The EMBO Journal Vol.18 No.19 pp.5234–5241, 1999
Apg10p, a novel protein-conjugating enzyme
essential for autophagy in yeast
Takahiro Shintani, Noboru Mizushima,
Yoko Ogawa1, Akira Matsuura2,
Takeshi Noda and Yoshinori Ohsumi3
Department of Cell Biology, National Institute for Basic Biology,
Okazaki 444-8585, Japan
1Present address: Department of Neurobiology, Graduate School of
Medicine, The University of Tokyo, Tokyo 113-0033, Japan
2Present address: Department of Life Science, Tokyo Institute of
Technology, Yokohama 226-8501, Japan
Autophagy is a cellular process for bulk degradation of
cytoplasmic components. The attachment of Apg12p, a
modifier with no significant similarity to ubiquitin, to
Apg5p is crucial for autophagy in yeast. This reaction
proceeds in a ubiquitination-like manner, and requires
Apg7p and Apg10p. Apg7p exhibits a considerable
similarity to ubiquitin-activating enzyme (E1) and is
on the other hand, shows no significant similarity to
other proteins whose functions are known. Here, we
show that after activation by Apg7p, Apg12p is
transferred to the Cys-133 residue of Apg10p to
form an Apg12p–Apg10p thioester. Cells expressing
Apg10pC133Sdo not generate the Apg12p–Apg5p conju-
indicate that Apg10p is a new type of protein-conjugat-
ing enzyme that functions in the Apg12p–Apg5p con-
Keywords: autophagy/protein conjugation/protein-
conjugating enzyme/Saccharomyces cerevisiae
Proteins are often modified by a variety of moieties in
cells: phosphates, carbohydrates, lipids or proteins, etc.
Such chemical processes, called post-translational modi-
fications, modulate protein function by altering protein
activity, stability, subcellular localization or interaction
with other molecules. A well characterized type of protein
modification is ubiquitination, which is involved in select-
ive degradation of short-lived proteins, quality control
of endoplasmic reticulum proteins, turnover of plasma
membrane proteins and so on (Varshavsky, 1997;
Bonifacino and Weissman, 1998; Ciechanover, 1998;
Hershko and Ciechanover, 1998). Ubiquitin, a highly
conserved 76-amino-acid-residue protein, is transferred to
target proteins to form isopeptide bonds between the
C-terminal glycine of ubiquitin and the ε-amino group of
a lysine residue in the target protein. Ubiquitination
involves sequential enzymatic reactions by several classes
© European Molecular Biology Organization
hydrolyzes ATP and forms an intermediate of ubiquitin
adenylate, followed by the binding of ubiquitin to its own
ubiquitin is next transferred to an active-site cysteine
residue of a ubiquitin-conjugating enzyme, E2, to form
the ubiquitin thioester in a similar fashion. At the final
step, which is often catalyzed by ubiquitin-protein ligase,
E3, ubiquitin is attached to a lysine residue of the
target protein via an isopeptide bond (Varshavsky, 1997;
Bonifacino and Weissman, 1998; Hershko and Ciechan-
In recent years, the ubiquitin-related proteins have been
discovered and some of them have been found to be
covalently attached to other proteins (Johnson and
Hochstrasser, 1997; Kamitani et al., 1997; Saitoh et al.,
(Ran-specific GTPase-activating protein) (Matunis et al.,
1996, 1998; Mahajan et al., 1997, 1998), PML (Kamitani
et al., 1998; Mu ¨ller et al., 1998), Sp100 (Sternsdorf et al.,
1997, 1999) and I-κBα (Desterro et al., 1998) were found
to be covalently modified by SUMO-1 (small ubiquitin-
related modifier-1). The ligation of SUMO-1 to RanGAP1
is crucial for its association with RanBP2, a component
of the nuclear pore complex. Modification of PML or
Sp100 by SUMO-1 is also important for the assembly of
subnuclear structures termed nuclear dots or PML nuclear
bodies (Duprez et al., 1999; Mu ¨ller and Dejean, 1999).
Smt3p, a yeast homolog of SUMO-1, is essential for
viability (Johnson and Blobel, 1997) and is likely to
contribute to cell cycle regulation (Li and Hochstrasser,
1999). The enzyme system involved in conjugation of
SUMO-1/Smt3p to other proteins has been identified
recently (Gong et al., 1997, 1999; Johnson et al., 1997;
Schwarz et al., 1998; Desterro et al., 1999; Okuma et al.,
1999). SUMO-1/Smt3p is activated by an Aos1–Uba2
heterodimer and conjugated to proteins by Ubc9. NEDD8
(neural precursor cell-expressed developmentally down-
regulated gene 8) and its yeast homolog Rub1p, showing
~50% identity to ubiquitin, are conjugated to cullin/
Cdc53p, a common subunit of the multifunctional SCF
ubiquitin ligase, in a ubiquitination-like manner. The
NEDD8 conjugation pathway requires at least two
enzymes: an E1-like heterodimeric activating enzyme,
APP-BP–hUba3, and an E2-like conjugating enzyme,
hUbc12 (Osaka et al., 1998; Gong and Yeh, 1999).
The activating and conjugating enzymes of the Rub1p
conjugation pathway have also been identified as Ula1p–
Uba3p and Ubc12p, respectively (Lammer et al., 1998;
Liakopoulos et al., 1998). Although substrates of Ubc9 and
Ubc12 are not ubiquitin, Ubc9 and Ubc12 are structurally
similar to the ubiquitin E2s.
Recently, we found a novel modifier essential for auto-
phagy (Mizushima et al., 1998a). Autophagy is a cellular
enzymes. Theubiquitin-activating enzyme, E1,
Protein-conjugating enzyme essential for autophagy
process essential for bulk degradation of cytoplasmic
lipids, nucleic acids and organelles are transported to the
vacuole inyeast by macroautophagyin responseto nutrient
starvation (Takeshige et al., 1992; Baba et al., 1994,
1995). When cells face nutrient starvation, cytoplasmic
components are sequestered non-selectively by double-
membrane-bound structures termed autophagosomes and
then targeted to the lysosome/vacuole to be degraded.
Autophagy plays a central role in protein turnover, which
may be important for cellular remodeling during develop-
ment and differentiation. Taking advantage of yeast
genetics, autophagy-deficient mutants of Saccharomyces
cerevisiae (apg and aut) have been isolated independently
by two laboratories (Tsukada and Ohsumi, 1993; Thumm
overlap with cvt mutants that are based on defects in
cytoplasm-to-vacuole targeting (Cvt) of aminopeptidase I
(Harding et al., 1996; Scott et al., 1996). The Cvt pathway
uses a similar mechanism to the autophagic pathway (Baba
et al., 1997). During analyses of APG gene products,
we found a novel protein conjugation system that is
indispensable for both autophagy and the Cvt pathway
(Mizushima et al., 1998a). Apg12p is a hydrophilic 186-
amino-acid-residue protein with no similarity to ubiquitin
or ubiquitin-related modifiers, which is covalently attached
to Apg5p. The Apg12p–Apg5p conjugate further associ-
ates with Apg16p to form the Apg12p–Apg5p–Apg16p
multimeric complex (Mizushima et al., 1999). The manner
of conjugation of Apg12p to Apg5p is ubiquitination-like:
a covalent linkage of the C-terminal glycine of Apg12p
to the lysine-149 side chain of Apg5p via an isopeptide
bond. This reaction requires at least two proteins: Apg7p
and Apg10p. Apg7p is similar to ubiquitin-activating
enzyme in its amino acid sequence around the ATP binding
site and the active-site cysteine residue, and has been
found to be an E1-like Apg12p-activating enzyme
(Mizushima et al., 1998a; Tanida et al., 1999). Apg10p,
on the other hand, shows no significant similarity with
other proteins whose functions are known. Here, we show
that Apg10p is a new type of protein-conjugating enzyme
that functions in the Apg12p conjugation pathway.
apg10-1 shows a defect in autophagy
We have isolated and characterized an apg10-1 mutant
(Tsukada and Ohsumi, 1993). Wild-type cells accumulated
autophagic bodies (ABs) in their vacuoles in a nitrogen-
starvationmedium containing1mM phenylmethylsulfonyl
fluoride (PMSF), which were observed under a light
microscope (Takeshige et al., 1992 and Figure 1A, wild
type). On the other hand, the apg10-1 cells did not
accumulate ABs (Figure 1A). Moreover, the apg10-1
mutation severely reduced the viability of the cells under
the starvation condition (Figure 1B). It was found that
Apg12p is covalently attached to Apg5p via an isopeptide
linkage in a ubiquitination-like manner and this conjuga-
tion is crucial for autophagy (Mizushima et al., 1998a).
The apg10-1 mutant completely lost the ability to generate
the Apg12p–Apg5p conjugate (Mizushima et al., 1998a
and Figure 1C). These data suggest that the defect of the
Fig. 1. Cloning of the APG10 gene and phenotype of ∆apg10 cell.
(A) Wild-type (YW5-1B), apg10-1 mutant (MT91-4-2) and ∆apg10
(TFD10-L1) cells were incubated in SD(–N) medium containing 1 mM
PMSF at 30°C. After incubation for 6 h, cells were observed under
light microscopy (Nomarski images). (B) Wild-type (closed circles),
apg10-1 mutant (closed triangles) and ∆apg10 (open circles) cells
were grown to 1 OD600/ml in YPD and then transferred to SD(–N)
medium. After incubation at 30°C for the times indicated, their
viability was determined by phloxine B staining (Tsukada and Ohsumi,
1993). (C) Cell lysates from wild-type cells with empty vector
pRS316 (lane 1) orHAAPG12 (lane 2), and also apg10-1 (lane 3) and
∆apg10 cells withHAAPG12 (lane 4) were subjected to Western
blotting analysis with anti-HA antibody (16B12). (D) Amino acid
sequence of Apg10p. Cysteine residues are indicated in bold.
apg10-1 mutant in autophagy is due to loss of the Apg12p–
Apg5p conjugate, and that Apg10p probably functions at
the Apg12p-conjugating step.
Isolation and sequence analysis of APG10
The APG10 gene was cloned by complementation of
reduced viability of apg10-1 mutant under starvation
conditions as described in Materials and methods. A
YCp50-based yeast genomic library (a gift from Dr Wada,
Osaka University) was introduced into apg10-1 cells, and
screening of ~10 000 transformants yielded plasmid 5-5C
containing an 8.0 kb genomic fragment. Subcloning of
the fragment of 5-5C revealed that a 1.2 kb XbaI–HindIII
fragment containing one open reading frame (ORF)
YLL042c was sufficient for complementation. The
YLL042c is a novel gene and encodes a hydrophilic
protein of 167 amino acids with a predicted molecular
mass of 19.8 kDa (Figure 1D).
To determine whether YLL042c was the authentic
APG10 gene, this ORF was disrupted by replacing with
a LEU2 gene. The disruptant exhibited the same phenotype
as the apg10-1 cell: (i) the autophagic bodies did not
accumulate in the vacuole during starvation (Figure 1A);
(ii) the viability of the disruptant decreased under the
starvation condition (Figure 1B); and (iii) the Apg12p–
Apg5p conjugate was not generated at all (Figure 1C).
Sequence analysis of the apg10-1 allele revealed that the
482nd nucleotide of the coding sequence was altered from
T.Shintani et al.
G to A, which led to translation termination at the 160th
amino acid residue. Furthermore, a diploid cell obtained
by crossing apg10-1 and ∆yll042c cells is also defective
in autophagy. We concluded therefore, that YLL042c was
the authentic APG10 gene.
The amino acid sequence of Apg10p did not provide
any insight into its function. A BLAST search, however,
revealed that it was homologous to a Caenorhabditis
elegans protein of unknown function (DDBJ/EMBL/Gen-
Bank accession No. Z54282) but exhibited no significant
similarity to ubiquitin-conjugating enzymes or ubiquitin-
Formation of an Apg12p–Apg10p thioester
To elucidate how Apg10p participates in the Apg12p–
Apg5p conjugation pathway, we first examined whether
Apg10p could interact with other components of this
pathway. In a two-hybrid assay (James et al., 1996), we
observed that Apg10p physically interacts with Apg7p
and Apg12p but not with Apg5p (Figure 2A). Next we
performed coimmunoprecipitation to confirm the Apg10p–
Apg12p interaction. Total cell lysates of ∆apg5 cells with
HAAPG10 and/orMycAPG12 (3? Myc-tagged construct)
on 2µ plasmids were immunoprecipitated with either anti-
HA or anti-Myc antibody. The resulting precipitates were
analyzed by Western blotting. When the cells expressed
bothHAApg10p andMycApg12p,MycApg12p was copreci-
pitated by pullingHAApg10p down with anti-HA antibody
(Figure 2B, lane 17), andHAApg10p was coprecipitated
indicate that Apg10p interacts with Apg12p in vivo. Under
non-reducing SDS–PAGE conditions, an additional 56
kDa band was also detected by both anti-HA and anti-
Myc antibodies (Figure 2B, lanes 8 and 9), whereas it
was not detected in cells harboring eitherHAAPG10 or
MycAPG12 plasmid. Furthermore, its molecular mass was
shifted from 56 to 61 kDa by changingMycAPG12 plasmid
to a 6? Myc-tagged construct (data not shown). This
difference in molecular mass just corresponds with a size
of 3? Myc. These findings show that the 56 kDa band
molecules conjugate 1:1 via a reducing reagent-sensitive
bond. Apg12p has three cysteine residues. Because the
substitutions of all three cysteine residues of Apg12p by
alanine did not affect the formation of the Apg12p–
Apg10p conjugate, the dithiothreitol (DTT)-sensitive bond
is probably a thioester bond between the C-terminal
glycine of Apg12p and a cysteine residue of Apg10p, but
is not a disulfide bond. Another DTT-sensitive 67 kDa
band was also detected by anti-Myc but not by anti-HA
antibody (Figure 2B, lanes 6, 8 and 9), suggesting that it
contained Apg12p but not Apg10p. We concluded, how-
ever, from several lines of evidence (described below)
that the 67 kDa complex is not involved in the Apg12p–
Apg5p conjugation pathway. Similar results were obtained
with the cells expressing bothHAApg10p andMycApg12p
from CEN plasmids (data not shown).
Next we examined the effects of Apg7p and Apg5p on
formation of the Apg12p–Apg10p conjugate. Apg7p has
already been found to function as an E1-like Apg12p-
activating enzyme by Mizushima et al. (1998a) and Tanida
et al. (1999). Apg7p probably activates Apg12p with
hydrolysis of ATP and subsequent transfer to its own thiol
MycApg12p (Figure 2B, lane 18). These results
MycApg12p, and these
Fig. 2. In vivo interaction of Apg10p with Apg12p. (A) Two-hybrid
assay for interactions of Apg10p with Apg5p, Apg7p and Apg12p.
Yeast cells (PJ69-4A) harboring the indicated plasmids were streaked
out on plates with medium lacking adenine to assay for interaction-
dependent activation of the ADE2 gene. (B) Total lysates from ∆apg5
cells withHAAPG10 and/orMycAPG12 on 2µ plasmid were
immunoprecipitated with anti-HA (mAb 16B12) or anti-Myc (mAb
9E10) antibody. Precipitates were analyzed by Western blotting using
anti-HA or anti-Myc antibody. Apg12p adduct of Apg10p is indicated
by d and an unidentified complex of 67 kDa by dd. The positions of
cross-reacting IgG heavy chain (H) and light chain (L) are indicated
group of the Cys-507. By analogy with ubiquitination, an
Apg12p covalently bound to the Cys-507 residue of Apg7p
can be transferred to a cysteine residue of a hypothetical
E2-like enzyme. Figure 3 shows that the Apg12p–Apg10p
conjugate was not formed in ∆apg7 cells, suggesting that
Apg12p is tranferred to Apg10p after Apg7p functions
(Figure 3, lanes 8 and 9). The non-covalent interaction
between Apg10p and Apg12p was also lost completely in
∆apg7 cells. Considering that Apg7p should activate the
C-terminus of Apg12p, the Apg7p-dependent formation
of the Apg12p–Apg10p conjugate suggests that Apg12p
and Apg10p are linked via a thioester bond. As stated
above, the interaction between Apg7p and Apg10p was
confirmed in the two-hybrid assay. Taken together, these
results suggest that an activated Apg12p is transferred
directly from Apg7p to an active-site cysteine residue
Protein-conjugating enzyme essential for autophagy
Fig. 3. Effects of the deletion of APG5 or APG7 on Apg12p–Apg10p
thioester formation. Coimmunoprecipitation was performed with wild-
type (KA311B), ∆apg5 (YNM122) or ∆apg7 (YTS12) cells harboring
HAAPG10 andMycAPG12 on 2µ plasmids. The resulting precipitates
were analyzed by Western blotting. The DTT-resistant Apg12p–Apg5p
conjugate is indicated by d.
When coimmunoprecipitation was carried out using
wild-type cells, Apg12p–Apg10p thioester was generated
as in the ∆apg5 cells, i.e. the deletion of Apg5p, a target
molecule of the Apg12p conjugation, did not promote
accumulation of the Apg12p–Apg10p conjugate (Figure
3, lanes 2 and 3). Overexpression of Apg5p also had no
effect on the formation of the Apg12p–Apg10p thioester
(data not shown). These results indicate that the amount
of Apg5p would not change the kinetics of Apg12p–
Apg10p thioester formation. Moreover, in wild-type cells,
the Apg12p–Apg5p conjugate was precipitated with anti-
Myc but not with anti-HA antibody (Figure 3B, lanes 3
and 12) providing evidence that Apg10p no longer associ-
ates with Apg12p after Apg12p is transferred to Apg5p.
The interaction between Apg10p and Apg5p was not
detected in the two-hybrid assay (Figure 2A). These data
suggest that Apg12p is not transferred directly from
Apg10p to Apg5p, and other factor(s) may be necessary
for the Apg10p-to-Apg5p transfer of Apg12p.
The DTT-sensitive 67 kDa band was even detected in
the ∆apg7 cells when immunoprecipitation was carried
out by anti-Myc antibody (Figure 3, lane 9), suggesting
that the 67 kDa complex could be generated without
activation of Apg12p. Moreover, the band appeared even
when an APG12∆G (C-terminal Gly deletion) construct,
which could conjugate to Apg5p no longer, was used
for immunoprecipitation instead of a wild-type APG12
construct (data not shown). Therefore, we concluded that
the 67 kDa complex did not participate in the Apg12p–
Apg5p conjugating reaction.
Apg12p is attached to the Cys-133 residue of
There are three cysteine residues in Apg10p (Figure
1D). To determine which cysteine residue contributes to
thioester formation, each cysteine residue of Apg10p was
substituted by serine using site-directed mutagenesis. First,
Fig. 4. Apg12p is transferred to the Cys-133 residue of Apg10p.
(A) Cell lysates from ∆apg10 cells (TFD10-L1) harboringHAAPG12
and each mutant form ofHAAPG10 on CEN plasmids were subjected
to Western blotting analysis with anti-HA antibody (mAb 16B12).
(B) Cell extracts from the wild-type cells (KA311B) harboring
MycAPG12 and mutatedHAAPG10 were subjected to
immunoprecipitation and the resulting precipitates were analyzed by
Western blotting with the rabbit anti-HA antibody.
we examined the abilities of the substitutes to generate
the Apg12p–Apg5p conjugate by Western blotting. The
Apg12p–Apg5p conjugate of 70 kDa was formed in
∆apg10 cells harboringHAAPG10 andHAAPG12 on CEN
plasmids (Figure 4A, lane 3). The substitution of Cys-26
or Cys-137 by Ser (C26S or C137S, respectively) in
Apg10p did not affect the generation of the conjugate at
all, whereas the substitution of Cys-133 by Ser (C133S)
resulted in a complete loss of the conjugation, although
Apg10pC133Swas detected in an amount comparable to
that of wild-type Apg10p (Figure 4A).
To investigate whether Apg12p was covalently attached
to Cys-133 of Apg10p by a thioester bond, coimmunopre-
cipitation was carried out using the C133S or C133A (the
substitution of Cys-133 by Ala) mutants (Figure 4B). As
compared with the positive control (Figure 4B, lanes 2
and 3), quite a small amount of Apg12p–Apg10pC133S
conjugate was detected (Figure 4B, lanes 5 and 6). This
conjugate was found to be resistant to the reducing reagent
(Figure 4B, lanes 14 and 15), which suggested that an
ester bond is formed between Apg12p and Apg10pC133S
instead of a thioester bond. The C133A mutation caused
a complete loss of the Apg12p–Apg10p conjugate even
in the absence of the reducing reagent (Figure 4B, lanes
8 and 9). These results indicate that Cys-133 is an active
center of Apg10p and contributes to thioester formation.
T.Shintani et al.
Fig. 5. The cysteine-133 residue of Apg10p is essential for autophagy
and the Cvt pathway. (A) Autophagic activities were measured by an
ALP assay (Noda and Ohsumi, 1998). YTS3 cells (∆apg10
PHO8::pho8∆60) harboring each mutant APG10 on CEN plasmids
were grown to 1 OD600/ml in SC medium lacking tryptophan and then
transferred to SD(–N) medium. Lysates from the cells after incubation
for 0 and 4 h were used for assay. Error bars indicate the SD of three
independent experiments. (B) Cell lysates from ∆apg10 cells (TFD10-
L1) harboring empty vector, pRS316 (lane 1), wild-typeHAAPG10
(lane 2),HAAPG10C26S(lane 3),HAAPG10C133S(lane 4) or
HAAPG10C137S(lane 5) on CEN plasmids were subjected to Western
blotting analysis with anti-API antiserum.
Both mutations prevented the interaction between the free
forms of Apg12p and Apg10p (Figure 4B, lanes 6 and 9).
affected autophagy and Cvt of proaminopeptidase I (pro-
API). Autophagic activities were measured using a bio-
processing of alkaline phosphatase Pho8∆60 (Noda et al.,
1995). In wild-type cells, Pho8∆60 was activated by
transferring the cells from a rich medium to a nitrogen-
Apg10pC133Sdid not activate Pho8∆60 even in the starva-
tion medium, while those expressing Apg10pC26Sor
Apg10pC137Sshowed normal autophagic activity (Figure
5A). These data indicate that the C133S mutation in
Apg10p causes a defect in autophagy. ProAPI is synthe-
sized in the cytoplasm and imported directly to the vacuole
by a mechanism closely related to the autophagic pathway,
and then processed to the mature form by proteinase B.
As expected, proAPI was not processed in the ∆apg10
cells expressing Apg10pC133S(Figure 5B), implying that
the formation of a thioester intermediate is also essential
for API transport.
Apg12p is a modifier protein with no significant similarity
to ubiquitin and ubiquitin-related modifiers. The covalent
binding of Apg12p to Apg5p is essential for autophagy
in yeast. The Apg12p–Apg5p conjugating reaction is
initiated by Apg7p, an Apg12p-activating enzyme, with
ATP hydrolysis (Mizushima et al., 1998a; Tanida et al.,
1999). Here, we have characterized Apg10p as an Apg12p-
conjugating enzyme acting after Apg7p in the Apg12p–
experiments revealed that a reducing reagent-sensitive
covalent adduct was formed between Apg12p and Apg10p
in an Apg7p-dependent manner (Figures 2B and 3). This
adduct is likely to be a thioester linked between the
C-terminal carboxy group of Apg12p and the active-site
cysteine-133 of Apg10p (Figure 4). These findings suggest
that the high-energy thioester bond between Apg12p and
Apg7p is transferred to Apg10p through transacylation.
The Apg12p transfer from Apg7p to Apg10p certainly
occurs directly because the interaction between Apg7p
and Apg10p was observed in the yeast two-hybrid assay
(Figure 2A). As shown above, Apg10p functions as an
‘E2’ in the Apg12p–Apg5p conjugation system.
On the other hand, we speculate that the transfer of
Apg12p from Apg10p to Apg5p, a final target molecule
of Apg12p, requires other protein(s) on the grounds of
several lines of evidence: (i) the interaction between
Apg10p and Apg5p was not detected by a two-hybrid
assay (Figure 2A); (ii) Apg12p did not interact with Apg5p
in the ∆apg7 or ∆apg10 cells with a two-hybrid assay but
did in wild-type cells (data not shown); (iii) Apg10p
no longer associated to the Apg12p–Apg5p conjugate
(Figure 3, lanes 2, 3, 11 and 12), which suggests that
neither Apg10p nor Apg12p contributes to the recognition
of Apg5p; and (iv) moreover, the changes in expression
level of Apg5p did not affect the kinetics of the Apg12p–
Apg10p thioester formation (Figure 3). The other factor(s)
that recognizes Apg5p may be essential for the transfer
of Apg12p from Apg10p to Apg5p. Our model for
an enzyme system of the Apg12p–Apg5p conjugation
pathway is shown in Figure 6. Apg7p hydrolyzes ATP
and forms the high-energy thioester bond between its
active-site cysteine and the C-terminus of Apg12p. The
activated Apg12p is transferred directly from Apg7p to
bond. Then Apg12p is transferred to the target protein
Apg5p, which may require the additional protein(s).
In the coimmunoprecipitation experiment, the non-
covalent interaction between Apg12p and Apg10p was
also observed, which required both Apg7p and Apg10p
activity (Figures 3 and 4B). This suggests that the non-
covalent association is dependent on Apg12p–Apg10p
thioester formation. Therefore, the non-covalent complex
may actually exist as one of the intermediate forms of the
Alternatively, the non-covalent complex may be due to
the unexpected cleavage of the thioester bond during the
preparation of samples.
As described above, the reaction mechanism for
Apg12p–Apg5p conjugation is closely related to those for
the ubiquitin or ubiquitin-related modifier system, and
Apg10p catalyzes the E2-like reaction in the Apg12p–
Apg5p conjugation pathway. A large number of E2s have
been identified widely in eukaryotes and comprise the
Ubc (ubiquitin-conjugating enzyme) family that is charac-
terized by conserved sequences, termed ‘E2 motifs’, con-
taining the active-site cysteine and a conserved His–Pro–
Asn tripeptide (Haas and Siepmann, 1997). It has been
found that some Ubcs are used for ubiquitin-related
Protein-conjugating enzyme essential for autophagy
Fig. 6. Model of the enzymatic pathway for the Apg12p–Apg5p conjugation system. Apg12p is activated by Apg7p with ATP hydrolysis; the
carboxy group of the C-terminal glycine of Apg12 is conjugated to the thiol group of the cysteine-507 residue of Apg7p via a high-energy thioester
bond (~). Subsequent transfer of Apg12p to the thiol group of the cysteine-133 residue of Apg10p results in the formation of the Apg12p–Apg10p
thioester. Finally, the C-terminus of Apg12p is covalently attached to the lysine-149 residue of Apg5p via an isopeptide bond, which may require
hypothetical Apg5p-recognizing protein(s).
Table I. Yeast strains used in this study
MATa ura3 leu2 trp1
MATa ura3 apg10-1
MATa ura3 leu2 trp1 ∆apg10::LEU2
MATα ura3 leu2 his3 trp1
MATα ura3 leu2 his3 trp1 ∆apg5::HIS3
MATα ura3 leu2 his3 trp1 ∆apg7::HIS3
MATa ura3 leu2 his3 trp1 ade2 lys2 PHO8::pho8∆60
MATa ura3 leu2 his3 trp1 ade2 lys2 PHO8::pho8∆60 ∆apg10::LEU2
MATa ura3 leu2 his3 trp1 gal4∆ gal80∆ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ
Tsukada and Ohsumi (1993)
Irie et al. (1993)
Mizushima et al. (1998a)
Noda et al. (1998)
James et al. (1996)
modifier, e.g. Ubc9 and Ubc12 for SUMO-1/Smt3p and
NEDD8/Rub1p, respectively (Gong et al., 1997; Johnson
and Blobel, 1997; Lee et al., 1998; Liakopoulos et al.,
1998; Osaka et al., 1998; Schwarz et al., 1998). In spite
of the similarity of the reaction mechanisms between the
Apg12p and ubiquitin systems, the Apg12p system uses
the protein with no significant sequence similarity to
Ubcs as an E2. Because the structure of Apg12p does not
resemble that of ubiquitin or ubiquitin-related modifier at
all, except for the C-terminal glycine, the protein structur-
ally unrelated to Ubcs, namely Apg10p, must be necessary
for the recognition of Apg12p and/or Apg7p. This is the
first example showing that the protein quite different from
Ubcs functions as an E2-like protein-conjugating enzyme.
A BLAST search revealed that Apg10p has a potential
C.elegans homolog of unknown function. In addition, the
EST fragments that are closely related to Apg10p are
also found in human (DDBJ/EMBL/GenBank accession
No. AA448080) and mouse (DDBJ/EMBL/GenBank
accession No. AA673136). The sequences around the
cysteine-133 residue are well conserved among these
proteins, supporting the suggestion that the cysteine-133
residue of Apg10p is the active site. Considering that the
Apg12p–Apg5p conjugation system is conserved from
yeast to human (Mizushima et al., 1998b), Apg10p should
also be widely distributed in eukaryotes. Recently, it was
reported that the Pichia pastoris homolog of Apg7p, Gsa7,
is required for microautophagy of peroxisomes and is able
to complement the defects in macroautophagy and API
transport in S.cerevisiae (Kim et al., 1999; Yuan et al.,
1999). These findings suggest that Apg10p may function
in peroxisome degradation as well as macroautophagy and
the Cvt pathway.
Materials and methods
Strains and media, genetic and molecular biological
The S.cerevisiae strains used in this study are listed in Table I. Yeast
cells were grown in yeast extract–peptone–dextrose (YPD) or synthetic
complete (SC) media. SD(–N) medium (0.17% yeast nitrogen base
without amino acids and ammonium sulfate and 2% glucose) was used
for nitrogen starvation. Standard genetic manipulations were performed
as described by Adams et al. (1998). DNA manipulations were performed
using standard methods (Sambrook et al., 1989).
Cloning, disruption and epitope tagging of APG10
APG10 was cloned by complementation of reduced viability of
apg10-1 in the SD(–N) medium as described previously (Funakoshi
et al., 1997; Kametaka et al., 1998). A yeast mutant MT91-4-2 was
transformed with a YCp50-based yeast genomic library and Ura?
transformants were replica-plated onto SD(–N) plates containing
10 µg/ml phloxine B, on which dead cells were stained red (Tsukada
and Ohsumi, 1993). White colonies were picked up and subjected to a
light microscopic observation to confirm the accumulation of autophagic
bodies in their vacuoles.
Plasmids were rescued from the candidate cells and one plasmid,
named 5-5C, was used for further analysis. Subcloning identified a
1.2 kb XbaI–HindIII fragment as the minimal region with comple-
The 2.4 kb XbaI–KpnI fragment was subcloned into pBluescript II
KS? (Stratagene) to generate pKSAPG10. The 2.0 kb SmaI–PstI LEU2
fragment from pJJ282 (Jones and Prakash, 1990) was cloned into PstI–
EcoRV-digested pKSAPG10 to generate pKSapg10::LEU2. The 4.24 kb
NheI–NcoI fragment from pKSapg10::LEU2 was used for transformation
of YW5-1B and TN125. The disruption of the APG10 gene was
confirmed by PCR.
C-terminal epitope-tagged APG10 (HAAPG10) was constructed by
inserting the DNA sequence encoding a repeated hemagglutinin
(HA)-epitope tag (2? HA) just before the stop codon. The tagged
HAAPG10 was confirmed to be functional by complementation of the
apg phenotype of the apg10-1 cell.
HAAPG10 was cloned into the XbaI and EcoRI sites of pRS316 and
pRS426 (Sikorski and Hieter, 1989) to generate pHA-APG10-316
and pHA-APG10-426, respectively. pHA-APG12-314 was described
previously (Mizushima et al., 1998a) and pMyc-APG12-424 was gener-
ated by subcloning of the DNA fragment encoding 3? Myc-tagged
Apg12p (Mizushima et al., 1998a) into the SpeI and XhoI sites of
pRS424. For the two-hybrid assay, the entire APG10 ORF was amplified
by PCR and inserted into the EcoRI site of pGBD-C1 (James et al.,
1996) to generate pGBD-APG10 that expresses the DNA binding domain
of Gal4p fused with Apg10p. pGAD-APG5 was created by insertion of
the entire APG5 ORF into the BamHI and PstI sites of pGAD-C1 to
fuse Apg5p to the C-terminal end of the Gal4p activation domain.
pGAD-APG7 contains the same APG7 fragment as pGBD-APG7 (Tanida
T.Shintani et al.
et al., 1999). pGAD-APG12 was described previously (Mizushima
et al., 1999).
Site-directed mutagenesis of APG10 was performed by PCR (Ho et al.,
CCAGT-3?; C133S-Fw, 5?-TTCCATCCATCCGATACATCATGTATA-
3?; C133S-Rv, 5?-CATGATGTATCGGATGGATGGAA-3?; C133A-Fw,
5?-TTCCATCCAGCCGATACATCATGTATA-3?; C133A-Rv, 5?-CATG-
ATGTATCGGCTGGATGGAA-3?; C137S-Fw, 5?-GATACATCATCTA-
TAGTAGGTGAC-3?; C137S-Rv, 5?-GTCACCTACTATAGATGATG-
TATC-3?. The mutated APG10 fragments were inserted into the XbaI
and EcoRI sites of pRS316 or pRS426. All mutations were verified by
Yeast cells harboringHAAPG10 and/orMycAPG12 on 2µ plasmid were
broken with glass beads in IP buffer [50 mM Tris–HCl pH 7.5, 150 mM
NaCl, 5 mM EDTA, 1 mM PMSF and 1? protease inhibitor mixture
(Complete™, Boehringer Mannheim)]. Cell debris was removed by
centrifugation at 5000 g for 5 min, and the protein concentration of the
resultant lysate was determined by Bradford’s method. After Nonidet
P-40 was added to the lysate to a final concentration of 1%, it was
incubated with protein G–Sepharose 4 Fast Flow (Amersham Pharmacia
Biotech) at 4°C for 1 h for preabsorption. The preabsorbed lysate
containing 5 mg of protein was incubated with or without 1 µl of mouse
monoclonal anti-HA (16B12, BAbCo) or anti-Myc (9E10) antibody at
4°C for 2 h and then 20 µl of protein G–Sepharose (50% slurry) were
added to it. After incubation at 4°C for 2 h, unbound proteins were
removed by three washes in 1 ml of IP buffer containing 1% Nonidet
P-40. The immunoprecipitated proteins were eluted by boiling in SDS
gel-loading buffer with or without 100 mM DTT. Proteins were separated
by SDS–PAGE and analyzed by immunoblotting with anti-HA (16B12),
anti-Myc antibody (9E10) or rabbit polyclonal anti-HA antibody
Western blotting analysis of whole yeast lysate
Cells were resuspended in 100 µl of 0.2 N NaOH, 0.5% 2-mercapto-
ethanol. After incubation for 15 min on ice, 1 ml of ice-cold acetone
was added and further incubated for 30 min at –20°C. After centrifugation
at 10 000 g for 5 min, the resulting pellets were resuspended in the
appropriate volume of SDS loading buffer and boiled for 5 min. Lysates
equivalent to 0.5 OD600cells were separated by SDS–PAGE and
electrotransferred to a polyvinylidene difluoride membrane (Millipore).
ase I antibody (a gift from Dr Klionsky) was used for immunodetection.
Development was performed by the ECL detection methods (Amersham
For measurement of autophagic activity, the alkaline phosphatase (ALP)
assay was performed as described previously (Noda and Ohsumi, 1998).
The two-hybrid assay was performed as described by James et al. (1996).
We would like to thank Dr Yoh Wada (Osaka University) for the yeast
genomic library and Dr Daniel J.Klionsky (University of California,
Davis) for anti-API antibody. This work was supported in part by Grants-
in-Aids for Scientific Research from the Ministry of Education, Science,
Sports and Culture of Japan. T.S. is a Research Fellow of the Japan
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Received June 21, 1999; revised August 2, 1999;
accepted August 11, 1999