1 PRESTO, Japan Science and Technology Corporation, Kawaguchi 332-0012,
Japan
2 Department of Cell Biology, National Institute for Basic Biology, 38
Nishigonaka, Myodaiji, Okazaki 444-8585, Japan
3 Department of Molecular Biomechanics, School of Life Science, The Graduate
University for Advanced Studies, Okazaki 444-8585, Japan
4 Department of Physiology, Kansai Medical University, Moriguchi 570-8506,
Japan
5 Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka
565-0871, Japan
6 National Institute of Advanced Industrial Science and Technology, Tokyo
135-0064, Japan
7 Department of Cell Genetics, National Institute of Genetics, Mishima 411-8540,
Japan
* Authors for correspondence (e-mail: nmizu{at}nibb.ac.jp; yohsumi{at}nibb.ac.jp)
Accepted 21 January 2003
![]() |
Summary |
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Key words: Autophagy, Apg12, Apg5, WD repeat
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Introduction |
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Although Apg12-Apg5 functions to elongate the isolation membrane, most
Apg12-Apg5 exists freely in the cytosol
(Mizushima et al., 2001;
Kuma et al., 2002
). In yeast,
the Apg12-Apg5 conjugate further interacts with a small coiled-coil protein,
Apg16 (Mizushima et al.,
1999
). Apg16 forms a homo-oligomer through its coiled-coil region.
As each Apg16 molecule interacts with Apg5, Apg16 homo-oligomers cross-link
multiple Apg12-Apg5 conjugates. As a result, Apg12-Apg5 and Apg16 form a
350 kDa protein complex thought to contain four sets of Apg12-Apg5 and
Apg16 (Kuma et al., 2002
). We
have demonstrated that the formation of this
350 kDa complex is essential
for autophagy. In addition, we determined that Apg16 is localized to the
preautophagosomal structure and is required for membrane targeting of Apg5
(Suzuki et al., 2001
). As yet,
no molecules structurally related to Apg16 have been found in other species
through database analysis. Because the structure and function of the
Apg12-Apg5 conjugate is well conserved in mammals, we postulated the existence
of a functional Apg16 counterpart. In this study, we demonstrate that, in
mammalian cells, Apg12-Apg5 forms an 800 kDa protein complex with a novel
WD-repeat protein. Because biochemical and morphological analyses suggested
that it is the mammalian counterpart of yeast Apg16, we named it Apg16L
(Apg16-like protein). The C-terminal WD-repeat domain, however, is found only
in Apg16L. Because the WD-repeat domain mediates protein-protein interactions,
Apg16L might function as a scaffold for a protein complex functioning in
autophagosome formation.
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Materials and Methods |
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Immunoprecipitation
Cells grown on 35 mm dishes were labeled with 0.4 mCi of
[35S]methionine/cysteine (NEN Life Science Products) for 2 hours
when indicated. After cells were lysed in lysis buffer (2% NP-40 in PBS
supplemented with protease inhibitors) for 20 minutes, the nuclear and
cellular debris was cleared by centrifugation. Immunoprecipitation was
performed using rabbit polyclonal anti-green-fluorescent-protein (GFP; MBL),
mouse monoclonal anti-FLAG (M2) (Sigma) or anti-hemagglutinin (HA) antibody
(16B12) (Babco) and protein-A/Sepharose or protein-G/Sepharose (Amersham
Biosciences). Immunoprecipitates were washed six times in lysis buffer and
eluted in SDS sample buffer. Proteins were separated by SDS-PAGE and analyzed
by either a bioimage analyser BAS2000 (Fuji Film) or immunoblotting as
described (Mizushima et al.,
1998b).
Protein purification and mass spectrometry
Total cell lysates were prepared from 10-20 15 cm dishes of ES cells. The
protein complex containing GFP-Apg5 was purified by passing the lysates over
an anti-GFP-antibody-coupled protein-A/Sepharose bead column. After extensive
washing, bound proteins were eluted in 0.1 M glycine-HCl (pH 2.5), separated
by SDS-PAGE and visualized with Coomassie Brilliant Blue or silver staining.
Following excision from the gels, proteins of 63 kDa and 71 kDa were digested
in situ with lysyl-endopeptidase or trypsin. The resultant peptides were
subjected to matrix-assisted laser desorption ionization (MALDI) mass
spectrometry (MS). Proteins were identified by database searching based on
their peptide masses. For further unambiguous protein identification, the same
bands were digested with lysyl-endopeptidase and then analysed by the direct
nano-liquid-chromatography tandem MS system
(Natsume et al., 2002) in
conjunction with searches of the NCBInr database using Mascot software.
Plasmids
The cDNA corresponding to the open reading frame of mouse Apg16L was
obtained by polymerase chain reaction of IMAGE consortium clone 1480862
(GenBank accession number AI037166). This fragment was then cloned into the
SalI site of pCI-neo (Promega), the XbaI site of
p3XFLAG-CMV-10 (Sigma) and the SalI sites of pEGFP-C1, pECFP-C1 and
pEYFP-C1 (Clontech) to generate pCI-Apg16L, pFLAG-Apg16L, pEGFP-Apg16L,
pECFP-Apg16L and pEYFP-Apg16L, respectively. The mouse Apg5 and rat LC3 cDNAs
were also subcloned into pECFP-C1 (pECFP-Apg5) and pEYFP-C1 (pEYFP-LC3). To
attain better expression in ES cells, the Apg16L, FLAG-Apg16L, EGFP-Apg16L,
ECFP-Apg16L and EYFP-Apg16L fragments were also subcloned into pCE-FL (a gift
from S. Sugano), a vector containing a cytomegalovirus enhancer and elongation
factor promotor. pApg5-HA and pEGFP-Apg5 constructs have been described
previously (Mizushima et al.,
1998b
; Mizushima et al.,
2001
). For yeast two-hybrid analysis, various regions of the
Apg16L
cDNA corresponding to amino acids 1-588 (full length), 1-276,
1-79, 72-276 and 219-588 were cloned into the SalI sites of pGBD-C1
and pGAD-C1. The mouse Apg5 cDNA was also cloned into the SmaI sites
of pGBD-C1 and pGAD-C1. For expression of glutathione-S-transferase
(GST)-tagged Apg16L
(219-588) in Escherichia coli, a cDNA
encoding amino acids 219-588 of Apg16L
was first cloned into the
SalI site of pENT1A from the GATEWAY cloning system (Invitrogen). A
GST-Apg16L (219-588) expression plasmid (pDEST15-Apg16LC) was then generated
according to the manufacturer's instructions.
Generation of antibody against mouse Apg16L
To generate an antibody against mouse Apg16L (p63C-2), pDEST15-Apg16LC was
transformed into BL21-SI competent cells (Invitrogen). Expression of
C-terminal half of Apg16L fused to GST was induced for 2 hours with 0.3 M
NaCl. The recombinant protein, contained in inclusion bodies, was separated by
SDS-PAGE and isolated as previously described
(Kuma et al., 2002). The eluted
protein was then used to immunize rabbits.
Reverse-transcription PCR
Total RNA isolated from mouse liver, brain, kidney, ES cells and HeLa cells
was subjected to reverse transcription using a ProSTARTM First-Strand
RT-PCR kit (Stratagene). A part of the Apg16L cDNA corresponding to exons 6-10
was amplified with primers p63-4Bam
(5'-ACGTGGATCCAGGAGGCGTCAAGCACGGCTG-3') and p63-22Sal
(5'-GAACGTGTCGACCTGGGGGACTGGGATGGAAGAGAC-3').
Yeast two-hybrid assay
The two-hybrid analysis was performed as described
(James et al., 1996). The
strain PJ69-4A was co-transformed with one of each of the pGBD and pGAD
plasmids. Transformants were selected on SC Trp Leu plates and
tested for growth on SC His Trp Leu plates containing 3
mM 3-amino-triazole (3-AT).
Differential centrifugation and gel filtration
The liver and brain of a C57BL/6N Crj mouse were homogenized in nine
volumes of ice-cold PBS supplemented with protease inhibitors. ES cells were
lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl and protease
inhibitors] by passing the solution through a polycarbonate filter with
5-µm pores (Whatman). After a preclearing step at 100 g for
5 minutes, lysates were subjected to low-speed centrifugation at 13,000
g for 20 minutes to generate a pellet (P13) fraction. The
resulting supernatant was further centrifuged at 100,000 g for
1 hour to generate pellet (P100) and supernatant (S100) fractions. The S100
fraction (0.3 mg protein in 200 µl) was then applied to a Superose 6
column (Amersham Biosciences) and eluted at a flow rate of 0.4 ml
min-1 with 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM DTT. 0.6 ml
fractions were then examined by immunoblotting. The column was calibrated with
gel filtration protein standards (Amersham Biosciences) containing
thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa) and albumin
(67 kDa).
Fluorescence microscopy
ES cells expressing protein fused to GFP, yellow fluorescent protein (YFP)
or cyan fluorescent protein (CFP) were directly observed with a Delta Vision
microscope system (Applied Precision Incorporation). For examination by
immunofluorescence microscopy, ES cells grown on gelatinized coverslips were
fixed and stained with an anti-mouse Apg16L antibody (200x dilution) and
a Cy5-conjugated goat anti-rabbit IgG antibody (Amersham Biosciences). Samples
were examined under a fluorescence laser scanning confocal microscope, LSM510
(Carl Zeiss) as previously described
(Yoshimori et al., 2000).
Electron microscopy
ES cells, grown on gelatinized plastic coverslips, were fixed for 2 hours
with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, and then
subjected to the pre-embedding silver-enhancement immunogold method for
immunoelectron microscopy using an antibody against GFP
(Mizushima et al., 2001).
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Results |
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As the 800 kDa complex is also detected in APG5-/-
mouse ES cells stably expressing GFP-fused Apg5 (clone GFP24)
(Mizushima et al., 2001
) (data
not shown), we identified additional subunits of this complex by
co-immunoprecipitation using an antibody against GFP. Anti-GFP
immunoprecipitates from total cell lysate of 35S-labeled GFP24
cells contained several specific proteins of 63 kDa, 71 kDa and 144 kDa, in
addition to GFP-Apg5 and Apg12/GFP-Apg5
(Fig. 1A). These proteins were
not present in precipitates from wild-type ES cells expressing GFP alone.
|
To identify these proteins, cell lysates derived from GFP24 ES cells were
passed over an anti-GFP antibody-coupled protein-A/Sepharose column. Bound
proteins were eluted and separated by SDS-PAGE
(Fig. 1B). Coomassie-stained
gel bands corresponding to p63 and p71 were digested in situ with
lysyl-endopeptidase or trypsin. MS protein identification revealed that both
p63 and p71 represent a novel protein (isoforms, see below), predicted by
several expressed sequence tags (ESTs; AI037166, BI687378, BB620083, AA982950,
BE3714456, BB660407, BB839395, BB853783 etc.)
(Fig. 2A). This protein
contains a coiled-coil region at the N-terminal region (amino acids 91-190)
and seven WD repeats, which are implicated in protein-protein interactions
(Smith et al., 1999), in the
C-terminus (outlined in black in Fig.
2A). Although this protein is much larger than yeast Apg16, the
N-terminal region (amino acids 106-208) demonstrated a weak but significant
homology with yeast Apg16 (amino acids 28-145) (22% identity and 43%
similarity) (Fig. 2B).
Therefore, we tentatively named it Apg16L (Apg16-like protein). Putative
Apg16L homologues are present in various eukaryotes, but are not present in
Saccharomyces cerevisiae (Fig.
2C).
|
Spliced isoforms of Apg16L
We generated polyclonal antibodies against the C-terminal region of mouse
Apg16L. Immunoblot analysis of mouse tissue demonstrated that Apg16L was
expressed ubiquitously (Fig.
3B, data not shown for heart, spleen, thymus, testis). The size of
Apg16L, however, differed among the tissues and cell lines tested. The liver,
kidney (Fig. 3B), spleen,
thymus and testes (data not shown) contained a major form of 63 kDa with a
minor form of 71 kDa. This expression pattern, consistent with the results of
immunoprecipitation and affinity purification using anti-GFP antibody
(Fig. 1), was also observed in
ES cells (Fig. 3B). By
contrast, a larger 75 kDa form was the most abundant species in brain,
skeletal muscle (Fig. 3B) and
heart (data not shown). Both the 63 kDa and 71 kDa proteins were the major
forms in HeLa cells. The genomic DNA sequence and the cDNA sequences of the
EST database suggested that the APG16L gene encodes multiple
isoforms. We confirmed by sequence analysis that at least three isoforms of
Apg16L were generated by alternative splicing events
(Fig. 3A). The major cDNA
present in the liver, designated Apg16L, lacks all of exons 8 (57 bp)
and 9 (48 bp). The minor cDNA present in the liver, designated Apg16Lß,
lacks exon 9. The major cDNA present in the brain, designated Apg16L
,
contains the complete sequences of both exons 8 and 9. The peptides encoded by
exons 8 and 9, deleted in Apg16L
and Apg16Lß, are located between
the coiled-coil region and WD repeat. Transient transfection of the
Apg16L
cDNA into HeLa cells increased the levels of p63
(Fig. 3B, lane 7). Therefore,
the p63 and p71 proteins affinity purified from ES cell lysates represented
Apg16L
and Apg16Lß, respectively. The reverse-transcription-PCR
pattern amplifying exons 8 and 9 is shown in
Fig. 3C.
|
Apg16L interacts with Apg5 and Apg16L itself
The isolation of mouse Apg16L from the Apg5 complex suggests that Apg16L
has a function similar to yeast Apg16. We thus examined the interaction of
Apg16L with Apg5. HeLa cells were transiently transfected with FLAG-tagged
mouse Apg16L and/or HA-tagged Apg5. Immunoprecipitation analysis using
anti-FLAG or anti-HA antibody revealed that Apg16L and Apg5 were specifically
co-precipitated (Fig. 4A). Both
Apg12-conjugated and unconjugated Apg5 were immunoprecipitated with the
anti-FLAG antibody, suggesting that Apg16L interacts directly with Apg5 but
not with Apg12. We determined the specific Apg5-binding domain of Apg16L using
a yeast two-hybrid assay. The extreme N-terminal region of Apg16L (amino acids
1-79), upstream of the coiled-coil region, was sufficient to interact with
Apg5 (Fig. 4C). This result is
consistent with the interaction of yeast Apg16 with yeast Apg5 through the
corresponding region (Mizushima et al.,
1999
). We also observed that the N-terminal region of Apg16L
(amino acids 1-276) homodimerizes (Fig.
4C), although we could not determined the region required in
greater detail. As yeast Apg16 forms homo-oligomers through its coiled-coil
domain, we speculate that the coiled-coil domain of mouse Apg16L also
self-associates. These data also demonstrated that the WD domain is not
required for either Apg5 interaction or homo-oligomerization.
|
Although we could not detect full-length Apg16L homo-dimerization by two-hybrid analysis, this self-association was demonstrated by co-immunoprecipitation experiments in wild-type ES cells transiently expressing both GFP- and FLAG-tagged Apg16L. When GFP-tagged Apg16L was precipitated with an anti-GFP antibody, FLAG-tagged Apg16L was co-precipitated (Fig. 4B) and vice versa (data not shown). Apg16L self-interaction does not require Apg5, because similar results were obtained when using APG5-/- ES cells (Fig. 4B). Thus, Apg16L probably forms a homo-oligomer.
Apg12-Apg5 and Apg16L form a 800 kDa protein complex
In yeast, Apg12-Apg5 forms a stable 350 kDa protein complex with Apg16
in the cytosol (Kuma et al.,
2002
). We therefore examined the composition of the mammalian
complex by probing for mammalian Apg12-Apg5 and Apg16L. As previously
reported, in wild-type ES cells, almost all Apg5 was conjugated to Apg12,
mainly recovered in the cytosolic fraction by differential centrifugation
analysis (Mizushima et al.,
2001
). Apg16L was also primarily recovered in 100,000
g supernatants of both APG5+/+ and
APG5-/- ES-cell homogenates, suggesting that most Apg16L
is present in the cytosol (Fig.
5A). Mouse tissues (liver, brain, kidney and testes) demonstrated
similar results (data not shown). Although the presence of physiological salt
concentrations in the lysis buffer is important in differential centrifugation
experiments using yeast cell lysates (Kuma
et al., 2002
), the centrifugation results for mammalian cells were
not affected by salt concentration (data not shown).
To determine the complex molecular mass, the 100,000 g
fraction was then subjected to gel filtration analysis using a Superose 6
column; subsequent immunoblotting of eluate fractions with anti-Apg5 and
anti-Apg16L antibodies determined the location of the complex. The Apg12-Apg5
conjugate from multiple tissues and cell lines eluted primarily in fractions
corresponding to 800 kDa, with an occasional minor peak observed at
400 kDa (Fig. 5B). This
result conflicted with the data obtained using yeast cells, in which
Apg12-Apg5 and Apg16 form a
350 kDa complex
(Kuma et al., 2002
). It is
unlikely that components other than Apg12, Apg5 and Apg16L are contained in
the mammalian
800 kDa complex, because other stoichiometric subunits were
not detected (Fig. 1). The
amount of p144 precipitated with Apg5 was small enough that it would not
contribute to the molecular mass of the complex. Considering the molecular
mass of mouse Apg12 (15 kDa), Apg5 (32 kDa) and Apg16L
(66 kDa), the
800 kDa complex probably includes eight sets of Apg12-Apg5·Apg16L,
whereas the
400 kDa minor complex contains four sets.
The elution pattern of all three isoforms of mouse Apg16L was similar to
that of Apg12-Apg5 (Fig. 5B):
Apg16L and Apg16Lß (liver and ES), and Apg16L
(brain).
Co-elution of Apg12-Apg5 and Apg16L indicated that most of the Apg12-Apg5
conjugate and Apg16L are contained within the
800 kDa protein complex.
All three Apg16L isoforms can be recruited into the complex. Monomeric
Apg12-Apg5 conjugate and Apg16L were not detected. Formation of the
800
kDa complex was not affected by nutrient conditions (data not shown).
In APG5-/- ES cells, this 800 kDa complex was not
formed. Apg16L was recovered as a single peak in fractions corresponding to
250 kDa, a size larger than the molecular mass of Apg16L
monomer
(Fig. 5B). We assume that the
250 kDa complex in the APG5-/- ES cells represents a
tetrameric Apg16L oligomer.
Apg16L localizes to autophagic isolation membrane
A small proportion of the cytosolic Apg12-Apg5 conjugate localizes to
autophagic isolation membranes, playing an essential role in the elongation of
the membrane. Thus, we examined the possible co-localization of Apg16L to the
isolation membrane with Apg12-Apg5. We generated ES cells stably co-expressing
combinations of fluorescently tagged Apg5, Apg16L and LC3. LC3, a mammalian
homologue of Aut7, serves as a molecular marker of autophagosomes
(Kabeya et al., 2000). Upon the
induction of autophagy by the withdrawal of serum and amino acids, YFP-LC3
associated with both isolation membranes and completely formed autophagosomal
membranes. As autophagosome size in ES cells is larger than in other cell
lines, LC3 labeling was often identified as a ring-shaped structure even when
using conventional fluorescent microscopy
(Fig. 6A, right). Apg5,
however, was present only on the isolation membranes enclosing the cytoplasm,
not on ring-shaped autophagosome structures
(Fig. 6A, middle)
(Mizushima et al., 2001
). The
subcellular localization of Apg16L was similar to Apg5; CFP-Apg5-positive
isolation membranes clearly co-labeled with YFP-Apg16L
(Fig. 6B). Although the
Apg16L-labeled structure was positive for LC3, Apg16L never co-localized with
the LC3-positive ring-shaped structures
(Fig. 6C).
|
Apg5 is localized not only to cup-shaped isolation membranes but also to
small crescent-shaped vesicles (Mizushima
et al., 2001). Immunoelectron microscopy revealed that Apg16L
associates extensively with small vesicles, considered to be isolation
membranes in the early stages of formation
(Fig. 7A,B). Although Apg16L
associated with the membrane throughout isolation membrane development, its
localization gradually became asymmetric, as observed for Apg5
(Mizushima et al., 2001
).
Apg16L was primarily found on the outer membrane of the cup-shaped isolation
membrane, with only a little on the inner membrane
(Fig. 7C). Apg16L was absent
from the completed autophagosomal membrane
(Fig. 7D) and autolysosomes
(data not shown). These results suggest that Apg16L, in conjunction with the
Apg12-Apg5 conjugate, localizes to the autophagic isolation membrane at the
beginning of elongation and dissociates from the membrane at the completion of
autophagosome formation.
|
Membrane targeting of Apg16L requires Apg5 but not Apg12
conjugation
To examine the membrane targeting of Apg16L further, we examined endogenous
Apg16L by indirect immunofluorescence microscopy. GFP-Apg5-expressing ES cells
were stained with an antibody against Apg16L. Apg16L staining co-localized
with the GFP-Apg5 dots. There were no nonspecific dots
(Fig. 8A), validating the
specificity of the antibody. Using this antibody, we determined the role of
Apg5 in the membrane association of Apg16L. Wild-type ES cells exhibited the
formation of numerous Apg16L dots after 1 hour of amino acid starvation
(Fig. 8C). By contrast, no
punctate spots were observed in APG5-/- ES cells
(Fig. 8D), suggesting that Apg5
is required for the membrane targeting of Apg16L.
|
We reported previously that Apg12 conjugation is not required for the
membrane targeting of Apg5 using APG5-/- ES cells stably
expressing a GFP-labeled, conjugation-defective Apg5K130R, in which
the Apg12 acceptor lysine residue was replaced (clone GKR-1)
(Mizushima et al., 2001).
Using these cells, we also examined the role of the Apg12-conjugation of Apg5
in the membrane association of Apg16L. In GKR-1 cells, the accumulating small
GFP-positive structures (autophagosome precursors) were well stained with
anti-Apg16L antibodies (Fig.
8B), suggesting that membrane targeting of Apg16L requires Apg5,
but not a covalent attachment with Apg12.
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Discussion |
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Multiple homologs of Apg16L in higher eukaryotes possess WD repeats in the
C-terminal region, which is absent from yeast Apg16. This motif, first
identified in the ß subunits of trimeric G proteins, forms a
ß-propeller structure, which creates a stable platform for simultaneous
interactions with multiple proteins (Smith
et al., 1999). This symmetrical structure usually consists of at
least four, and typically seven, repeats. Because the WD domain of Apg16L is
not involved in either the interaction with Apg5 or homo-oligomerization, it
is likely that unknown protein(s) interact with the WD domain. Purification of
the
800 kDa complex, however, demonstrated that Apg12, Apg5 and Apg16L
are the main components (Fig.
1). Therefore, the putative WD-domain-binding protein might
interact with only a small population of the total Apg12-Apg5·Apg16L
complex, or might interact with the complex transiently. One attractive idea
is that this protein might be a receptor for the Apg12-Apg5·Apg16L
complex on the isolation membrane. Alternatively, the Apg12-Apg5·Apg16L
complex might interact with this protein in the cytosol. The p144 protein
(Fig. 1) remains a good
candidate; we are now attempting both its purification and identification.
Binding of p144 might then promote membrane association of the complex.
S. cerevisiae Apg16 lacks the C-terminal WD-repeat domain. Paz3,
an Apg16 homolog in Pichia pastoris, also lacks this region
(Mukaiyama et al., 2002) (Y.
Sakai, personal communication). Higher-eukaryotic Apg16L might contain a WD
domain because the putative binding partners of this domain might mediate
additional autophagic machinery specific to higher eukaryotes. The several
differences in autophagosome formation between yeast and mammalian cells might
be controlled by this WD-repeat-containing region. Yeast autophagosomes are
generated from a single preautophagosomal structure in the perivacuolar
region, whereas several autophagosomes can be generated at the same time from
multiple sites in mammalian cells. The size variation of autophagosomes is
also much larger in mammalian cells than in yeast cells. Therefore, higher
eukaryotes might have developed extra machinery creating these inherent
differences from yeast in autophagosome formation. In addition, an
unidentified WD-repeat protein corresponding to the C-terminal half of Apg16L
might function together with Apg16 in yeast. Genome sequencing revealed that
yeast has at least 60 WD-repeat proteins, many of unknown function. It is also
possible that a short segment at the extreme C-terminal region (downstream of
the coiled-coil region) of yeast Apg16 could exert a function corresponding to
that of the Apg16L WD domain.
The mammalian Apg12-Apg5·Apg16L complex (800 kDa) is much
larger than the yeast complex (
350 kDa). Because it is unlikely that
other molecules are involved in the formation of this mammalian complex
(Fig. 1), we speculate that the
800 kDa complex contains eight sets of Apg12-Apg5·Apg16L, whereas
the
400 kDa minor complex (Fig.
5B) contains four sets. If so, the minor
400 kDa complex
might contain equivalent components to the yeast complex; most of these
complexes would then doubly associate in mammalian cells.
Apg16L is expressed in different isoform patterns in mouse depending on the
tissue. We identified three isoforms produced by alternative splicing. We
could not discern any differences among these isoforms in complex formation;
each can be incorporated into the 800 kDa complex. The possibility
remains, however, that the Apg16L isoforms differ in additional functions.
The intracellular localization of Apg16L is exactly the same as that of
Apg5, indicating that the Apg12-Apg5·Apg16L complex localizes to the
autophagosome precursors, remaining there until autophagosome formation is
completed. In yeast, the membrane targeting of Apg5 requires Apg16
(Suzuki et al., 2001). We
thought that Apg16 might function as a membrane anchor for Apg5. Apg16L,
however, is unable to associate with membranes in Apg5-deficient ES cells.
Therefore, Apg16L alone is not sufficient for membrane association. Apg12
conjugation of Apg5, however, is dispensable for the membrane targeting of
Apg5 and Apg16L. Thus, the interaction between Apg5 and Apg16L is necessary
and sufficient for the binding of Apg16L to the isolation membranes. Apg12
conjugation, although it is dispensable for membrane association, is required
for the involvement of the Apg5·Apg16L complex in isolation membranes
elongation (Mizushima et al.,
2001
).
Identification of Apg16L not only acquires a missing Apg homolog but also gives additional clues pertinent to understanding the molecular mechanism of autophagy. The future identification of putative WD-domain-binding proteins will provide valuable information about the function of the Apg12-Apg5·Apg16L complex in autophagosome formation and possible higher-eukaryote-specific activities of these proteins.
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Acknowledgments |
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