From the Department of Biochemistry, Juntendo
University School of Medicine, Tokyo 113-8421, § Department of Bioscience, Teikyo University of Science and
Technology, Yamanashi 409-0193, and ¶ Department of Cell
Biology, National Institute for Basic Biology,
Okazaki 444-8501, Japan
Received for publication, August 24, 2000, and in revised form, December 21, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apg7p/Cvt2p, a protein-activating enzyme, is
essential for both the Apg12p-Apg5p conjugation system and the Apg8p
membrane targeting in autophagy and cytoplasm-to-vacuole targeting in
the yeast Saccharomyces cerevisiae. Similar to the
ubiquitin-conjugating system, both Apg12p and Apg8p are activated by
Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an
E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred
to Apg3p, another E2-like enzyme, followed by conjugation with
phosphatidylethanolamine. Evidence is presented here that Apg7p
forms a homodimer with two active-site cysteine residues via the
C-terminal region. The dimerization of Apg7p is independent of the
other Apg proteins and facilitated by overexpressed Apg12p. The
C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is
neither an ATP binding domain nor an active-site cysteine essential for
its E1 activity. The deletion of its carboxyl 40 amino acids (residues
591-630 out of 630 amino acids) results in several defects of not only
Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for
Apg8p. These results suggest that Apg7p forms a homodimer via the
C-terminal region and that the C-terminal region is essential for both
the activity of the E1 enzyme for Apg12p and Apg8p as well as the
formation of an E1-E2 complex for Apg8p.
Autophagy is responsible for the bulk of intracellular protein
degradation in the lytic organelles, lysosome/vacuole (1, 2). When
cells exist under conditions of nutrient starvation, the cytoplasmic
components are nonselectively sequestered into autophagosomes,
double-membrane structures, and are subsequently targeted to the
lysosome/vacuole for degradation. The entire process is conserved
through eukaryotes from yeast to mammals. Unique membrane dynamics are
observed in the process of autophagy. In the case of the yeast,
Saccharomyces cerevisiae, cytoplasmic components are
nonselectively surrounded by membranes, which on expansion and
completion, give rise to an autophagosome (3, 4). Autophagosomal membranes are morphologically distinct from any other known organellar membranes (3). The outer membrane of the autophagosome fuses with the
vacuolar membrane (5). The inner membrane structure, which is referred
to as an autophagic body, is released into the lumen (3, 4). Finally,
the cytoplasmic components within an autophagic body are degraded in
the vacuole (3). In the case of dynamic autophagosomal
membrane-formation and fusion with the vacuole (lysosome in mammals),
the molecular mechanism for this process remains unknown.
Several autophagy-defective (apg and aut) mutants
have been isolated via the application of yeast genetics (6, 7). These apg and aut mutants genetically overlap with most
cvt mutants, which have defects in the
cytoplasm-to-vacuole targeting
(Cvt) pathway of aminopeptidase I (Refs. 8-10; for a review, see Ref. 11), indicating that the mechanism for autophagy and the Cvt pathway
share some common features. Recently, some of the characteristics of
individual APG gene products have been elucidated.
APG1/AUT3 encodes a protein kinase (12, 13).
Apg13p is phosphorylated and interacts with Apg1p and Vac8p (14, 15).
Apg6p/Vps30p forms a complex with Apg14p and is localized on as-yet
unidentified membrane structures (16). Aut9p/Apg9p is an integral
membrane protein that is required for both the Cvt and autophagic
pathways and is localized on large perivacuolar punctate structures
(17, 18).
Of the APG gene products characterized thus far, the Apg12p
modification system and the
Apg8p/Aut7p1
membrane-targeting system have been the subjects of considerable attention in that they function as protein modifiers similar to ubiquitin (for reviews, see Refs. 19-22). In the case of autophagy, Apg12p binds covalently to Apg5p (23, 24). In this conjugation system,
Apg7p and Apg10p function as E1 and E2 enzymes, respectively (24-26).
After Apg12p-Apg5p conjugation, Apg16p is assembled with the conjugate,
resulting in a high molecular weight Apg12p·Apg5p·Apg16p complex
(27), which is essential for the subsequent formation of
autophagosomes. A second modifier, Apg8p, is processed by a novel
cysteine protease, Apg4p/Aut2p, and Apg7p and Apg3p/Aut1p are essential
for Apg8p lipidation (conjugation with phosphatidylethanolamine), suggesting that Apg7p and Apg3p are, respectively, E1 and E2 enzymes for Apg8p (28-30). The expression of Apg8p is enhanced by starvation, and Apg8p is associated with autophagosomal membranes under conditions of starvation and with Cvt vesicles under conditions of active growth
(31, 32). Apg8p also interacts with two ER-to-Golgi vesicular-soluble N-ethylmaleimide factor attachment
protein receptors (v-SNAREs), Bet1p and Sec22p, in addition to a
vacuolar t-SNARE (Vam3p) and a v-SNARE (Nyv1p) (33).
Considering the fact that the function of Apg7p as a unique E1 enzyme
for two substrates, Apg7p promises to be a key enzyme for the
functional divergence or correlation between the Apg12p and Apg8p
pathway. Furthermore, in view of the functional relationship between
the Apg12p, Apg7p, and Apg12p-Apg5p conjugate, it would be of interest
to examine the differential intracellular distribution of Apg7p,
Apg12p, Apg8p, and Apg5p (24, 31). Apg7p is mainly present in the
cytoplasm (26, 34). Apg12p is distributed in the cytoplasm as well as
on a membrane compartment, whereas Apg5p, Apg8p, and Apg12p-Apg5p are
localized only on the membrane compartment(s). These findings suggest
that a mechanism for the targeting of Apg12p to the Apg5p-localized
membrane during the Apg12p-Apg5p conjugation reaction might well be
operative. The present study shows that Apg7p forms a homodimer via the
C-terminal region with two active-site cysteines and that the
C-terminal region is essential not only for interaction with Apg12p and
Apg8p, an activity of the E1 enzyme, but also for interaction with
Apg3p in the formation of an E1-E2 complex.
Strains, Media, Materials, and Molecular Biological
Techniques--
Escherichia coli strain DH5 Plasmid Construction--
Plasmids used in this study are also
listed in Table I. For two-hybrid screening, the bait plasmid
(pGBD-APG7), which encodes the Apg7p fused in-frame to the C-terminal
end of the GAL4-DNA binding domain, has been described previously (26).
pGAD-APG7
To express HA-Apg7p and Myc-Apg7p, the DNA sequence that encodes a
repeated hemagglutinin (HA) epitope tag and a triplet-repeated Myc
epitope tag was inserted just before the start codon of the APG7 gene, and the resulting DNA fragments were cloned into
the pGem-T vector. To construct galactose-inducible expression plasmids for these epitope-tagged Apg7ps, the DNA fragments were introduced into
the BamHI and SalI sites of pESC-LEU and pESC-URA
vectors (Stratagene), resulting in pESCL-HAAPG7, pESCL-MycAPG7,
pESCU-HAAPG7, and pESCU-MycAPG7, respectively. For the deletion of the
C-terminal region of Apg7p, polymerase chain reaction was performed
with appropriate primers, and the resulting product was inserted into the BamHI-SalI sites of pESC-LEU and pESC-URA
vectors (Stratagene) to generate pESCL-HAAPG7
The APG3 and APG8 open reading frames in which
the stop codon was excluded were amplified by polymerase chain reaction
using a yeast genomic library and appropriate oligonucleotides that incorporated a BamHI site on the 5'-primer and a
SalI site on the 3'-primer
(5'-CGGATCCATTATCATGATTAGATCTAC-3', 5'-AGTCGACCCAACCTTCCATGGTATAGT-3' for APG3, 5'-AGGATCCAGAGACATGAAGTCTACATT-3',
5'-AGTCGACCCTGCCAA ATGTATTTTCTC-3' for APG8), and the
amplified DNA fragments were cloned into pGem-T vector (Promega),
resulting in pGEM-APG3 and pGEM-APG8, respectively. The
BamHI-SalI fragments of pGEM-APG3 and pGEM-APG8
were introduced into the BamHI-SalI sites of
pGAD-C1, thus generating pGAD-APG3 and pGAD-APG8, respectively. For the expression of Myc-Apg3p, the BamHI-SalI fragments
of pGEM-APG3 were inserted into the BamHI-SalI f
site of pESC-URA vector (Stratagene), resulting in pESCU-APG3Myc. For
the expression of the N-terminal FLAG-Apg8p, the DNA sequence,
which encodes for a FLAG epitope tag, was inserted just before the
start codon of the APG8 gene, and the resulting sequence was
introduced into the BamHI and SalI sites of
pESC-URA vector (Stratagene), resulting in pESCU-FLAGAPG8.
Two-hybrid Experiment--
The improved two-hybrid system was
performed as described previously (36). A host strain, PJ69-4A
(MAT a trp1 leu2 ura3 his 3 gal4 Chemical Cross-linking--
Yeast cells that express Myc-Apg7p
were harvested and converted to spheroplasts in 1 M
sorbitol with 10 mg/ml zymolyase 100T. The spheroplasts were lysed with
ice-cold PNE buffer (10 mM potassium phosphate, pH 7.4, 1%
Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and protease inhibitors) and sonicated for a short time. The lysate was
then centrifuged at 10,000 × g for 5 min at 4 °C to
remove debris. The supernatant was incubated with 5 mM
disuccinimidyl suberate for 30 min at 30 °C. Free reactive groups of
disuccinimidyl suberate were quenched by incubation with 50 mM Tris-Cl, pH 7.5, for 15 min at 30 °C.
Glycerol Gradient Centrifugation--
Yeast spheroplasts were
prepared in the presence of 1 M sorbitol, lysed with
ice-cold PNE buffer, and sonicated for a short time. The lysates were
centrifuged at 10,000 × g for 5 min at 4 °C to
remove cell debris. The supernatant was subjected to glycerol density
centrifugation in 10-40% glycerol in 10 mM potassium
phosphate, pH 7.4, 1% Nonidet P-40, and 150 mM NaCl. After
centrifugation at 200,000 × g for 18 h at 4 °C
in a Beckman SW-41 rotor, the gradient was separated into 18 fractions
of 600 µl. Each fraction was analyzed by immunoblotting with an
anti-Myc antibody (9E10) or an anti-HA antibody (16B12). 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.
Immunoblotting--
Immunoblots were performed using a
previously described protocol (26). Briefly, yeast cells harboring the
appropriate plasmids were suspended in 100 µl of 0.2 N
NaOH that contained 0.5% 2-mercaptoethanol. After incubation for 15 min on ice, 1 ml of acetone at Immunoprecipitation--
The spheroplasts in 1 M
sorbitol were lysed by sonication. 800 µl of PNE buffer was added to
the lysate, and the mixture was centrifuged at 10,000 × g for 5 min at 4 °C to remove debris. The supernatant was
precleared with 50 µl of protein A-agarose (20% slurry, Santa Cruz
Biotechnology, Santa Cruz, CA). 50 µl of protein A-agarose and 1 µg
of anti-HA antibody (F7, Santa Cruz Biotechnology, Santa Cruz, CA) was
then added to the lysate, and the mixture was rotated for 12 h at
4 °C. The immunoprecipitate-bead complex was washed five times with
ice-cold PNE buffer. The complex was then boiled for 5 min in SDS
sample buffer in the presence of Apg12p-activating Enzyme, Apg7p, Forms a
Homodimer--
Considering the divergence in the localization of
Apg7p, Apg12p, and the Apg12p-Apg5p conjugate, the possibility
of targeting machinery for Apg12p to an Apg5p-associated membrane
becomes an important issue. Previous studies indicated that Apg7p
interacts more potently with Apg12p than with Apg10p (25, 26, 39). Therefore, it is possible that Apg7p-interacting protein(s) functions as targeting machinery in an Apg12p-dependent manner. To
investigate the Apg12p-dependent Apg7p-interacting protein,
a yeast two-hybrid screening was carried out using a tester strain that
overexpressed HA-Apg12p on a 2-µm plasmid with Apg7p as the bait. Of
1 × 106 independent clones, two positive candidates
were isolated. DNA-sequencing analysis indicated that both inserts
encode the C-terminal region of Apg7p (residues 262-630 out of 630 amino acids, Apg7p
To confirm the interaction of Apg7p homooligomer, the
coimmunoprecipitation with Myc- and HA-tagged Apg7p proteins was
employed. Myc-Apg7p and HA-Apg7p were coexpressed in the
apg7
We next employed a cross-linking experiment using a chemical
cross-linker, disuccinimidyl suberate. The lysate of the
apg7
Glycerol density gradient ultracentrifugation also indicates that Apg7p
forms a homodimer. Total cell lysates of the apg7
The Apg7p-Apg7p interaction was first found in the presence of excess
Apg12p. Thus, it is possible that an endogenous Apg12p mediates the
interaction of Apg7p. To investigate this possibility, we examined the
cross-linking experiment of Myc-Apg7p in the apg12 The C-terminal Region of Apg7p Is Essential for Apg7p
Dimerization--
The issue arises as to the nature of the essential
domain for the formation of Apg7p-homodimer. Motif analysis of the
amino acid sequence of Apg7p showed that no potential dimerization
motifs such as a coiled-coil or a leucine zipper exist on the molecule. To determine the region of Apg7p that is essential for dimer formation, systematic deletion analyses were performed. The original clone isolated (Apg7p
The location of the essential domain within the C-terminal 123 amino
acids (residues 508-630) is also an open question. ClustalW analysis
revealed that a C-terminal region containing 40 amino acids (residues
591-630) has a significant homology with the equivalent region of
mammalian Apg7p homologs and that it shows a weak homology with the
equivalent region of Uba1p (Fig.
3A) (40). The relevance of
this region (C40 region) to Apg7p-Apg7p interaction was thus investigated. In a two-hybrid analysis, the deletion of the C40 region
from Apg7p results in the complete loss of its ability to interact with
full-length Apg7p (Fig. 1, APG7 The C-terminal Region Is Also Essential for Interactions of Apg7p
with Two Substrates, Apg12p and Apg8p--
What is the importance of
the Apg7p-dimerization? Apg7p
These defects in the interactions of Apg7p
We next examined the effect of the C40 region of Apg7p on the Cvt
pathway. Aminopeptidase I (API) is synthesized as the pro-form (proAPI)
in the cytoplasm, transferred to the vacuole by a mechanism that is
closely related to the autophagic pathway, and then processed to the
mature form in the vacuole. In the case of the apg7 The C-terminal Region of Apg7p Is Necessary for the Formation of
E1-E2 Complex for Apg8p--
Recently, a comprehensive analysis of
protein-protein interactions in the yeast S. cerevisiae by
two-hybrid screening has indicated that Apg7p interacts not only with
Apg12p and Apg8p but also with Apg3p (Fig.
5A) (39). Recent findings
revealed that Apg3p is an E2 enzyme for Apg8p. Thus far, no report has appeared on the formation of an E1-E2 complex in a protein modification system similar to ubiquitylation. Then we first investigated whether Apg3p is coimmunoprecipitated with Apg7p. Both HA-Apg7p and Apg3p-Myc were coexpressed under the control of a galactose-inducible promoter in
the apg7
Since the C40 region of Apg7p is essential for its interaction with the
two substrates, it is probable that this region is essential for the
interaction of Apg7p with Apg3p, too. To investigate this possibility
further, a coimmunoprecipitation experiment was performed in the
apg7 The evidence presented herein indicates that Apg7p is a unique
protein-activating enzyme that is capable of forming a homodimer and is
essential for the two substrates (Apg12p and Apg8p). These characteristics have not been reported for other E1 enzymes.
Furthermore, the Apg7p is able to form a stable E1-E2 complex. The
dimerization occurs independently of other APG gene products
examined thus far, supporting the possibility that Apg7p interacts with
itself without the need for any other factors. The deletion of the C40 region of Apg7p results in the loss of Apg7p dimerization. It is
surprising that the C40 region is also essential for the interaction of
Apg7p with two substrates, Apg12p and Apg8p, even though the mutant
Apg7p During starvation-induced autophagy, Apg8p became localized on the
forming autophagosomal membranes (31). Our recent findings suggest that
both Apg7p and Apg3p function as E1 and E2 enzymes, respectively, which
is necessary for Apg8p to target the autophagosomal membranes (29, 30).
We therefore reason that multimer complexes that are formed during the
two enzymatic reactions catalyzed by the Apg7p homodimer may play a key
role in autophagy. An Apg7p-related scheme is shown in Fig.
6A. First, Apg7p undergoes
homodimer formation (Fig. 6A, I). In one route of
the next step, the Apg7p homodimer, which forms an enzyme-substrate
conjugate with Apg12p via a thiol ester bond (Fig. 6A,
IIa), functions as an E1 enzyme, which is essential for
subsequent Apg12p-Apg5p conjugation (Fig. 6A,
IIIa). Similarly, in the other route (Fig. 6A,
IIIb), Apg7p, which forms an enzyme-substrate conjugate with
Apg8p, also functions as an E1 enzyme, which is essential for
subsequent Apg8p-phophatidylethanoamine conjugation. The interaction of
Apg3p (E2) with Apg7p (E1) may facilitate the effective targeting of
Apg8p to autophagosomal membranes as well as to ER-to-Golgi vesicles
(Fig. 6A, IIIb) (28-32). How do these
interactions correlate with the functions of these APG gene
products? A key to revealing this question would be an interaction of
Apg12p with Apg3p, which has been reported by a comprehensive
two-hybrid screening (39). This suggests that Apg12p plays an important
role in Apg8p targeting on autophagosomal membranes. At present, it is
difficult to completely explain the functional correlation between
Apg12p modification system and Apg8p-membrane targeting. Further
analyses to clarify Apg12p versus Apg3p interaction will be
necessary.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, the host
for plasmids and protein expression, was grown in Luria Broth medium in
the presence of the required antibiotics (35). The S. cerevisiae apg7
mutants and PJ69-4A used in this study are
listed in Table I. The apg mutant strains have been described previously (6). All yeast strains
were cultured in a rich medium (YPD: 1% yeast extract, 2%
polypeptone, 2% glucose, 20 mg/liter adenine, 20 mg/liter tryptophan, 20 mg/liter 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. (36). The nitrogen starvation medium contained 0.17% yeast nitrogen base without amino acids, ammonium sulfate, and 2% glucose. For the galactose-inducible expression of proteins in the yeast, SSG medium (0.67% yeast nitrogen base without amino acids, 0.2% sucrose, and 2% galactose) was employed. The solid medium contained 2% Bacto agar. Standard genetic and molecular biological techniques were performed as described by
Kaiser et al. (36) and Ausubel et al. (35). The
polymerase chain reaction was performed with a program temperature
control system PC-701 (ASTEC, Fukuoka, Japan). The DNA sequence was
determined using an ABI 373A DNA sequencer (PE Applied Biosystems,
Foster City, CA). Restriction enzymes were purchased from TOYOBO
(Osaka, Japan) and New England Biolabs (Beverly, MA). Oligonucleotides were synthesized by ESPEC oligo service (Ibaraki, Japan).
Two-hybrid screening was performed using the system described by James
et al. (37). The expression of a protein under the control
of a galactose-inducible promoter was performed after the
manufacturer's recommended protocol (Stratagene, La Jolla, CA). pRS
series vectors were generous gifts from P. Hieter, and the pGAD-C1
vector, pGBD-C1 vector, and PJ69-4A strain were generous gifts from P. James (37, 38). pGem-T vector was purchased from Promega (Madison, WI). A series of pESC vectors was purchased from Stratagene.
Yeast strains and plasmids used in this study
N containing APG7, which encodes the C-terminal
half (amino acids 262-630 out of 630 amino acids), was isolated in the
two-hybrid screening. Parts of the APG7-encoding deletion of
the C- and N-terminal regions were amplified by polymerase chain
reaction and inserted into the BamHI-SalI sites
of pGAD-C1. pGAD-APG7 contains the same insert sequences as pGBD-APG7.
pGAD-APG12 has been described previously (26).
C40,
pESCL-MycAPG7
C40, pESCU-HAAPG7
C40, and pESCU-MycAPG7
C40, respectively.
gal80
GAL2-ADE2 LYS2::GAL1-HIS3
met2::GAL7-lacZ), has been created that contains three
easily assayed reporter genes, each under the control of a different
inducible promoter. The ADE2 gene is under the control of
the GAL2 promoter, and HIS3 gene is under the
control of the GAL1 promoter. As a result, this strain is
extremely sensitive to weak interactions and eliminates nearly all
false positives using simple plate assays, i.e. a strong interaction of the examined gene proteins suppresses both
Ade
and His
auxotrophy of PJ69-4A, whereas
a weak interaction suppresses only His
auxotrophy.
20 °C was added, and the incubation
was continued for an additional 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 sample buffer
and boiled for 5 min. Lysates equivalent to
0.5-A600 cells were separated by SDS-PAGE and
transferred to a polyvinylidene difluoride membrane (Millipore). Mouse
monoclonal anti-HA antibody (16B12), anti-Myc antibody (9E10), or
rabbit anti-API antibody (a gift from Prof. Klionsky) was used for
immunodetection. Development was performed by the ECL Plus detection
methods (Amersham Pharmacia Biotech).
-mercaptoethanol to elute proteins
and centrifuged at 10,000 × g for 5 min at 4 °C.
The supernatant was subjected to SDS-PAGE, transferred to a
polyvinylidene difluoride membrane, and analyzed by immunoblotting with
anti-HA (F7), anti-Myc (9E10), or anti-FLAG antibody (M2, Sigma).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N261) (Fig. 1,
APG7
N261, PJ69-4A [APG12
2 µ]). An interaction between Apg7p
N261 and Apg7p
was observed without Apg12p being expressed (Fig. 1,
APG7
N261, PJ69-4A), although
overexpressed Apg12p significantly enhanced the interaction. Similarly,
the interaction of wild-type Apg7p with itself was also observed in the
absence of overexpressed Apg12p (Fig. 1, APG7 wild
type).
View larger version (11K):
[in a new window]
Fig. 1.
Two hybrid analysis of self interaction of
Apg7p. The domain structure of wild-type Apg7p is schematically
represented on the top. The black box shows ATP binding
domain (residues 331-336 out of 630 amino acids), and Cys-507 is the
active-site cysteine. In a two-hybrid assay, a tester strain, PJ69-4A,
contains two reporter genes under the control of a different inducible
promoter, and its growth phenotype is shown according to the
auxotrophy: +++, cells grew well on the SD-Ade and SD-His plates
(colony size was about 1.5 mm after incubation at 30 °C for 3 days
on an SD-Ade plate); ++, cells grew on SD-Ade and SD-His plate
(colony-size was about 0.5 mm after incubation at 30 °C for 3 days
on an SD-Ade plate and about 1.5 mm after incubation at 30 °C for 3 days on an SD-His plate); +, cells grew on SD-His plate (colony size is
about 0.5 mm after incubation at 30 °C for 7 days on SD-His plate);
, cells did not grow on SD-His plate after incubation at 30 °C for
7 days. As host strains, a tester PJ69-4A strain overexpressing Apg12p
(PJ69-4A [APG12, 2µ]) and a tester PJ69-4A
strain (PJ69-4A) were used. Constructs fused with GAD of
wild type and five deletion mutants of Apg7p are shown. The
number in the parentheses of each construct
indicates the amino acid number of Apg7p.
mutant under the control of a galactose-inducible
promoter, which suppresses both Apg
and Cvt
phenotypes of the apg7
mutant (data not shown). Cell
lysates of the transformant were prepared, and HA-Apg7p was
immunoprecipitated with an anti-HA antibody. When galactose was added
to the medium, HA-Apg7p was immunoprecipitated with the anti-HA
antibody (Fig. 2A, lane
4). At the same time, Myc-Apg7p was also coimmunoprecipitated (Fig. 2A, lanes 2). Essentially the same results
were obtained by coimmunoprecipitation with an anti-Myc antibody (data
not shown).
View larger version (44K):
[in a new window]
Fig. 2.
Homodimer formation of Apg7p.
A, coimmunoprecipitation (IP) of Myc-Apg7p with
HA-Apg7p. A total lysate of the apg7 cells expressing
both Myc-Apg7p and HA-Apg7p was prepared, and HA-Apg7p was
immunoprecipitated with the anti-HA antibody. The sediments were
analyzed by immunoblotting (IB) using anti-Myc (lane
2) or anti-HA (lane 4) antibodies. B, Apg7p
dimer formed by chemical cross-linking. Myc-Apg7p was expressed in the
apg7
cells, and the cell lysate was treated with 5 mM disuccinimidyl suberate (DSS), as described
under "Experimental Procedures." Myc-Apg7p was recognized by
immunoblotting with an anti-Myc antibody. C, sedimentation
analysis of Apg7p. Cell lysate was prepared from the apg7
cells expressing Myc-Apg7p and separated by centrifugation through
10-40% glycerol gradients. Fractions were collected from the bottom
of the gradients and assayed for the presence of Myc-Apg7p by
immunoblotting using an anti-Myc antibody (9E10). The positions of
marker proteins in the gradients are indicated on the top (both as
molecular mass, kDa; and sedimentation values,
S). BSA, bovine serum albumin. D,
cross-linking of Apg7p with DSS in the extracts prepared from several
apg mutants.
cells, which express Myc-Apg7p, was prepared in the
presence or absence of disuccinimidyl suberate and separated by
SDS-PAGE, and the Myc-Apg7p was subsequently identified by immunoblot
with an anti-Myc antibody. In the absence of the cross-linker, a 78-kDa band was observed that corresponds to monomeric Apg7p. When the cell
lysate was treated with the cross-linker, the intensity of the monomer
band decreased significantly, and a higher molecular mass band (about
160 kDa), the size of which corresponded to a dimer, appeared (Fig.
2B).
cells
overexpressing Myc-Apg7p were subjected to a 10-40% glycerol density
gradient centrifugation. The resulting fractions were analyzed by
SDS-PAGE, and the Myc-Apg7p was subsequently identified by
immunoblotting with an anti-Myc antibody. Myc-Apg7p was collected in
fractions 12-15 and mainly sedimented with a sedimentation coefficient
of ~7.4 S (Fig. 2C, fraction 13). The
overexpression of Apg12p along with the Apg7p resulted in a higher
concentration of Apg7p in fraction 13 (data not shown).
mutant. Even in the apg12
mutant, a higher molecular mass
band corresponding to Apg7p homodimer (about 160 kDa) appeared in the presence of a chemical cross-linker as in wild-type (Fig.
2D). The dimerization of Apg7p was further investigated in
other apg mutants. As in wild-type and the
apg12
cells, a higher molecular weight band of Apg7p
(about 160 kDa) appeared depending on the chemical cross-linker in
other apg mutants (apg1c apg2,
apg3, apg4, apg5, apg6,
apg8, apg9, apg10, apg13,
and apg14 mutants) (representative data are presented in
Fig. 2D, and the data on the other mutants are not shown).
These results indicate that Apg7p forms a homodimer with itself without
the participation of other APG gene products.
N261) lacks N-terminal 261 amino acid residues, suggesting that a region that is proximal to the C terminus may be
important for dimerization. Since Apg7p
N261 still possesses both an
ATP-binding site and an active-site cysteine residue, we first deleted
the catalytic domain and examined the resultant construct
(Apg7p
N507) to determine whether it is capable of binding to
full-length Apg7p. Apg7p
N507 interacts with full-length Apg7p, indicating that the C-terminal portion (residues 508-630 out of 630 amino acids), which contains neither an ATP binding domain nor an
active-site cysteine, is sufficient for interaction with the
full-length Apg7p (Fig. 1, APG7
N507).
C40),
suggesting that the C40 region is essential for Apg7p dimerization. An
attempt was made to determine whether the C40 region is sufficient for interaction with full-length Apg7p in a two-hybrid system. However, it
was found that cells expressing only GBD-C40 were able to grow in
selection plates. Therefore, further analyses on the C40 region itself were not pursued, and efforts were concentrated on Apg7p
C40. A coimmunoprecipitation assay confirmed the loss of interaction of
Apg7p with Apg7p
C40. Both wild-type HA-Apg7p and Myc-Apg7p
C40 were coexpressed in the apg7
cells. The cell lysate was
immunoprecipitated with the anti-HA antibody, and Myc-Apg7p and
Myc-Apg7p
C40 were examined by immunoblot with the anti-Myc antibody.
Myc-Apg7p
C40 was expressed at a level similar to that of wild-type
Myc-Apg7p in a galactose-dependent manner (Fig.
3B, lanes 4 and 2). Wild-type Myc-Apg7p was coimmunoprecipitated with HA-Apg7p (Fig. 3C,
lane 2). In contrast, the mutant Apg7p
C40, was not
coimmunoprecipitated with HA-Apg7p (Fig. 3C, lane
6). These results indicate that the C40 region of Apg7p is
required for the formation of the Apg7p homodimer.
View larger version (37K):
[in a new window]
Fig. 3.
Effects of C-terminal deletion of Apg7p on
Apg7p dimerization. A, amino acid sequences of the
C-terminal region of yeast Apg7p (ScAPG7c; residues 581-630
out of 630 amino acids) compared with those of the equivalent regions
of human Apg7p (HsAPG7c; residues 654-703 out of 703 amino
acids), murine Apg7p (MmAPG7c; residues 649-698 out of 698 amino acids), and yeast Uba1p (ScUBA1c; residues 985-1024
out of 1024 amino acids) using the ClustalW program (24, 39, 40, 42).
B, a mutant protein, Myc-Apg7p C40 was expressed at the
same level of wild-type Myc-Apg7p. The apg7
cells were
transformed with both pESCL-MycAPG7 and pESCU-HAAPG7 (lane
2) or both pESCL-MycAPG7
C40 and pESCU-HAAPG7 (lane
4), respectively. After galactose induction, total lysates from
the transformants were prepared and analyzed by immunoblotting
(IB) using an anti-Myc antibody (9E10). C, total
lysates of the apg7
cells, which express a set of
wild-type Myc-Apg7p and wild-type HA-Apg7p (lanes 1-4) or a
set of mutant Myc-Apg7p
C40 and wild-type HA-Apg7p (lanes
5-8) were prepared, and HA-protein was immunoprecipitated with an
anti-HA antibody (IP: Anti-HA). The resulting
sediment was analyzed by SDS-PAGE, and epitope-tagged proteins were
detected by immunoblot using anti-HA or anti-Myc antibodies
(IB, Anti-Myc or Anti-HA).
C40 still contains both an ATP binding
domain and an active-site cysteine residue but has a defect relative to
the formation of Apg7p homodimer. We hypothesized that the dimerization
of Apg7p may be somehow correlated with the E1 activity of Apg7p for
the Apg12p and Apg8p. It would be interesting to determine whether
Apg7p
C40 is able to bind to Apg12p and Apg8p. The interaction of
Apg7p
C40 with Apg12p was first investigated using a two-hybrid
analysis. Surprisingly, the interaction of Apg7p
C40 with Apg12p was
completely abolished compared with the interaction of wild-type Apg7p
and Apg12p (Fig. 4A,
Prey APG12). Furthermore, the loss of interaction of
Apg7p
C40 resulted in a defect in the formation of the Apg12p-Apg5p
conjugate. No Apg12p-Apg5p conjugate was present in the
apg7
mutant expressing Apg7p
C40, whereas the conjugate
was present in the mutant expressing the wild-type Apg7p (Fig.
4B, lane 4 and 2). Similarly, a loss of interaction of Apg7p
C40 with Apg8p was also detected using a
two-hybrid assay (Fig. 4A, Prey APG8). This was
further confirmed via a coimmunoprecipitation experiment. A lysate of
apg7
cells coexpressing HA-Apg7p
C40 and FLAG-Apg8p was
prepared, and HA-Apg7p was immunoprecipitated with an anti-HA antibody.
FLAG-Apg8p in the sediment was identified by immunoblot with the
anti-FLAG antibody. No FLAG-Apg8p was coimmunoprecipitated with
HA-Apg7p
C40, whereas the FLAG-Apg8p was coimmunoprecipitated with
wild-type HA-Apg7p (Fig. 4C, lanes 4 and
2). These results indicate that the C40 region of Apg7p is
essential for the interaction of Apg7p with the two substrates.
View larger version (43K):
[in a new window]
Fig. 4.
Effects of C-terminal deletion of Apg7p on
the interaction of Apg7p with two substrates, Apg12p and Apg8p.
A, summary of the interactions of deletion mutants of Apg7p
with Apg12p and Apg8p by an improved two-hybrid system. Cells
expressing both GBD-fused Apg7p variants and GAD-Apg12p (APG12) or
GAD-Apg8p (APG8) were assayed for interaction-dependent
activation of the ADE2 gene and HIS3 gene as
described in Fig. 1. B, the formation of the Apg12p-Apg5p
conjugate. The apg7 cells harboring pAPG12HA-426 and
either pESCL-MycAPG7 or pESCL-MycAPG7
C40 were cultured in the
presence (+) or absence (
) of galactose and lysed as described under
"Experimental Procedures." The conjugate was recognized by
immunoblot using anti-HA antibody. pESCL-MycAPG7/pAPG12HA-426: the
lysate of the apg7
cells, which express both wild-type
Myc-Apg7p and HA-Apg12p; pESCL-MycAPG7
C40/pAPG12HA-426: the lysate
of the apg7
cells expressing both Myc-Apg7p
C40 and
HA-Apg12p. C, coimmunoprecipitation of Apg8p with Apg7p.
Total lysates from the apg7
cells, which express
FLAG-Apg8p and either HA-Apg7p or HA-Apg7p
C40 in the presence (+) or
absence (
) of galactose were immunoprecipitated (IP) with
an anti-HA antibody. Precipitates were analyzed by immunoblotting
(IB) using anti-HA or anti-FLAG antibody. Lanes 1 and 2, the apg7
cells expressing FLAG-Apg8p
and HA-Apg7p; lanes 3 and 4, the
apg7
cells expressing FLAG-Apg8p and
HA-Apg7p
C40. IgG HC, heavy chain of IgG; IgG LC, light chain
of IgG. D-E, effects of the deletion of the C-terminal
region of Apg7p on the accumulation of autophagic bodies
(D), the viability under nitrogen starvation conditions
(E), and cytoplasm-to-vacuole targeting of aminopeptidase I
(F). In D, cells grown to early logarithmic phase
in MVD + Ura medium were transferred to nitrogen starvation medium in
the presence of phenylmethylsulfonyl fluoride and incubated for 8 h at 30 °C. Representative Nomarski images of the cells are shown.
In E, cells were plated on nitrogen starvation medium
containing 10 µg/ml phloxine B and incubated at 30 °C for 3 days.
Inviable cells were stained red (gray in monochrome),
whereas viable cells were not stained (white in monochrome).
apg7
, the apg7
cells carrying control
vector, pRS314; wild type, the apg7
cells carrying
pAPG7Myc-314;
C40, the apg7
cells carrying
pAPG7
C40-314. In F, cell lysates from the
apg7
cells harboring pRS314 (lane 1),
pAPG7Myc-314 (lane 2), or pAPG7
C40 (lane 3)
were subjected to immunoblotting analysis with anti-API
antiserum.
C40 with two substrates
will result in pleiotropic defects of the autophagic and Cvt pathways.
In yeast, autophagy is induced by a variety of starvation conditions,
and its progression is easily monitored by means of light microscopy;
autophagic bodies accumulate in the vacuoles of wild-type cells under
conditions of nitrogen starvation, and the detection of these
autophagic bodies is facilitated by phenylmethylsulfonyl fluoride, a
protease inhibitor that blocks their degradation (3). In the vacuoles
of apg7
cells expressing wild-type Apg7p, a significant accumulation of autophagic bodies was observed, whereas the
apg7
cells expressing Apg7p
C40 failed to accumulate
autophagic bodies in the vacuole, as was the case of the
apg7
cells carrying a control vector (Fig.
4D, wild type,
C40, and
apg7
). A defect in autophagy results in a loss of
viability under conditions of starvation (6). The colony color of the
apg7
mutant expressing Apg7p
C40 turned a pink color
under nitrogen starvation conditions, as evidenced by phloxine
B-staining, similar to the apg7
mutant, whereas that of
the apg7
mutant expressing wild-type Apg7p was not
stained (Fig. 4E,
C40, apg7
, and wild type).
cells expressing wild-type Apg7p, proAPI was processed to the mature form
(Fig. 4F, lane 2). In contrast, in the case of
apg7
cells expressing mutant Apg7p
C40, the mature form
of API was not detected, and proAPI accumulated, similar to that of the
apg7
cells (Fig. 4F, lane 3 and
1). Thus, the loss of function of Apg7p as the result of the
deletion of the C40 region of Apg7p results in a defect in the Cvt
pathway. These results indicated that the C40 region of Apg7p is
essential for its E1 function in both the autophagic and Cvt pathways.
cells, and the cell lysate was
immunoprecipitated with an anti-HA antibody. Apg3p-Myc in the sediment
was recognized by immunoblot with an anti-Myc antibody. Apg3p-Myc was
coimmunoprecipitated with HA-Apg7p (Fig. 5B, lane
2). The coimmunoprecipitation of Apg3p with Apg7p occurs in the
apg12
mutant (data not shown). This result indicates that
the Apg7p interacts with Apg3p to form an E1-E2 complex.
View larger version (30K):
[in a new window]
Fig. 5.
Effects of the deletion of C-terminal region
of Apg7p on the formation of E1-E2 complex for Apg8p.
A, summary of interactions of Apg3p with Apg7p variants.
Cells expressing both GBD fused Apg7p-variants (Bait) and
GAD-Apg3p (Prey APG3) were examined for
interaction-dependent activation of the ADE2
gene and HIS3 gene. B, inability of Apg3p to
coimmunoprecipitate with mutant Apg7p C40. Total lysates from
apg7
cells expressing either HA-Apg7p or HA-Apg7p
C40
and Apg3p-Myc in the presence (+) or absence (
) of galactose were
immunoprecipitated (IP) with an anti-HA antibody. The
sediments were analyzed by immunoblotting (IB) using an
anti-HA or anti-Myc antibody. pESCL-HAAPG7/pESCU-APG3Myc, the
apg7
cells, which express HA-Apg7p and Apg3p-Myc;
pESCL-HAAPG7
C40/pESCU-APG3Myc, the apg7
cells, which
express HA-Apg7p
C40 and Apg3p-Myc. IgG HC, heavy chain of
IgG.
cells expressing both HA-Apg7p
C40 and Apg3p-Myc. No Apg3p-Myc was coimmunoprecipitated with HA-Apg7p
C40, whereas the Apg3p-Myc was coimmunoprecipitated with wild-type HA-Apg7p
(Fig. 5B, lanes 4 and 2). This finding
was confirmed by a two-hybrid analysis (Fig. 5A).
Furthermore, as is the case with the interaction of Apg12p and Apg8p,
Apg3p does not interact with Apg7p
N507 (Fig. 5A). These
results indicated that the C-terminal region of Apg7p is essential for
the formation of the E1-E2 complex, Apg7p·Apg3p.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C40 monomer still contains an ATP binding domain and an active
site cysteine. The C40 region of Apg7p is also essential for the
formation of the E1-E2 complex, Apg7p·Apg3p. The C-terminal 123 residues (residues 508-630), which contain the C40 region, are
sufficient for interaction with full-length Apg7p. Combining these
data, we conclude that homodimer formation via the C-terminal region is
important for enzyme-substrate interaction and the formation of an
E1-E2 complex.
View larger version (23K):
[in a new window]
Fig. 6.
Hypothetical scheme of the Apg7p homodimer
and domain structure of the E1 enzymes are shown. A,
the dimerization of Apg7p is essential for further reaction.
Considering the fact that the C-terminal region of Apg7p, but neither
Apg12p, Apg8p, nor Apg3p, interacts directly with wild-type Apg7p, the
formation of Apg7p homodimer is necessary for interactions with
substrates and E2 enzyme (I). After Apg7p dimerization,
Apg12p(s) is activated by the Apg7p homodimer (IIa).
Subsequent transfer of Apg12p to Apg10p results in the formation of the
Apg12p-Apg10p intermediate. Finally, Apg12p is covalently attached to
Apg5p via an isopeptide bond (IIIa). When the Apg7p
homodimer interacts with Apg8p, Apg8p is activated by the Apg7p
homodimer (IIb). Apg3p transiently interacts with Apg7p
homodimer to effectively form Apg8p-Apg3p intermediate. Finally, Apg8p
targets to an autophagosome (IIIb). PE,
phosphatidylethanolamine. B, Box V is proposed as the
essential domain for the homodimerization of Apg7p and Uba1p. The box
V, which contains a cluster of acidic amino acids, is essential for
homodimerization of Apg7p and the formation of E1-E2 complex. The
C-terminal region of Uba1p has a similarity with the box V within
Apg7p, and Uba1p will also then form a homodimer. UBI,
ubiquitin.
The human Apg7p/Cvt2p/Gsa7p homolog has been reported (41). The issue of whether or not the mammalian homolog forms a homodimer is of great interest. Several lines of observations suggest that the mammalian Apg7p homolog also is capable of entering into a homodimer formation. (i) The Apg12p conjugation system is conserved from yeast through mammalian cells (42). (ii) The human Apg7p homolog is an authentic protein-activating enzyme for human Apg12p (43). (iii) The C40 region of S. cerevisiae Apg7p is highly conserved among human Apg7p and mouse Apg7p (Fig. 4A). We are now investigating the possibility of homodimer formation in mammalian Apg7p homologs by means of cross-linking experiments and glycerol density gradient centrifugation.
Several E1 enzymes, Uba1p for ubiquitin, Aos1p-Uba2p for Smt3p,
Ula1p-Uba3p for Rub1p, Apg7p for Apg12p, and Uba4p for Urm1p have been
characterized in yeast (26, 44-51). According to Johnson et
al. (46) and Liakopoulos et al. (49), four similarity
boxes (I~IV) exist that are preserved in the E1 enzymes. Apg7p
contains an ATP binding domain within the box I and an active-site
cysteine within the box III, but no domains similar to the box II and
the box IV are present within Apg7p (Fig. 6B) (26). We
propose the C40 region of Apg7p as box V to be an essential domain for
homodimer formation. Box V contains a cluster of acidic amino acids.
Interestingly, the C-terminal region of Uba1p has some similarity with
the C40 region of Apg7p. In an earlier study, Ciechanover et
al. (52) report the purification of a mammalian homolog of Uba1p
by "covalent affinity" chromatography and conclude that the
purified enzyme is composed of two subunits. It is therefore possible
that Uba1p also forms a homodimer via its C-terminal region, box V. The
absence of a box V in two other E1 enzymes (Aos1p-Uba2p and
Ula1p-Uba3p) suggests that they are functional when the exist as a
heterodimer with only one active-center cysteine. It is therefore
important to understand how the divergence in molecular composition of
protein-activating enzyme (E1) can be correlated with functional
divergence in various cellular activities.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank P. James (University of Wisconsin) for providing strains and plasmids, N. Mizushima (National Institute for Basic Biology) for providing plasmids, D. J. Klionsky (University of California, Davis) for providing the anti-API antibody, M. Kiyooka for technical assistance, and K. Ishidoh, J. Ezaki, and D. Muno (Juntendo University) for helpful discussions.
![]() |
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. kominami@med.
juntendo.ac.jp.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M007737200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Apg8p, Aut7p/Apg8p; Apg3p, Aut1p/Apg3p; Apg3-myc, myc-tagged Apg3p; API, aminopeptidase I; proAPI, pro-form of API; Cvt, cytoplasm-to-vacuole; GAD, GAL4-activating domain; GBD, GAL4 DNA binding domain; HA, hemagglutinin epitope-tagged; PAGE, polyacrylamide gel electrophoresis; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Seglen, P. O., and Bohley, P. (1992) Experientia (Basel) 48, 158-172 |
2. | Dunn, W. A., Jr. (1994) Trends Cell Biol. 4, 139-143[CrossRef] |
3. | Takeshige, K., Baba, M., Tsuboi, S., Noda, T., and Ohsumi, Y. (1992) J. Cell Biol. 119, 301-311[Abstract] |
4. | Baba, M., Takeshige, K., Baba, N, and Ohsumi, Y. (1994) J. Cell Biol. 124, 903-913[Abstract] |
5. | Baba, M., Osumi, M., and Ohsumi, Y. (1995) Cell Struct. Funct. 20, 465-471[Medline] [Order article via Infotrieve] |
6. | Tsukada, M., and Ohsumi, Y. (1993) FEBS Lett. 333, 169-174[CrossRef][Medline] [Order article via Infotrieve] |
7. | Thumm, M., Egner, R., Koch, B., Schlumpberger, M., Straub, M., Veenhuis, M., and Wolf, D. H. (1994) FEBS Lett. 349, 275-280[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Harding, T. M.,
Hefner-Gravink, A.,
Thumm, M.,
and Klionsky, D. J.
(1996)
J. Biol. Chem.
271,
17621-17624 |
9. |
Scott, S. V.,
Hefner-Gravink, A.,
Morano, K. A.,
Noda, T.,
Ohsumi, Y.,
and Klionsky, D. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12304-12308 |
10. |
Baba, M.,
Osumi, M.,
Scott, S. V.,
Klionsky, D. J.,
and Ohsumi, Y.
(1997)
J. Cell Biol.
139,
1687-1695 |
11. | Klionsky, D. J., and Ohsumi, Y. (1999) Annu. Rev. Cell Dev. Biol. 15, 1-32[CrossRef][Medline] [Order article via Infotrieve] |
12. | Matsuura, A., Tsukada, M., Wada, Y., and Ohsumi, Y. (1997) Gene 192, 245-250[CrossRef][Medline] [Order article via Infotrieve] |
13. | Straub, M., Bredschneider, M., and Thumm, M. (1997) J. Bacteriol. 179, 3875-3883[Abstract] |
14. | Funakoshi, T., Matsuura, A., Noda, T., and Ohsumi, Y. (1997) Gene 192, 207-213[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Scott, S. V.,
Nice, I. I. I., D. C.,
Nau, J. J.,
Weismann, L. S.,
Kamada, Y.,
Keizer-Gunnink, I.,
Funakoshi, T.,
Veenhuis, M.,
Ohsumi, Y.,
and Klionsky, D. J.
(2000)
J. Biol. Chem.
275,
25840-25849 |
16. |
Kametaka, S.,
Okano, T.,
Ohsumi, M.,
and Ohsumi, Y.
(1998)
J. Biol. Chem.
273,
22284-22291 |
17. |
Noda, T.,
Kim, J.,
Huang, W.-P.,
Baba, M.,
Tokunaga, C.,
Ohsumi, Y.,
and Klionsky, D. J.
(2000)
J. Cell Biol.
148,
465-480 |
18. |
Lang, T.,
Reiche, S.,
Straub, M.,
Bredschneider, M.,
and Thumm, M.
(2000)
J. Bacteriol.
182,
2125-2133 |
19. | Varshavsky, A. (1997) Trends Biochem. Sci. 22, 383-387[CrossRef][Medline] [Order article via Infotrieve] |
20. | Bonifacino, J. S., and Weissman, A. M. (1998) Annu. Rev. Cell Dev. Biol. 14, 19-57[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Ciechanover, A.
(1998)
EMBO J.
17,
7151-7160 |
22. | Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425-479[CrossRef][Medline] [Order article via Infotrieve] |
23. | Kametaka, S., Matsuura, A., Wada, Y., and Ohsumi, Y. (1996) Gene 178, 139-143[CrossRef][Medline] [Order article via Infotrieve] |
24. | Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M. D., Klionsky, D. J., Ohsumi, M., and Ohsumi, Y. (1998) Nature 395, 395-398[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Shintani, T.,
Mizushima, N.,
Ogawa, Y.,
Matsuura, A.,
Noda, T.,
and Ohsumi, Y.
(1999)
EMBO J.
18,
5234-5241 |
26. |
Tanida, I.,
Mizushima, N.,
Kiyooka, M.,
Ohsumi, M.,
Ueno, T.,
Ohsumi, Y.,
and Kominami, E.
(1999)
Mol. Biol. Cell
10,
1367-1379 |
27. |
Mizushima, N.,
Noda, T.,
and Ohsumi, Y.
(1999)
EMBO J.
18,
3888-3896 |
28. |
Lang, T.,
Schaeffeler, E.,
Bernreuther, D.,
Bredschneider, M.,
Wolf, D. H.,
and Thumm, M.
(1998)
EMBO J.
17,
3597-3607 |
29. |
Kirisako, T.,
Ichimura, Y.,
Okada, H.,
Kabeya, Y.,
Mizushima, N.,
Yoshimori, T.,
Ohsumi, M.,
Takao, T.,
Noda, T.,
and Ohsumi, Y.
(2000)
J. Cell Biol.
151,
263-275 |
30. | Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., and Ohsumi, Y. (2000) Nature. 408, 488-492[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Kirisako, T.,
Baba, M.,
Ishihara, N.,
Miyazawa, K.,
Ohsumi, M.,
Yoshimori, T.,
Noda, T.,
and Ohsumi, Y.
(1999)
J. Cell Biol.
147,
435-446 |
32. |
Huang, W.-P.,
Scott, S. V.,
Kim, J.,
and Klionsky, D. J.
(2000)
J. Biol. Chem.
275,
5845-5851 |
33. |
Legesse-Miller, A.,
Sagiv, Y.,
Gluzman, R.,
and Elazar, Z.
(2000)
J. Biol. Chem.
275,
32966-32973 |
34. |
Kim, J.,
Dalton, V. M.,
Eggerton, K. P.,
Scott, S. V.,
and Klionsky, D. J.
(1999)
Mol. Biol. Cell
10,
1337-1351 |
35. | Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1995) Short Protocols in Molecular Biology , 3rd Ed. , John Wiley & Sons, Inc., New York |
36. | Kaiser, C., Michaelis, S., and Mitchell, A. (1994) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
37. |
James, P.,
Halladay, J.,
and Craig, E. A.
(1996)
Genetics
144,
1425-1436 |
38. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |
39. | Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi-Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J. M. (2000) Nature 403, 623-627[CrossRef][Medline] [Order article via Infotrieve] |
40. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |
41. |
Yuan, W.,
Stromhaug, P. E.,
and Dunn, W. A., Jr.
(1999)
Mol. Biol. Cell
10,
1353-1366 |
42. |
Mizushima, N.,
Sugita, H.,
Yoshimori, T.,
and Ohsumi, Y.
(1998)
J. Biol. Chem.
273,
33889-33892 |
43. |
Tanida, I.,
Tanida-Miyake, E.,
Ueno, T.,
and Kominami, E.
(2001)
J. Biol. Chem.
276,
1701-1706 |
44. | McGrath, J. P., Jentsch, S., and Varshavsky, A. (1991) EMBO J. 10, 227-236[Abstract] |
45. |
Dohmen, R. J.,
Stappen, R.,
McGrath, J. P.,
Forrova, H.,
Kolarov, J.,
Goffeau, A.,
and Varshavsky, A.
(1995)
J. Biol. Chem.
270,
18099-18109 |
46. |
Johnson, E. S.,
Schwienhorst, I.,
Dohmen, R. J.,
and Blobel, G.
(1997)
EMBO J.
16,
5509-5519 |
47. |
Johnson, E. S.,
and Blobel, G.
(1997)
J. Biol. Chem.
272,
26799-26802 |
48. |
Johnson, E. S.,
and Blobel, G.
(1999)
J. Cell Biol.
147,
981-994 |
49. |
Liakopoulos, D.,
Doenges, G.,
Matuschewski, K.,
and Jentsch, S.
(1998)
EMBO J.
17,
2208-2214 |
50. |
Lammer, D.,
Mathias, N.,
Laplaza, J. M.,
Jiang, W.,
Liu, Y.,
Callis, J.,
Goebl, M.,
and Estelle, M.
(1998)
Genes Dev.
12,
914-926 |
51. |
Furukawa, K.,
Mizushima, N.,
Noda, T.,
and Ohsumi, Y.
(2000)
J. Biol. Chem.
275,
7462-7465 |
52. |
Ciechanover, A.,
Elias, S.,
Heller, H.,
and Hershko, A.
(1982)
J. Biol. Chem.
257,
2537-2542 |