Molecular Biology of the Cell
Vol. 10, 1367–1379, May 1999
Apg7p/Cvt2p: A Novel Protein-activating Enzyme
Essential for Autophagy
Isei Tanida,* Noboru Mizushima,†Miho Kiyooka,‡Mariko Ohsumi,‡
Takashi Ueno,* Yoshinori Ohsumi,†and Eiki Kominami*§
*Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan;
†Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan; and
‡Department of Bioscience, Teikyo University of Science and Technology, Yamanashi 409-0193, Japan
Submitted November 3, 1998; Accepted February 16, 1999
Monitoring Editor: Randy W. Schekman
In the yeast Saccharomyces cerevisiae, the Apg12p–Apg5p conjugating system is essential
for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable
to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant
similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the
function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was
coimmunoprecipitated with c-myc–tagged Apg7p. A two-hybrid experiment confirmed
the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent.
Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overex-
pressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with
Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507of
Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine
residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p–
Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507Ashowed
defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These
results indicated that Apg7p functions as a novel protein-activating enzyme necessary for
Autophagy is the process of bulk degradation of cy-
toplasmic components by the lysosomal/vacuolar sys-
tem (Seglen and Bohley, 1992; Dunn, 1994). The phe-
nomenon is dramatically enhanced under nutrient
starvation conditions. In the initial step of the macro-
autophagy, a cup-shaped membrane sac surrounds
cytosolic components to form an autophagosome
(Baba et al., 1994). The outer membrane of the auto-
phagosome fuses with a lysosome/vacuole (Baba et
al., 1995). A transient single-membrane structure, the
autophagic body, is released into the lumen and sub-
sequently degraded in the lysosome/vacuole. Al-
though biochemical and cell–biological approaches
have revealed several aspects of autophagy in mam-
malian cells, the molecular mechanism of autophagy
In the yeast Saccharomyces cerevisiae, macroautoph-
agy was first described by Takeshige et al. (1992), and
the process is similar to that in higher eukaryotes
(Baba et al., 1994, 1995). Taking advantage of yeast
genetics, autophagy-defective mutants (14 apg mu-
tants and 9 aut mutants) have been isolated in two
different laboratories (Tsukada and Ohsumi, 1993;
Thumm et al., 1994; Harding et al., 1996). Surprisingly,
most of the apg and aut mutants overlap genetically
and phenotypically with some cvt mutants, which
have defects in the cytoplasm-to-vacuole targeting of
aminopeptidase I (API)1. These results suggest that the
APG and AUT gene products function even in a veg-
§Corresponding author. E-mail address: firstname.lastname@example.org.
1Abbreviations used: Ac.No., accession number; API, aminopep-
tidase I; GAL4AD, GAL4 activation domain; GAL4BD, GAL4
DNA binding domain; HA, hemagglutinin.
© 1999 by The American Society for Cell Biology 1367
CVT12, APG15/CVT11, and CVT17/AUT5) (Harding et
al., 1996; Scott et al., 1996). Several APG and AUT genes
have been identified, and most encode novel proteins
APG12, APG13, APG14, AUT1, AUT2, and AUT7) (Ka-
metaka et al., 1996; Funakoshi et al., 1997; Matsuura et
al., 1997; Schlumpberger et al., 1997; Straub et al., 1997;
Kametaka et al., 1998; Lang et al., 1998; Mizushima et
al., 1998a). Biochemical characterization of the gene
products has revealed some molecular aspects of au-
tophagy: Apg1p/Aut3p is a novel protein kinase
(Matsuura et al., 1997; Straub et al., 1997). Apg6p/
Vps30p and Apg14p form a protein complex (Ka-
metaka et al., 1998). Aut2p interacts with Aut7p/
Apg8p, a homologue of rat microtubule-associated
protein light chain 3 (Lang et al., 1998).
Recently, we found that the Apg12p–Apg5p conju-
gation system is essential for autophagy (Mizushima
et al., 1998a). Apg12p has no significant homology to
ubiquitin or ubiquitin-related modifiers; however,
Apg12p is conjugated to Apg5p through an isopeptide
bond between the C-terminal Gly residue of Apg12p
and the Lys149residue of Apg5p. Ubiquitination is a
posttranslational modification to present the degrada-
tion signal for proteolytic attack by 26S proteasomes
(reviewed by Finley and Chau, 1991; Hershko and
Ciechanover, 1992; Hershko, 1996; Hochstrasser,
1996a,b; Haas and Siepmann, 1997; Varshavsky, 1997).
Ubiquitin is activated by a ubiquitin-activating en-
zyme (E1) in an ATP-dependent manner in which a
thioester bond is formed between the C terminus of
ubiquitin and a Cys residue within the E1 enzyme
(Ciechanover et al., 1982; Haas et al., 1982). The ubiq-
uitin is transferred from the E1 enzyme to a Cys
residue within a ubiquitin-conjugating enzyme (E2).
Finally, ubiquitin is covalently attached to a target
protein by an isopeptide linkage directly from E2 or by
a ubiquitin-protein ligase (E3) (Hershko et al., 1983;
Bartel et al., 1990; Scheffner et al., 1993, 1995; Peters et
al., 1996; Zachariae et al., 1996).
Recent discoveries have revealed that the ubiquitin-
related modifiers other than ubiquitin play essential
roles in eukaryotes (reviewed by Johnson and Hoch-
strasser, 1997; Saitoh et al., 1997; Dolan, 1998; Hoch-
strasser, 1998). A mammalian ubiquitin-related pro-
tein, SUMO-1 [small ubiquitin-related modifier; also
called GMP1, PIC1, UBL1, or sentrin (Boddy et al.,
1996; Matunis et al., 1996; Okura et al., 1996; Shen et al.,
1996)] is covalently attached to the RanGAP1 and PML
proteins (Matunis et al., 1996; Mahajan et al., 1997;
Muller et al., 1998). This posttranslational modification
affects the subcellular localization of these proteins
(Mahajan et al., 1998; Matunis and Blobel, 1998; Muller
et al., 1998). A yeast SUMO-1 homologue, Smt3p, is
activated by an E1-like heterodimer Aos1p/Uba2p
(Dohmen et al., 1995; Johnson et al., 1997). The E2
enzyme for Smt3p and SUMO-1 are Ubc9p and its
mammalian homologue (Gong et al., 1997; Johnson
and Blobel, 1997; Lee et al., 1998; Schwarz et al., 1998).
Another family of ubiquitin-related proteins is RUB1
and NEDD8 (Kumar et al., 1993; Callis et al., 1995;
Hochstrasser, 1996; Kamitani et al., 1997; Lammer et
al., 1998; Liakopoulos et al., 1998). A major substrate of
RUB1/NEDD8 is Cdc53p/Cullin in yeast and mam-
malian cells, which play an essential role in regulating
the cell cycle (Lammer et al., 1998; Liakopoulos et al.,
1998; Osaka et al., 1998). In Arabidopsis thaliana, the
auxin response depends on the RUB1 modification of
nuclear proteins (del Pozo et al., 1998). RUB1 and
NEDD8 are activated by E1-like heterodimers: Ula1p
(Enr2p)/Uba3p in yeast, an APP-BP1 (a 59-kDa
man UBA3 homologue in humans, and AXR1/ECR1
in A. thaliana (Leyser et al., 1993; Chow et al., 1996; del
Pozo et al., 1998; Liakopoulos et al., 1998; Osaka et al.,
1998). The E2 enzyme for RUB1 and NEDD8 are
Ubc12p and its mammalian homologue (del Pozo et
al., 1998; Liakopoulos et al., 1998; Osaka et al., 1998).
These findings strongly suggest that there must be
E1- and E2-like enzymes for the Apg12p–Apg5p con-
jugation system in yeast. Candidates in the Apg12p
conjugation system are Apg7p/Cvt2p and Apg10p. In
apg7 and apg10 mutants, no Apg12p–Apg5p conjugate
is observed, indicating that Apg7p and Apg10p play
indispensable roles in the Apg12p conjugation system
(Mizushima et al., 1998a). A region of Apg7p (residues
322 to 407 out of 633 amino acids) shows significant
homology to the corresponding region of a ubiquitin-
activating enzyme, Uba1p, although the other regions
show no homology (McGrath et al., 1991; Mizushima et
al., 1998a). We took particular interest in Apg7p/
Cvt2p and investigated functions of Apg7p through
both biochemical and molecular biological techniques.
In this study, we provide several lines of evidence
showing that Apg7p is an Apg12p-activating enzyme.
MATERIALS AND METHODS
Strains, Media, Materials, and Genetic Techniques
Escherichia coli strain DH5? as the host for plasmids and protein
expression was grown in Luria Broth medium in the presence of
antibiotics as required (Ausubel et al., 1995). The S. cerevisiae strains
and plasmids used in this study are listed in Table 1. All yeast
strains were cultured in rich medium (YPD, pH 5.0: 1% yeast
extract, 2% polypeptone, 2% glucose, 20 mg/l adenine, 20 mg/l
tryptophan, 20 mg/l uracil, and 50 mM succinate/NaOH, pH 5.0),
MVD medium (0.67% yeast nitrogen base without amino acids,
0.5% casamino acids, and 2% glucose), or SD medium (0.67% yeast
nitrogen base without amino acids, 2% glucose, and appropriate
amino acids as described by Kaiser et al. ). Nitrogen-starvation
medium contained 0.17% yeast nitrogen base without amino acids
and ammonium sulfate and 2% glucose. Solid medium contained
2% Bacto agar. Standard genetic and molecular biological tech-
niques were performed as described by Kaiser et al. (1994) and
I. Tanida et al.
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Novel E1 Enzyme Essential for Autophagy
Vol. 10, May 19991379