From the Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan
Received for publication, October 23, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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Autophagy is a process that involves the bulk
degradation of cytoplasmic components by the lysosomal/vacuolar system.
In the yeast, Saccharomyces cerevisiae, an autophagosome is
formed in the cytosol. The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole. The autophagic body is subsequently degraded by
vacuolar hydrolases. Taking advantage of yeast genetics,
apg (autophagy-defective) mutants were
isolated that are defective in terms of formation of autophagic bodies
under nutrient starvation conditions. One of the APG gene
products, Apg12p, is covalently attached to Apg5p via the C-terminal
Gly of Apg12p as in the case of ubiquitylation, and this conjugation is
essential for autophagy. Apg7p is a novel E1 enzyme essential for the
Apg12p-conjugation system. In mammalian cells, the human Apg12p homolog
(hApg12p) also conjugates with the human Apg5p homolog. In this study,
the unique characteristics of hApg7p are shown. A two-hybrid experiment indicated that hApg12p interacts with hApg7p. Site-directed mutagenesis revealed that Cys572 of hApg7p is an authentic active site
cysteine residue essential for the formation of the
hApg7p·hApg12p intermediate. Overexpression of hApg7p enhances
the formation of the hApg5p·hApg12p conjugate, indicating that hApg7p
is an E1-like enzyme essential for the hApg12p conjugation system.
Cross-linking experiments and glycerol-gradient centrifugation analysis
showed that the mammalian Apg7p homolog forms a homodimer as in yeast
Apg7p. Each of three human Apg8p counterparts, i.e. the
Golgi-associated ATPase enhancer of 16 kDa, GABAA
receptor-associated protein, and microtubule-associated protein light
chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant
hApg7pC572S to form a stable intermediate via an ester
bond. These results indicate that hApg7p is an authentic
protein-activating enzyme for hApg12p and the three Apg8p homologs.
Post-translational modifications regulate the functions and
localization of target proteins, resulting in many significant intracellular events. One unique modification is the covalent attachment of modifier proteins, ubiquitin, ubiquitin-related proteins
(SUMO-1/Smt3p and NEDD-8/RUB1), and Apg12p (for reviews, see Refs.
1-6). The enzymatic processes of these modifications have been
intensively studied in ubiquitylation. Ubiquitin forms conjugates with
a target protein via a three step mechanism. First, ubiquitin is
activated at its C-terminal Gly by the ubiquitin-activating enzyme
(UBA1, E1 enzyme) to form a conjugate with the active site Cys in the
E1 enzyme via a thiol ester bond. Next, ubiquitin is transferred from
the E1 enzyme to one of several ubiquitin-conjugating enzymes (UBCs, E2
enzymes). In the last step, ubiquitin is attached to a Lys within the
target protein via an isopeptide bond. This step is often catalyzed by
a member of the ubiquitin-protein ligase family, an E3 enzyme. The
reaction mechanism is basically common for each modifier protein.
Autophagy is a process of bulk degradation of cytoplasmic components by
the lysosomal/vacuolar system (5, 7, 8). In the initial step of
macroautophagy, a cup-shaped membrane sac surrounds cytosolic
components to form an autophagosome (9). The outer membrane of the
autophagosome fuses with a lysosome/vacuole (10). Taking advantage of
yeast genetics,
apg1 and
aut mutants were isolated as autophagy-defective mutants in
the yeast, Saccharomyces cerevisiae (12, 13). Surprisingly, most of the apg mutants overlap genetically with
cvt mutants, which have a defect in the cytoplasm-to-vacuole
targeting of aminopeptidase I, indicating that these genes function in
a unique transport system under vegetative growth conditions in
addition to starvation conditions (14-16). A novel modifier protein,
Apg12p, was discovered as an APG gene product (11). Apg12p
shows little homology to ubiquitin, but it is covalently attached to
Apg5p via the C-terminal Gly of Apg12p as in the case of
ubiquitylation. In this conjugation reaction, Apg7p and Apg10p function
as E1- and E2-like enzymes for Apg12p, respectively (11, 17-19). After
the formation of the Apg12p· Apg5p conjugate, Apg16p attaches to
Apg5p forming an Apg12p·Apg5p·Apg16p complex for autophagy (20).
Unlike other modifier-conjugation systems, the unique character of the
Apg12p-conjugation system is that it plays indispensable roles in the
formation of membrane structures, including autophagosomes and
Cvt-vesicles.
Apg7p, an authentic E1-like enzyme essential for Apg12p, plays an
indispensable role in the initial step of the conjugation system,
whereas the enzyme shows slight homology to other E1 enzymes (18).
Apg7p interacts with Apg8p/Aut7p and Aut1p/Apg3p in addition to Apg12p
(21, 22).2 The dimerization
of Apg7p via the C-terminal region is essential for these interactions,
suggesting that Apg7p forms multimeric complexes with these
proteins.2 Apg8p/Aut7p is localized on autophagosomes and
Cvt-vesicles (23). The AUT1/APG3 gene is a multicopy
suppressor of the apg8/aut7 mutant (24). Apg8p/Aut7p also
interacts with two ER-to-Golgi v-SNAREs (Bet1p and Sec22p) and vacuolar
t- and v-SNAREs (Vam3p and Nyv1p, Ref. 25). Furthermore, more recent
findings suggest that Apg8p/Aut7p, Aut1p/Apg3p, and Apg7p comprise a
second protein-conjugation system indispensable for autophagy and Cvt
pathways (22, 26). The second modifier is Apg8p/Aut7p, and Apg7p and
Aut1p/Apg3p are corresponding E1- and E2-like enzymes. These results
suggest that Apg7p, because it is involved in two distinct conjugation systems, is a key enzyme for membrane formation and the targeting of
autophagosomes and Cvt vesicles.
In mammalian cells, several homologs of yeast APG gene
products have been reported. hApg12p conjugates with hApg5p
(first identified as an apoptosis-specific protein), suggesting that the Apg12p conjugation system exists even in human cells (27, 28).
There are three candidates for mammalian Apg8p/Aut7p homologs, GATE-16
(Golgi-associated ATPase enhancer of 16 kDa), GABARAP (GABA
receptor-associated protein), and MAP-LC3 (microtubule-associated protein light chain 3) (25, 29-33). GATE-16 was first identified as a
ganglioside expression factor, but was recently characterized as a
soluble transport factor. GATE-16 interacts with NSF and the Golgi
v-SNARE GOS-28 (33). The mRNA of GATE-16 is expressed ubiquitously
but at significantly higher levels in brain tissue. The interaction of
yeast Apg8p/Aut7p with two ER-to-Golgi v-SNAREs was proven as a
functional GATE-16 homolog (25). GABARAP interacts with
GABAA receptors, cytoskeleton, and gephyrin, suggesting
functional importance in brain or neuronal cells (31, 34). MAP-LC3
copolymerizes with tubulin and is a component of the MAP-1 complex,
which is composed of light chains 1, 2, and 3 and heavy chains (29,
30). Rat MAP-LC3 is localized on autophagosomal membranes, suggesting that rat MAP-LC3 is also a functional Apg8p/Aut7p homolog (35). These
results suggest that mammalian Apg8p/Aut7p homologs have divergent
functions in mammalian cells, especially in neuronal cells. For Apg7p,
there are several clones in the EST database, and the sequence of
hApg7p has been determined to be homologous to Pichia pastoris
GSA7, which is essential for microautophagy (36). However, as yet,
there is no biochemical evidence that hApg7p is an E1-like enzyme for
hApg12p. There is a further question; which of MAP-LC3, GATE-16, and
GABARAP is an authentic substrate for hApg7p? In this report, we show
that hApg7p conjugates with hApg12p and all three Apg8p/Aut7p homologs
as hApg7p substrate intermediates in mammalian cells.
Animals--
Male Wistar rats (250-300 g) were maintained in an
environmentally controlled room (lights on from 7:00 AM to 20:00 PM)
for at least 2 weeks before experiments. All rats were fed a standard pellet laboratory diet and tap water ad labitum during this period.
Strains, Media, Materials, and Molecular Biological
Techniques--
Escherichia coli strain DH5 Plasmid Construction and Site-directed Mutagenesis--
Based on
the DNA sequence of the human APG7/GSA7 homolog
(GenbankTM/EBI accession number AF094516), we cloned an
open-reading frame of the human GSA7/APG7 cDNA by polymerase chain
reaction with high fidelity (36, 40), introduced the amplified DNA fragment into the SalI-NotI site of pBluescriptII
(SK+), and designated the resultant plasmid for pSKhAPG7 plasmid. To
construct an expression plasmid as hApg7p, a
KpnI-NotI fragment (~2.5 kilobase pairs) of the
pSKhAPG7 plasmid was introduced into the pEGFP-N1 vector (CLONTECH) and designated pCMV-hAPG7.
To obtain a DNA fragment containing an open-reading frame of the human
APG12 homolog, GATE-16, and GABARAP, polymerase chain reaction was performed with specific primers for their open-reading frames with high fidelity using a human brain cDNA library as a
template, and the amplified fragment was introduced into pGEM-T vector
(pGEM-hAPG12, pGEM-hGATE-16, and pGEM-hGABARAP). The isolated DNA
fragments were introduced into pEGFP-C1 to express GFP fusion proteins (pGFP·hApg12p, pGFP·hGATE-16, and pGFP·hGABARAP).
Cys572 within hApg7p was replaced by Ser, mutagenized by
the Gene-Editor in vitro site-directed mutagenesis system
(PROMEGA) with an oligonucleotide (hAPG7CS;
5'-CGGACCTTGGACCAGCAGAGCACTGTGAGTCGTCCAGG-3') according to the
manufacturer's protocol. The expression plasmid for mutant
Apg7pC572S was constructed as in the case of pCMV-hAPG7 and
was designated pCMV-hAPG7C572S.
Antibodies--
A polyclonal antibody against a synthetic
polypeptide (VVAPGDSTRDRTL) corresponding to residues 550-571 of
hApg7p was raised in Japanese white rabbits (anti-hApg7p). The antibody
was affinity-purified by chromatography on immobilized
hApg7p-peptide-Sepharose. For the preparation of antibody against
murine Apg12p homolog (mApg12p), rabbits were immunized with a
maltose-binding protein·mApg12p fusion protein. The antibody to
mApg12p was purified by affinity chromatography on maltose-binding
protein·mApg12p-immobilized-Sepharose. The polyclonal anti-GFP
antibody was purchased from CLONTECH.
Cloning of hMAP-LC3 cDNA--
The advanced BLAST search
program from the National Center for Biotechnology Information was used
to search for homologs in the human and mouse EST database. Based on
the DNA sequence of EST clones, we performed rapid amplification of the
5'-cDNA ends in Marathon-Ready cDNA
(CLONTECH) by polymerase chain reaction with high
fidelity (40). The amplified DNA fragment was introduced into pGEM-T
vector, and the DNA sequence was determined. The DNA sequences of all
five independent clones were identical, and the predicted amino acids
of the clones show significant homology to rat and murine MAP-LC3. To
express GFP·hMAP-LC3 in HEK293 cells, the cloned DNA fragment was
introduced into pEGFP-C1 vector (pGFP·hMAP-LC3).
Expression of hApg Proteins in HEK293 Cells--
HEK293 cells
were maintained in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum. For transfection, 2 × 105 cells
were seeded on 60-mm dishes. After incubation for 24 h at
37 °C, the cells were transfected with a mixture of 2.5 µg of
plasmid DNA and 12 µl of FuGene-6. For cotransfection, 1 µg of each
plasmid was used. The transfectant was harvested after incubation for
an additional 48 h. 1 × 106 cells were washed
with 1 ml of phosphate-buffered saline, and resuspended in 200 µl of
phosphate-buffered saline containing Complete protease inhibitor
mixture (Roche Diagnostics). The cell suspension was lysed by
sonication for 10 s at 4 °C. Proteins in the lysate were
separated by reducing or nonreducing SDS-PAGE and transferred to a
polyvinylidene difluoride membrane (Millipore, Bedford, MA). Immunoblot
analysis was performed with anti-hApg7p and -GFP antibodies
(CLONTECH), and the blots were developed by an
enhanced chemiluminescence system (Amersham Pharmacia Biotech).
Glycerol Gradient Centrifugation--
Livers were isolated from
Wistar male rats, passed through a stainless steel mesh, and suspended
in 5 volumes of 5 mM Tes-NaOH, pH 7.5, 0.3 M
sucrose. The homogenate was centrifuged at 100,000 × g
for 1 h, and the supernatant was used as the cytosol fraction. Cytosol (0.4 ml) was loaded onto an 11.5-ml linear glycerol gradient (10-40%) in 20 mM Tes-NaOH, pH 7.5, 0.15 M
NaCl and centrifuged at 151,000 × g for 15 h
(Beckman SW-41 rotor). Fractions of 0.7 ml were collected from the
bottom of the tubes. Rat Apg7p was immunoprecipitated from each
fraction with anti-hApg7p and subjected to immunoblotting analysis,
because hApg7p cross-reacts with rat Apg7p. Authentic thyroglobulin
(670 kDa, 19 S), catalase (220 kDa, 11.2 S), aldolase (158 kDa, 7.4 S),
and bovine serum albumin (67 kDa, 4.3 S) were used as internal S-value
standards. Two-hybrid analysis was performed as described by James
et al. (39).
The Human Apg7p/Cvt2p/Gsa7p Homolog Is an E1-like
Protein-activating Enzyme for hApg12p--
In yeast, S. cerevisiae, Apg7p is a protein-activating enzyme for Apg12p (18).
If hApg7p is a protein-activating enzyme essential for the
hApg12p·hApg5p conjugation system, hApg12p will interact with hApg7p.
We first examined the interaction between hApg7p and hApg12p by a
two-hybrid experiment (Fig. 1). We
constructed yeast expression plasmids of GAL4BD-fused hApg7p
(GAL4BD·hApg7p) and GAL4AD-fused hApg12p (GAL4AD·hApg12p) and
expressed both fusion proteins in a yeast tester strain
(trp1-901 leu2-3, 112 LYS2::GAL1-HIS3). The
tester strain expressing both GAL4AD·hApg12p and GAL4BD·hApg7p grew
well on selection plate (SD-Trp-Leu-His plate), whereas strains expressing GAL4AD and GAL4BD, GAL4AD·hApg12p and GAL4BD, or GAL4AD and GAL4BD·hApg7p did not (Fig. 1). These results indicate that hApg12p interacts with hApg7p.
To investigate whether hApg7p forms an enzyme-hApg12p intermediate, we
employed site-directed mutagenesis of a predicted active site cysteine
residue within hApg7p. Based on a homology search between yeast and
human Apg7p, we predicted that the active site cysteine residue within
hApg7p must be Cys572 (Fig.
2A). If an active site
cysteine residue within an E1-enzyme is changed to serine, an
O-ester bond will be formed instead of a thiol ester bond.
Therefore, we changed Cys572 within hApg7p to Ser by
site-directed mutagenesis and expressed both the mutant
hApg7pC572S and GFP-fused hApg12p (GFP·hApg12p) in HEK293
cells (Fig. 2B). Cell lysates expressing both proteins were
prepared and analyzed by SDS-PAGE. hApg7p was recognized by immunoblot
with anti-hApg7p antibody. Wild-type hApg7p and mutant
hApg7pC572S were both expressed well in HEK293 cells (Fig.
2B, wild and C572S, hApg7p of ~80
kDa). When both hApg7pC572S and GFP·hApg12p were
expressed in HEK293 cells, a higher molecular mass band consistent with
a stable GFP·hApg12p·hApg7pC572S intermediate (~140
kDa) appeared in addition to the band of ~80 kDa for free hApg7p
(Fig. 2B, C572S). This higher molecular mass band
was also recognized by immunoblotting with anti-GFP antibody in the
presence or absence of reducing reagent (data not shown). These results
indicate that hApg12p is an authentic substrate for hApg7p.
If hApg7p is an E1-like enzyme in the hApg12p-conjugation system, it is
possible that the overexpression of hApg7p will influence the
conjugation of hApg12p with hApg5p. To investigate this possibility, we
expressed both hApg7p and GFP·hApg12p in HEK293 cells, metabolically labeled the cells with 35S-labeled Met and Cys and prepared
a cell lysate. hApg12p was immunoprecipitated with anti-mApg12p
antibody, and the precipitates were analyzed by SDS-PAGE and
autoradiography. When GFP·hApg12p alone was expressed, GFP·hAPG12p
itself was immunoprecipitated with anti-mApg12p antibody (Fig.
2C, vector). In cells expressing both
GFP·hApg12p and hApg7p, a high molecular weight peptide corresponding to the hApg5p·GFP·hApg12p conjugate was immunoprecipitated in addition to GFP·hApg12p with anti-mApg12p antibody (Fig.
2C, pCMV-hAPG7). The formation of the
hApg5p·GFP·hApg12p conjugate was further confirmed by a second
immunoprecipitation using anti-hApg5p antibody (data not shown). The
overexpression of mutant hApg7pC572S did not enhance the
conjugation (data not shown). These results indicate that hApg7p is an
authentic protein-activating enzyme essential for the human
Apg12p-conjugation system.
hApg7p Forms a Homodimer--
Komatsu et al.
2 have found that yeast Apg7p forms a homodimer via the
C-terminal region. Considering the functional homology between yeast
and human Apg7p, it is likely that hApg7p will also form a homodimer.
To investigate this possibility, we conducted a cross-linking
experiment. A HEK293 cell lysate expressing hApg7p was prepared and
treated with a noncleavable cross-linker, disuccinimidyl suberate.
After cross-linking, the lysate was analyzed by SDS-PAGE, and hApg7p
was detected by immunoblotting with anti-hApg7p antibody. Before
treatment with the cross-linking reagent, hApg7p was detected in the
cell lysate as a band corresponding to ~80 kDa (Fig.
3A, pCMV-hAPG7,
DSS
We next analyzed endogenous Apg7p in rat liver cytosol by glycerol
density gradient ultracentrifugation using the cross-reactivity of the
anti-hApg7p antibody with rat Apg7p. The cytosolic fraction of a rat
liver homogenate was prepared and subjected to a 10-40% glycerol
density gradient centrifugation. Rat Apg7p was immunoprecipitated with
anti-hApg7p antibody. The resulting precipitates were analyzed by
SDS-PAGE, and rat Apg7p was identified by immunoblotting with anti-hApg7p antibody. Rat Apg7p was collected in fractions 11-14 and
sedimented mainly with a sedimentation coefficient of ~7.4 S (Fig.
3B, fraction 13). A two-hybrid experiment also
indicated that hApg7p interacts with itself (data not shown). Therefore we conclude that mammalian Apg7p forms a homodimer similar to yeast Apg7p.
All Three hApg8p Proteins (MAP-LC3, GATE-16, and GABARAP), Are
Substrates of hApg7p--
Recent findings have indicated that yeast
Apg7p also functions as an activating enzyme for Apg8p and is essential
for Apg8p targeting to autophagosomal membranes (22, 26).2
A BLAST search suggested that there are at least three Apg8p homologs,
GATE-16 (human), GABARAP (human, mouse, and rat), and MAP-LC3 (rat) in
mammalian cells. A BLAST search of the human EST database suggested
that there are human MAP-LC3 homologs (GenbankTM/EBI
accession numbers AI365977, AA476809, and AI382200). hMAP-LC3 was
isolated from a human brain cDNA library by rapid amplification of
the 5'-cDNA ends according to the information obtained from the
BLAST search. The amino acid sequence of hMAP-LC3 shows 95.9% identity
with its rat counterpart. The C-terminal regions of the three hApg8p
proteins show significant homology to yeast Apg8p. Considering the
significant homology between human and yeast Apg8p, these proteins may
also be substrates for Apg7p.
To investigate whether these Apg8p homologs interact with hApg7p as
substrates, we first performed a coimmunoprecipitation experiment.
hMAP-LC3, hGATE-16, and hGABARAP were expressed as GFP fusion proteins
together with hApg7p in COS7 cells. Cell lysates expressing both hApg7p
and a GFP fusion protein were prepared. The GFP fusion proteins were
immunoprecipitated using anti-GFP antibody. The precipitates were
analyzed by SDS-PAGE, and hApg7p was recognized by immunoblotting using
anti-hApg7p antibody. hApg7p coimmunoprecipitated with GFPhGATE-16,
GFPhGABARAP, and GFPhMAP-LC3 but not with GFP alone (Fig.
4A). The results indicate that
hGATE-16, hGABARAP, and hMAP-LC3 interact with hApg7p.
We next examined the formation of stable conjugates of mutant
hApg7pC572S with hGATE-16, hGABARAP, and hMAP-LC3 via an
O-ester bond. hApg7pC572S was coexpressed with
GFPhGATE-16, GFPhGABARAP, or GFPhMAP-LC3 in COS7 cells, and the cell
lysates were analyzed by SDS-PAGE under reducing conditions. The
GFP·hApg8p homologs were recognized by immunoblotting with anti-GFP
antibody. A high molecular weight band corresponding to a stable
hApg7pC572S substrate intermediate was detected in cell
lysates expressing both hApg7pC572S and a GFP·Apg8p
homolog, but not in cells expressing both wild-type hApg7p and
GFP·Apg8p homologs, indicating that a stable conjugate is formed
between hApg7pC572S and the Apg8p homologs (Fig.
4B). These results indicate that all three hApg8p proteins,
hGATE-16, hGABARAP, and hMAP-LC3, are authentic substrates for hApg7p.
In this study, we showed that the human Apg7p homolog is an
authentic E1-like enzyme for the hApg12p conjugation system and that
hGATE-16, hGABARAP, and hMAP-LC3 are substrates for hApg7p. GATE-16 as
a soluble transport factor interacts with NSF and GOS-28, is localized
in the Golgi, and is expressed in the largest amount in brain (33).
GABARAP is GABAA receptor-associated protein that
colocalizes with the GABAA receptor in cultured cortical neurons and interacts with gephyrin (31, 32, 34). MAP-LC3 is localized
on autophagosomal membranes (35). Considering the divergent functions
and intracellular localizations of the three Apg8p homologs, it is
surprising that all three human Apg8p homologs are substrates for
hApg7p. Because yeast Apg7p plays an indispensable role in autophagy
and the Cvt transport of aminopeptidase I, mammalian Apg7p must also be
essential for autophagy and other forms of membrane transport, common
phenomena involving the formation of cup-shaped and/or elongated
membrane structures.
Because MAP-LC3 is localized on autophagosomal membranes in rat
liver as in yeast Apg8p (35), at least two substrates, MAP-LC3 and
hApg12p, play major roles in autophagy in mammalian cells. At present,
there has been no report of a mammalian Cvt-like pathway. Considering
the strong expression of GATE-16 and GABARAP in brain and neuronal
cells, a Cvt-like pathway and/or other membrane transport pathways in
which GATE-16 and GABARAP function as protein modifiers may also exist
in these tissues. It is difficult to explain how hApg7p distinguishes
the four substrates and regulates the multiple interactions among the
substrates. There must be some regulatory factors associated with the
hApg7p homodimer to form multimeric complexes. Further candidates
related to hApg7p will be sought by a two-hybrid experiment using a
human brain cDNA library and coimmunoprecipitation of rat Apg7p
with anti-hApg7p antibody in several rat tissues.
At present, the target proteins of GATE-16, GABARAP, and MAP-LC3 remain
unknown. There is no report that these proteins conjugate with other
proteins. We have recognized no targeting protein with which MAP-LC3
forms a conjugate. Kabeya et al. (35) reported that MAP-LC3
is processed to several forms in cultured mammalian cells, so there may
exist some unknown mechanism. Further biochemical analysis of hApg8p is necessary.
It has become more and more evident that the Apg machinery plays an
important role in at least brain and cardiac and skeletal muscles.
Clinical and biochemical analyses of a group of severe inheritable
neurodegenerative disorders, neuronal ceroid-lipofuscinosis, have
suggested that lysosomal degradation via autophagy occurs actively
during neuronal development (for a review see Ref. 41). Furthermore,
clinical, genetic and biochemical analyses of X-linked vacuolar
cardiomyopathy, myopathy in humans and LAMP-2-deficient mice have
indicated that autophagic processes play an indispensable role in
normal mammalian bodies (42, 43). In addition, autophagy is activated
by apoptotic signaling in sympathetic neurons (44). In view of the
ubiquitous distribution of hApg12p and human Apg8p homologs, GATE-16,
GABARAP, and MAP-LC3, and the multiple reactivity of hApg7p with these
different substrates, it is possible that mammalian Apg7p plays an
essential role in various stages of development and apoptosis in
addition to autophagy. We are now investigating the possible
tissue-specific functions of hApg7p using APG7 gene-knockout mice. These studies will contribute to the understanding of the physiological functions of mammalian Apg7p.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells, the
host for plasmids and protein expression, were grown in Luria Broth
medium in the presence of antibiotics as required (37). Standard
genetic and molecular biological techniques were performed as described
(37, 38). The yeast strain for two hybrid experiments was cultured in
SD medium (0.67% yeast nitrogen base without amino acids, 2% glucose,
and appropriate amino acids) as described previously (38). Protein was
determined by BCA protein assay following the manufacturer's protocol
(Pierce, Rockford, IL). The polymerase chain reaction was performed
with a programmed temperature control system PC-701 (ASTEC, Fukuoka,
Japan). The DNA sequence was determined with an ABI 373A DNA sequencer
(PE Biosystems, Foster City, CA). DNA plasmid was transfected into
mammalian cells with FuGene-6 transfection reagent according to the
manufacturer's protocol (Roche Diagnostics, Mannheim, Germany).
Restriction enzymes were purchased from TOYOBO (Osaka, Japan) and New
England BioLabs (Beverly, MA). Oligonucleotides were synthesized by the
ESPEC oligo-service (Ibaraki, Japan). pGAD-C1 vector, pGBD-C1 vector,
and PJ69-4A strain were kind gifts from P. James (39). pcDNA3 was
purchased from Invitrogen (Carlsbad, CA), pGEM-T was from PROMEGA
(Madison, WI), pEGFP-C1 and pEGFP-N1 were from
CLONTECH, and pBluescriptII (SK+) was from
Stratagene (La Jolla, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
hApg7p interacts with the hApg12p in
vivo. pGBD-hAPG7 (TRP1) and pGAD-hAPG12
(LEU2) were transformed into PJ69-4A cells (trp1 leu2
LYS2::GAL1-HIS3) to express GAL4BD· hApg7p and
GAL4AD·hApg12p, respectively. pGAD-C1 and pGBD-C1 were used as
controls. Cells were plated on SD-Trp-Leu plate (positive
control) and SD-Trp-Leu-His plate (selective condition) and incubated
at 30 °C for 3 days. PJ69-4A cell strains counterclockwise from the
right carried pGBD-C1 and pGAD-C1 (GBD GAD),
pGBD-C1 and pGAD-hAPG12 (GBD GAD-hApg12p), pGBD-hAPG7 and
pGAD-C1 (GBD·hApg7p GAD), pGBD-hAPG7 and pGAD-hAPG12
(GBD·hApg7p GAD-hApg12p). A strain expressing both
GAL4BD·hApg7p and GAL4AD·hApg12p grew well on the SD-Trp-Leu-His
plate, indicating that hApg7p interacts with hApg12p.
View larger version (19K):
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Fig. 2.
hApg7p conjugates with hApg12p to form a
hApg7p·hApg12p intermediate and enhances the formation of the
hApg5p·hApg12p conjugate. A, comparison of the region
containing the active site cysteine residue between hApg7p and yeast
Apg7p/Cvt2p. The region of hApg7p (Human APG7; 548-598 of
703 amino acids) was compared with the corresponding region of yeast
Apg7p (Yeast APG7; 483-533 of 630 amino acids). The
predicted active site cysteine residue of hApg7p and the authentic
active site cysteine residue of yeast Apg7p are underlined.
Identical amino acids are indicated by asterisks.
B, formation of a stable
hApg7pC572S·GFPh·Apg12p conjugate in HEK293 cells. The
expression plasmid for hApg12p fused to GFP under the control of
cytomegalovirus-enhancer and promoter was constructed (pGFP·hApg12p).
The pGFP·hApg12p plasmid was cotransfected with pCMV-hAPG7
(wild) or pCMV-hAPG7C572S (C572S) into HEK293
cells. Lysates of the transfectant were prepared as described under
"Experimental Procedures," and analyzed by reducing and nonreducing
SDS-PAGE. hApg7p was recognized by immunoblot with
anti-hApg7p antibody. The hApg7pC572S·GFP·hApg12p
stable intermediate was recognized in the presence or absence of
reducing reagent. hApg7p, free hApg7p;
hApg7p-GFPhApg12p, hApg7pC572S·GFP·hApg12p
intermediate. C, enhancement of the formation of
hApg5p·GFP·hApg12p conjugate by overexpression of wild-type hApg7p.
HEK293 cells expressing both GFP·hApg12p and wild-type hApg7p were
metabolically labeled with [35S]Met and Cys. The cells
were lysed, and hApg12p was immunoprecipitated with anti-mouse Apg12p
antibody. The precipitate was analyzed by SDS-PAGE and subjected to
autoradiography. Both pGFP·hAPG12 and one of pCMV-hAPG7
(pCMV-hAPG7) or vector control was transfected into HEK293
cells. When wild-type hApg7p was expressed in HEK293 cells, an
hApg5p·GFP·hApg12p conjugate was recognized
(hApg5p-GFPhApg12p conjugate) in addition to GFP·hApg12p
(GFP-hApg12p).
). After cross-linking, the amount of this 80-kDa band
was decreased, and a broad band at ~160 kDa appeared (Fig. 3A, pCMV-hAPG7, DSS+).
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Fig. 3.
Mammalian Apg7p forms a homodimer.
A, appearance of an hApg7p band at ~160 kDa in addition to
a band at an ~80 kDa with cross-linking. Cells expressing hApg7p were
lysed, and the lysate was treated with 5 mM of
disuccinimidyl suberate, as described under "Experimental
Procedures." hApg7p was recognized by SDS-PAGE followed by
immunoblotting with anti-hApg7p antibody. B, endogenous rat
Apg7p homodimer in rat liver. The cytosolic fraction was prepared from
rat liver and was separated by centrifugation through a 10-40%
glycerol gradient. Fractions were collected from the bottom of the
gradients and assayed for the presence of rat Apg7p by immunoblotting
using anti-hApg7p antibody. The positions of marker proteins in the
gradient are indicated above the blot. kDa, molecular mass;
S, sedimentation value.
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[in a new window]
Fig. 4.
GATE-16, GABARAP, and MAP-LC3 are also
substrates of hApg7p. A, coimmunoprecipitation of
hApg7p with GATE-16, GABARAP, and MAP-LC3. Expression plasmids for
hApg7p and GFP fusion proteins were cotransfected into COS7 cells. Cell
lysates were prepared, and the GFP fusion proteins were
immunoprecipitated (IP) with anti-GFP antibody
( GFP). The precipitates were analyzed by
SDS-PAGE, and hApg7p was recognized by immunoblot with anti-hApg7p
antibody. All GFP fusion proteins immunoprecipitated well (data not
shown). GFPhGATE-16, pGFP·hGATE-16 plasmid;
GFPhGABARAP, pGFP·hGABARAP plasmid;
GFPhMAP-LC3, pGFP·hMAP-LC3 plasmid;
GFP, pGFP-C1 vector, hApg7p wild, pCMV-hAPG7
plasmid. Pairs of plasmids for cotransfection are indicated as plus
(+). Whereas hApg7p did not coimmunoprecipitate with GFP alone, it
coimmunoprecipitated with GFP·hGATE16, GFP·hGABARAP, and
GFP·hMAP-LC3. B, formation of
hApg7pC572S·hApg8p intermediates. Cells expressing hApg7p
and a GFP fusion protein were lysed and analyzed by SDS-PAGE. GFP
fusion proteins were recognized by immunoblot with anti-GFP antibody.
hApg7p wild+, cells expressing wild-type hApg7p;
hApg7p C572S+, cells expressing mutant
hApg7pC572S; GFPhGATE-16, cells coexpressing
GFP·hGATE-16 with hApg7p; GFPhGABARAP, cells coexpressing
GFP·hGABARAP with hApg7p; GFPhMAP-LC3, cells coexpressing
GFP·hGATE-16 with hApg7p. hApg7pC572S·GFP fusion
protein intermediates are indicated (conjugate) in addition
to GFP fusion proteins (GFP fusion). The asterisk
indicates a nonspecific band that reacted with the anti-GFP
antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Y. Ohsumi, T. Yoshimori, T. Noda, N. Mizushima, Y. Ichimura, K. Kirisako (National Institute for Basic Biology), D. J. Klionsky (University of California, Davis), and M. Komatsu (Juntendo University) for important discussions and information; P. James (University of Wisconsin) for providing strains and plasmids; and K. Ishidoh, J. Ezaki, and D. Muno (Juntendo University) for helpful discussions.
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FOOTNOTES |
---|
* This work was supported in part by Grants-in-aid 12780543 (to I. T.), 09680629 (to T. U.), and 12470040 (to E. K.) for Scientific Research, Grants-in-aid 12146205 (to E. K.) for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan, and The Science Research Promotion Fund from the Japan Private School Promotion Foundation (to E. K.).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 correspondence should be addressed: Dept. of Biochemistry,
Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo
113-8421, Japan. Tel.: 81-3-5802-1031; Fax: 81-3-5802-5889; E-mail:
kominami@med.juntendo.ac.jp.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.C000752200
2 M. Komatsu, I. Tanida, T. Ueno, M. Ohsumi, Y. Ohsumi, and E. Kominami, submitted manuscript.
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ABBREVIATIONS |
---|
The abbreviations used are:
apg and
APG, yeast autophagy mutant and wild-type genes;
Apgp, expression products from the APG gene;
Cvt, cytoplasm-to-vacuole targeting;
GABARAP, -aminobutyric acid
receptor-associated protein;
GAL4AD, GAL4 activation domain;
GAL4BD, GAL4 DNA binding domain;
GATE-16, Golgi-associated
ATPase enhancer of 16 kDa;
GFP, green fluorescent protein;
h, human;
m, murine;
MAP-LC3, microtubule-associated protein light chain 3;
NSF, N-ethylmaleimide-sensitive fusion protein;
SNARE, soluble
NSF attachment protein receptor;
PAGE, polyacrylamide gel
electrophoresis;
EST, expressed sequence tag;
HEK, human embryonic
kidney cells;
UBA, ubiquitin-activating enzyme;
UBC, ubiquitin-conjugating enzyme. Tes,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid;
ER, endoplasmic reticulum.
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