepatocytes even under nutrient starvation
conditions. These data indicate that the cleavage of p44 to produce p35
is an artificial event, irrelevant to intra-autolysosomal proteolysis.
It is likely that the homogenization of the liver causes the partial
destabilization of BHMT in the absence of substrates or products, which
enhances the susceptibility of the enzyme to cytosolic protease(s). In
view of the data showing that p35 is also associated with lysosomal
membranes (Fig. 3), p35 may have an affinity for cell membranes.
It should be noted that p32 is the first example of a limit-digested
intermediate of a sequestered protein found in autolysosomes. Inhibition of cysteine proteinases by leupeptin elicits the
accumulation of autolysosomes holding various sequestered proteins, the
substrates of autophagy, in their lumen, and these substrates can be
recovered in the autolysosomal sediment fraction (23). As far as we
have examined, none of these sequestered substrates have been found as
limit-digested fragments. In other words, the sequestered proteins have
the same mobilities in SDS-PAGE as their cytoplasmic counterparts. It
is therefore difficult to use sequestered autophagy substrates as
autolysosomal markers unless the autolysosomes can be clearly separated
from other cytoplasmic components. However, the fact that p32
accumulation is strictly dependent on leupeptin/E64c indicates that its
accumulation also parallels the accumulation of autolysosomes. As p32
can be easily and clearly distinguished from its cytoplasmic precursor
(p44) in immunoblots, p32 in the cell homogenate can be used as a
compelling autolysosomal marker. Immunoblot analysis using
-p32-10R
and
-p32-5R confirms this idea and allowed us to develop a new,
simple biochemical assay for starvation-induced autophagy as
demonstrated by experiments performed with cultured hepatocytes (Fig.
6). When hepatocytes are incubated with KRB buffer to induce autophagy,
p32 begins to accumulate, increases for 4 h after starvation, and
reaches a plateau. The accumulation of p32 is totally dependent on
E64d. Rapamycin, which enhances autophagy by inhibiting S6 kinase in both mammalian and yeast cells (37, 38), induces the accumulation of
p32 in hepatocytes incubated with Williams E/10% FCS, the conditions under which autophagic activity is thought to be suppressed to basal
levels. The accumulation is completely blocked by 3-methyladenine and
wortmannin, which have been shown to inhibit autophagosome formation
(34, 35). Bafilomycin, which has been recently demonstrated to inhibit
the maturation of autophagosomes to autolysosomes by inhibiting the
vacuolar H+ pump (36), also blocks the accumulation. In
summary, the p32 level corresponds directly to the activity of
autolysosome formation or autolysosomal proteolysis when cells are
incubated with E64d. Conversely, the absence of p32 accumulation means
that either the formation of autophagosomes or the maturation of
autophagosomes to autolysosomes is inhibited. The method is therefore
useful for the primary screening of those effectors that specifically affect formation and maturation steps of autophagosomes or of genetic
defects in autophagy. BHMT is abundantly expressed in hepatocytes and
hepatocyte-derived cell clones, but expression levels in other cells
are relatively low. The construction of a stable transformant will be
helpful for reproducing the E64d-dependent accumulation of
p32, because lysosomes have a common cassette of cysteine proteinases
among various mammalian cells. Thus, the present method of monitoring
p32 has general applicability to the study of autophagy.
 |
FOOTNOTES |
*
This work was supported by Grant-in-aid for Scientific
Research 09680629 and Grant-in-aid for Scientific Research on Priority Areas (Intracellular Proteolysis) 08278103 from the Ministry of Education, Science, Sports and Culture of Japan and the Science Research Promotion Fund from the Japan Private School Promotion Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom all correspondence should be addressed: Dept. of
Biochemistry, Juntendo University School of Medicine, Bldg. 9, Rm. 913, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: 813-5802-1031; Fax:
813-5802-5889; E-mail: kominami{at}med.juntendo.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
FCS, fetal calf serum;
PAGE, polyacrylamide gel
electrophoresis;
CI-M6PR, cation-independent mannose 6-phosphate
receptor;
E64c, (+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butylcarbamoyl]-2-oxiranecarboxylic
acid;
E64d, ethyl-(+)-(2S,3S)-3-[(S)-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-2-oxiranecarboxylate;
Caps, 3-cyclo- hexylaminopropanesulfonic acid;
Tes, N-tris(hydroxymethyl)-2aminoethanesulfonic acid;
PVDF, polyvinylidene fluoride;
BiP, immunoglobulin heavy chain binding
protein;
BHMT, betaine homocysteine methyltransferase;
-p44-10R, antibody raised against the NH2terminal 10 amino acid
residues (APIAGKKAKR) of the intact subunit of BHMT;
-p35-10R, antibody raised against the NH2-terminal 10 amino acid
residues (YVAEKISGQK) of the 35-kDa fragment of BHMT;
-p35-5R, antibody raised against the NH2-terminal 5 amino acid
residues (YVAEK) of the 35-kDa fragment of BHMT;
-p32-10R, antibody raised against the NH2-terminal 10 amino acid
residues (KISGQKVNEA) of the 32-kDa fragment of BHMT;
-p32-5R, antibody raised against the NH2-terminal 5 amino acid
residues (KISGQ) of the 32-kDa fragment of BHMT;
KRB, Krebs-Ringer
bicarbonate;
RACE, rapid amplification of cDNA ends;
RT, reverse
transcriptase;
PCR, polymerase chain reaction.
 |
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