From the Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, September 7, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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Gln3p is a nitrogen catabolite
repression-sensitive GATA-type transcription factor. Its nuclear
accumulation was recently shown to be under the control of TOR
signaling. Gln3p normally resides in the cytoplasm. When cells are
starved from nitrogen nutrients or treated with rapamycin, however,
Gln3p becomes translocated into the nucleus, thereby activating the
expression of genes involved in nitrogen utilization and transport. To
identify other genes under the control of Gln3p, we searched for the
Gln3p-binding GATAA motifs within 500 base pairs of the promoter
sequences upstream of the yeast open reading frames in the
Saccharomyces Genome Database. APG14, a gene
essential for autophagy, was found to have the most GATAA motifs. We
show that nitrogen starvation or rapamycin treatment rapidly causes a
more than 20-fold induction of APG14. The expression of
APG14 is dependent on Gln3p; deletion of Gln3p severely
reduced its induction by rapamycin, whereas depletion of Ure2p caused its constitutive expression. However, overexpression of
APG14 led to only a slight increase in autophagy in
nitrogen-rich medium. Therefore, these results define a signaling
cascade leading to the expression of APG14 in response to
the availability of nitrogen nutrients and suggest that the regulated
expression of APG14 contributes to but is not sufficient
for the control of autophagy.
In response to nutrient starvation conditions, particularly
nitrogen starvation, autophagy acts as an emergency measure to generate
an internal supply of nutrients. It may also serve to reduce
energy-consuming cellular activities, such as protein synthesis, by
nonselectively delivering cytosolic materials to the lysosome (higher
eukaryotes) or vacuole (yeast) for degradation (reviewed in Ref. 1).
Failure to undergo autophagy severely compromises the viability of
cells during starvation. In addition, autophagy is also involved in
selectively removing aged organelles such as mitochondria under normal
growth conditions in higher eukaryotes. During autophagy, a portion of
the cytoplasm is sequestered by a double- or multilayered membrane
structure referred to as the autophagosome or autophagy body.
Autophagosomes are then transported to and fused with the lysosome or
vacuole, resulting in the eventual degradation of cytoplasmic materials
by the lysosomal or vacuolar proteases (reviewed in Ref. 1).
Genetic approaches have been undertaken to identify the players
involved in autophagy. A number of autophagy (APG or
AUT) genes have been identified that are required for
autophagy (2, 3). In a separate genetic screen for genes involved in
cytoplasm to vacuole targeting, it was found that there is an overlap
of many common components between the cytoplasm to vacuole targeting and autophagy pathways (4). Emerging biochemical evidence indicates that the autophagy genes are involved in various steps in the formation
and delivery of autophagosomes. For example,
Apg6p/Vps30p/Vpt30p/Lph7p and Apg14p form a peripheral
membrane-associated complex. No autophagosomes were observed in the
vacuoles of apg6 Autophagy has long been morphologically associated with a variety of
human disorders. Elevated autophagy is seen in several human
degenerative diseases such as Alzheimer's and Parkinson's (9, 10). In
contrast, autophagy is significantly reduced in many cancer cells
(reviewed in Ref. 11). Beclin 1 is a Bcl-2-interacting protein and the human homolog of Apg6p. It complements the autophagic but not the vacuolar protein-sorting function of Apg6p in yeast (12).
beclin 1 is frequently deleted in sporadic breast and ovarian cancers, and its expression level is significantly decreased in
human breast epithelial carcinoma cell lines and tissues (12, 13).
Overexpression of beclin 1 leads to elevated autophagy, reduced proliferation of cancer cells, inhibition of in
vitro clonigenicity, and tumorigenesis in nude mice (12). Taken
together, these results indicate that the Apg6p-Apg14p complex plays an important role in the regulation of autophagy.
Rapamycin is a macrocyclic immunosuppressive antibiotic that inhibits
the growth of eukaryotic cells. The targets of rapamycin (TOR) genes,
TOR1 and TOR2, were initially isolated for their dominant mutations causing rapamycin resistance (14-16). It was later
shown that FKBP12-rapamycin directly binds to TOR and that their
dominant rapamycin-resistant mutations at a conserved serine residue,
Ser-1972 in Tor1p or Ser-1975 in Tor2p, disrupt the binding of
FKBP12-rapamycin (17-19). The binding of FKBP12-rapamycin to TOR is
mediated by a small 12-kDa domain called the FKBP12-rapamycin-binding (FRB1) domain (18,
20). The three-dimensional structure of the FKBP12-rapamycin-FRB
complex shows that the conserved serine residue is located in the
hydrophobic rapamycin-binding pocket of FRB (21). A substitution of the
conserved serine with a bulky amino acid prevents the binding of
rapamycin to the hydrophobic pocket. TOR proteins are highly conserved
evolutionarily and belong to the ataxia telangiectasia mutated
(ATM)-related kinase family (reviewed recently in refs. 22 and 23). The
yeast TOR proteins have protein serine and threonine kinase activities
(24-26). TOR has emerged as an important regulator of
nutrient-mediated signaling. Rapamycin treatment causes phenotypes
typical of starvation responses, including severely reduced protein
synthesis (27, 28), glycogen accumulation (27), and autophagy (29).
Rapamycin treatment or mutations in both TOR1 and
TOR2 leads to autophagy in the absence of starvation (29).
In addition, rapamycin treatment also causes elevated autophagy in
mammalian cells, indicating that the ability of TOR to regulate
autophagy is conserved.
Gln3p is a nitrogen catabolite repression (NCR)-sensitive GATA-type
transcription factor. It is required for the expression of genes
involved in the transport and utilization of nitrogen compounds
(reviewed in Ref. 30). It normally resides in the cytoplasm. The Gln3p
pathway has been recently shown to be under the control of TOR
(31-34). When cells are growing in poor nitrogen sources or under
starvation, Gln3p rapidly accumulates in the nucleus (31, 32), thereby
activating the transcription of NCR-sensitive genes such as general
amino acid permease (GAP1) and glutamine synthetase
(GLN1) (31-34). TOR directly binds to and phosphorylates
Gln3p, leading to the cytoplasmic retention of Gln3p (31). The
inhibition of TOR by rapamycin causes the dephosphorylation and nuclear
accumulation of Gln3p (31, 32). Ure2p is a pre-prion protein
genetically identified as an inhibitor of Gln3p (reviewed in Ref. 30).
Ure2p binds to Gln3p and appears to inhibit the dephosphorylation step
of Gln3p (31). The depletion of Ure2p also leads to the
dephosphorylation and nuclear accumulation of Gln3p (31, 32). TOR
signaling is also implicated in the regulation of several other
transcription factors. It binds to Gat1p, Gzf3p, Dal80p, Dal81p, and
Dal82p (31). Rapamycin treatment also causes nuclear accumulation of
Gat1p, Msn2p, and Msn4p (32).
In this study, we performed a DNA pattern search for the potential
target genes of Gln3p. We found that APG14 is among three genes with the largest number of Gln3p-binding motifs (GATAA). We
further showed that the inhibition of TOR by rapamycin or
nitrogen starvation leads to the rapid induction of APG14 in
a Gln3p-dependent manner. Interestingly, the overexpression
of APG14 itself only caused a slightly increased level of
autophagy. These observations defined a signaling cascade
leading to the expression of APG14 in response to nitrogen
starvation. They showed that regulated expression of APG14
may contribute to but is not sufficient for the control of autophagy.
Strains and Plasmids--
The strains and plasmids were
FM391 (MATa his Northern Blot Analysis--
Exponential wild type and
mutant yeast cultures were treated with 200 nM rapamycin or
drug vehicle. Aliquots of yeast cultures were withdrawn at different
times. For nitrogen and carbon starvation, logarithmic cells in the
synthetic complete medium were switched to synthetic complete minus
nitrogen sources or carbon sources, and samples were withdrawn at
different times. Total yeast RNAs were prepared using the freezing
phenol extraction method (39). Twenty µg of total yeast RNA samples
were separated on denaturing agarose gels, transferred onto nylon
filters, hybridized to 32P-labeled DNA probes, and detected
by a phosphorimaging device (Bio-Rad).
Western Blot Analysis--
Log-phase yeast cells were
harvested and lysed with glass beads in disruption buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) plus a mixture of protease
inhibitors (Roche Molecular Biochemicals) by vortexing. For
Western blot analysis, 10 µg of protein samples were used for gel
electrophoresis and detected by ECL (Amersham Pharmacia Biotech) with
monoclonal antibody 9E10.
Microscopy--
Exponential wild type and mutant yeast cells
were treated with rapamycin or a drug carrier (methanol) in the
presence or absence of 1 mM phenylmethylsulfonyl fluoride.
Aliquots of yeast cultures were withdrawn and analyzed for the
appearance of autophagosomes by a Zeiss Axioplan 2 microscope equipped
with a Nomarski objective lens and a SPOT digital camera system
(Diagnostic Instruments, Inc.). Consistent with a previous finding
(36), we found that phenylmethylsulfonyl fluoride treatment alone did
not cause autophagy but significantly enlarged the size of
autophagosomes, which is convenient for the analysis of autophagy.
Alkaline Phosphatase Assay--
The assay basically followed
that described by Noda et al. (38). Yeast cells were
harvested in phosphatase buffer (PB, 250 mM Tris, pH 9.0, 10 mM MgSO4, and 10 µM
ZnSO4), disrupted with glass beads, and centrifuged at
10,000 × g for 20 min. The protein concentrations of
the yeast extracts were measured by the Bradford assay (Bio-Rad). Fifty
µg of protein were incubated with 25 mM Recent gene expression profiling analysis has revealed that the
inhibition of TOR by rapamycin leads to a rapid global change in the
expression of NCR-sensitive genes as well as other diverse yeast genes
(31, 33, 34). Therefore, it is possible that Gln3p plays a broader role
in the control of gene expression beyond NCR-sensitive genes. To
identify new target genes for Gln3p, we carried out a pattern search
for the Gln3p-binding motif GATAA in 500 base pairs of the
5'-untranslated region (UTR) of yeast open reading frames. This
approach was inspired by the observation that
Gln3p-dependent genes often contain multiple GATAA motifs in their 5'-UTR (reviewed in Ref. 30). MEP2,
APG14, and YOR142w are found to each contain
eight GATAA motifs, the largest number in the entire yeast genome (The
APG14 5'-UTR is shown in Fig. 1a). MEP2 encodes
for ammonia transporter previously shown to be under the control of
Gln3p (31). In addition, two other Gln3p-dependent, NCR-sensitive genes, AMD2 (amidase) and DAL5
(allantoate permease), contain seven GATAA motifs. Therefore, multiple
GATAA motifs in the 5'-UTR appear to be a good indicator for potential
Gln3p-dependent genes. TOR has been linked to the control
of autophagy and regulation of Gln3p. APG8 was also recently
shown to be inducible by nitrogen starvation (7, 35) and rapamycin
(Ref. 33 and Footnote 2). Thus
APG14 is a likely Gln3p target gene and therefore is controlled by TOR signaling. To confirm this, we performed Northern blot analysis of logarithmically growing cells in YPD treated with 200 nM rapamycin. In the absence of rapamycin, the
transcript of APG14 was essentially undetectable. However,
rapamycin treatment a caused rapid increase in APG14
expression that peaked within 10 min and gradually declined to the
basal level after 60 min (Fig. 1b). In contrast, the
expression of ACT1 remained largely unchanged. Even at its
maximal expression, the level of APG14 transcripts remained
very low. A common problem with recent gene profiling technologies is
that they often fail to detect low-abundant transcripts. This may
explain why several recent studies were unable to reveal the induction
of APG14 by rapamycin (31, 33, 34). A closer look at the
500-base pair 5'-UTR region of APG14 revealed the open
reading frame for OPY1 at the
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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or apg14
cells (5). Thus
the Apg6p-Apg14p complex appears to be involved in the initial step(s)
of autophagy. Apg6p has a dual role in both autophagy and vacuolar
protein sorting, whereas Apg14p appears to be designated solely for
autophagy. Overexpression of Apg14p can suppress the defect of
apg6
in autophagy but not vacuolar protein sorting (5).
Apg8p/Aut7p/Cvt5p forms a complex with Apg4p/Aut2p, a microtubule-binding protein (6, 7). Mutations in APG4 or APG8 cause the accumulation of autophagosomes in the
cytoplasm, suggesting that the Apg4p-Apg8p complex is responsible for
the delivery of autophagosomes to the vacuole. Once autophagosomes are
fused with the lysosome/vacuole, vacuolar acidification by the vacuolar
H+-ATPase is required for the activity of vacuolar
proteases (e.g. Cps1p and Prb1p) to break down the cytosolic
materials into small molecules such as amino acids, resulting in the
eventual disappearance of autophagosomes (reviewed in Refs. 1 and
8).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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1, leu2
0,
met15
0, ura3
0) (a gift from
M. Johnston) and SZy159 (FM391,
ure2
::KanMX) (33), SZy415 (FM391,
pho8
60), and SZy426 (SZy159,
pho8
60). The PHO8 in the SZy
strains was replaced with pho8
60 by inserting the plasmid pTN9 (a gift from Dr. Y. Ohsumi) into the PHO8
locus and excised out as previously described (38). The
APG14 open reading frame was cloned into pAUD6 plasmid with
a Myc epitope tag in front of the starting codon ATG.
-naphthyl
phosphate (Sigma) at 30 °C for 30 min. The reactions were stopped by
the addition of 0.5 ml of 2 M glycine-NaOH (pH 11.0) and
measured by the fluorescence intensity of emissions at 472 nm
after excitation at 345 nm.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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238 position. After taking
the OPY1 sequences into consideration, only two of the eight
GATAA sequences actually fall between OPY1 and
APG14. It remains to be determined whether these two GATAA
sequences are sufficient for the regulation of APG14.
View larger version (72K):
[in a new window]
Fig. 1.
Identification of APG14 as a
candidate Gln3p target gene. a, search for GATAA motifs in
the 5'-UTR of APG14. The shaded sequence
indicates part of the open reading frame of OPY1.
b, rapamycin rapidly induces the expression of
APG14. Logarithmically growing cells in YPD were incubated
without or with 200 nM rapamycin. Aliquots of cells were
withdrawn at different times for Northern blot analysis with probes
derived from APG14. c, log-phase yeast cells
expressing the wild type TOR1, the rapamycin-resistant
TOR1(S1972I), or the kinase-inactive
TOR1(S1972I,D2294E) were incubated in 200 nM
rapamycin. Aliquots of cells were withdrawn at different times
for Northern blot analysis with probes derived from APG14
and ACT1. WT, wild type. RR, rapamycin-resistant.
KD, kinase deficient.
To confirm that the above rapamycin effects were attributable to the inhibition of TOR, we examined the expression of APG14 in cells carrying a plasmid-borne wild type TOR1 or a dominant rapamycin-resistant (RR) TOR1RR (18). Rapamycin caused elevated expression of APG14 in cells carrying the wild type TOR1 but not the TOR1RR cells (Fig. 1c). Thus the inhibition of TOR by rapamycin is directly responsible for the induction of APG14. Because phosphorylation of Gln3p by TOR is important for the cytoplasmic retention and inhibition of Gln3p, we also examined whether an intact TOR kinase domain is required for the repression of APG14. In this experiment, wild type yeast cells carrying a plasmid-borne TOR1(S1972I,D2294E) were used (Fig. 1c). TOR1(S1972I, D2294E) contains two independent point mutations: the S1972I mutation in the FKBP12-rapamycin-binding (FRB) domain that prevents the binding of FKBP12-rapamycin and the D2294E mutation located at a critical site of the kinase catalytic domain that completely abolishes the kinase activity of Tor1p (18). Even though FKBP12-rapamycin is unable to bind to Tor1p(S1972I,D2294E), Tor1p(S1972I,D2294E) failed to repress the induction of APG14 in the presence of rapamycin (Fig. 1c). Thus an intact kinase domain of TOR is required for the normal repression of APG14.
TOR has been implicated in the control of a large number of
transcription factors, including Gln3p, Gat1p, Gzf3p, Dal80p, Dal81p,
Dal82p (31), Msn2p, and Msn4p (32). Because Gln3p, Dal80p, and Gat1p
are GATA-type transcription factors, we asked whether they are involved
in the expression of APG14. We found that the deletion of
GLN3 severely reduced APG14 expression in the
presence of rapamycin (Fig. 2). A
residual expression of APG14 in the absence of Gln3p appears to be
mediated by Gat1p (data not shown). Ure2p is the inhibitor of Gln3p in
the presence of preferred nitrogen sources. Rapamycin or the deletion
of URE2 leads to the nuclear accumulation of Gln3p and
constitutive expression of several Gln3p-dependent genes
such as GAP1 and GLN1 (31, 32). Similar to
GAT1 and GLN1, the ure2 mutation
led to the constitutive expression of APG14 in the absence
of rapamycin (Fig. 2). Rapamycin treatment did not further increase
APG14 expression in the ure2
strain (Fig. 2).
Taken together, Gln3p is primarily responsible for mediating the
induction of APG14 by rapamycin.
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The starvation of either nitrogen or carbon sources causes autophagy
(reviewed in Ref. 1). We next examined the effect of deprivation of
nitrogen nutrients on APG14. Similar to rapamycin treatment,
we found that nitrogen starvation rapidly induced the expression of
APG14 (Fig. 3). However,
glucose starvation had no discernible change in the transcript levels
of APG14 (Fig. 3). Similarly, switching from glucose to a
nonfermentable carbon source also failed to induce the expression of
APG14 (data not shown). These findings are consistent with
our observation that glucose starvation did not significantly cause
autophagy in the strains examined in this study (data not shown). Thus
nitrogen, but not carbon, starvation causes elevated expression of
APG14.
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The accumulation of autophagosomes in the yeast vacuole is a hallmark
for cells undergoing autophagy and has been used as a convenient
microscopic measurement of autophagy (36, 37). Because rapamycin
activates both Gln3p and autophagy, we investigated whether the
activation of Gln3p is sufficient for autophagy. In this experiment, we
chose to use the ure2 strain that contains the active
form of Gln3p. Similar to rapamycin treatment, the deletion of
URE2 caused the accumulation of a large number of autophagosomes in nearly all the cells in a mid-log phase culture in
YPD (Fig. 4, a and
b). To confirm the above observation, we also performed a
Pho8
60p-based alkaline phosphatase assay (38). The alkaline
phosphatase Pho8p is normally localized on the vacuolar membrane.
Pho8
60p is instead localized in the cytoplasm because of the
truncation of its transmembrane region and can only be delivered into
the vacuole by autophagy. Once in the vacuole, Pho8
60p can be
processed by vacuolar proteases and becomes the active form that can be
detected by the alkaline phosphatase activity assay (38). As previously
reported (29), alkaline phosphatase activity increased 2.9-fold by
rapamycin treatment in the wild type yeast (Fig. 4c). The
ure2
mutation also caused an ~2.3-fold increase in
alkaline phosphatase activity over that in the wild type strain (Fig.
4c). Therefore, the activation of Gln3p by URE2 deletion appears to cause autophagy at a level comparable with that by
rapamycin.
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Apg14p forms a heterodimer with Apg6p. A recent study shows that
overexpression of beclin 1, the human homolog of Apg6p,
caused elevated autophagy in human breast carcinoma cells (12).
However, the level of APG6 remained largely unchanged in the
presence and absence of rapamycin or nitrogen starvation (data
not shown). Because APG14 is highly inducible by
rapamycin, we examined whether the overexpression of APG14
has any effect on autophagy. We engineered APG14 under the
control of the alcohol dehydrogenase (ADH) promoter on a 2 µM plasmid. We found the presence of high steady state levels of APG14 mRNA (Fig.
5a) and protein (Fig.
5b) in cells carrying the ADH-APG14 but
not in the vector control plasmid. The APG14 mRNA
level is at least 5- to 10-fold more than the endogenous APG14 mRNA induced by rapamycin (data not shown). In
yeast cells expressing Apg14p, however, there was only a slight
increase in the autophagy level over the control cells (Fig. 5,
c and d). This is in contrast to the
rapamycin-treated wild type cells or the untreated ure2
cells (Fig. 4). Therefore, the induction of APG14 does not
appear to be sufficient for autophagy. Other factor(s) under the
control of the Gln3p pathway must also be involved.
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DISCUSSION |
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Autophagy is a cellular process that nonselectively delivers
cytosolic materials to the lysosome (higher eukaryotes) or vacuole (yeast) for degradation (reviewed in Ref. 1). It can generate an
internal supply of nutrients and is essential for cell viability during
starvation. Because autophagy nonselectively degrades many cytosolic
materials such as ribosomes, it is not desirable under normal growth
conditions. Not surprisingly, autophagy is a highly regulated process;
it occurs at very low levels when cells are growing in nutrient-rich
medium but at high levels during starvation. Cancer cells appear to
have developed a strategy to gain growth advantage by deleting
beclin1/APG6 and decreasing their autophagy levels
(12). Therefore, it is important to understand the control of
APG genes and the regulatory circuit of autophagy.
Biochemical and genetic evidence indicates that the APG
genes play critical roles in the formation and transport of
autophagosomes (reviewed in Ref. 1). Mutations in these genes prevent
yeast from forming autophagosomes or delivering them to the vacuole
during starvation. A recent study establishes that TOR plays an
important role in the control of autophagy (29). Rapamycin treatment or
compromise in both TOR1 and TOR2 genes leads to
high levels of autophagy in the absence of starvation. In this study,
we discovered that the 500-base pair upstream region of
APG14 is highly abundant in Gln3p-binding sites (Fig.
1a). Further investigation shows that APG14 is
indeed highly inducible by nitrogen starvation or rapamycin treatment
(Figs. 1b and 3). The expression of APG14 is primarily under
the control of Gln3p; the deletion of GLN3 severely
reduced APG14 expression by rapamycin, whereas activation of
Gln3p by the ure2 mutation led to the constitutive
expression of APG14. Our results also show that the
inhibition of TOR kinase activity is necessary for the induction of
APG14.
Apg6p and Apg14p form a peripheral membrane-bound complex (5). Earlier
studies indicate that this protein complex is critical for autophagy.
The deletion of either APG6 or APG14 completely inhibits autophagy. In contrast, the overexpression of beclin 1, the human homolog of Apg6p, promotes autophagy in human breast carcinoma cells (12). Apg6p has a dual function in both autophagy and
vacuolar sorting in yeast. In contrast, Apg14p is specific for
autophagy (5). Unlike mammalian cells, we found that the transcript
level of APG6 remained unchanged during starvation or
rapamycin treatment. In contrast, APG14 is highly induced
under these conditions. Because the overexpression of beclin
1 leads to a significant increase in autophagy, we examined the
effect of APG14 overexpression on autophagy in yeast. Unlike
the ure2 mutation or rapamycin treatment, which leads to
autophagy in virtually all cells (Fig. 4) (29), we found that
APG14 overexpression only slightly increased autophagy (Fig.
5, c and d). Thus additional factor(s) appear to
contribute to the onset of autophagy. Apg8p may be one such protein.
The abundance of APG8 increased approximately 8-fold
following the starvation of nitrogen nutrients (7, 35). The expression
of APG8 also increased similarly as a result of rapamycin
treatment (data not shown) (33). We also found that Gln3p is
responsible for the expression of APG8 (data not shown). Apg8p interacts with Apg4p, a microtubule-associated protein (6). The
apg4
and apg8
mutations cause the
accumulation of autophagosomes in the cytoplasm, suggesting that Apg4p
or Apg8p is responsible for the delivery of the autophagosomes to the
vacuole (6, 7). Therefore, autophagy may require the simultaneous
induction of several components of the autophagy apparatus.
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ACKNOWLEDGEMENTS |
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We are grateful to M. Johnston and Y. Ohsumi for strains and plasmids, D. Dean and J. Skeath for the use of microscopes, W. Yokoyama for the use of a fluorescence spectrophotometer, and other members of the Zheng laboratory for discussions. We also thank the reviewer for pointing out the presence of OPY1 in the 5'-end of APG14.
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FOOTNOTES |
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* This work was supported by Grant 5R01CA77668 from the National Cancer Institute of the National Institutes of Health, a HHMI New Investigator Award, and a start-up fund from Washington University School of Medicine (to X. F. Z.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) NC_001134.
These authors contributed equally to this work.
§ A Coleman Foundation Scholar. To whom correspondence should be addressed: Tel.: 314-747-1884; Fax: 314-747-2797; E-mail: zheng@pathology.wustl.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008162200
2 T.-F. Chan, P. G. Bertram, W. Ai, and X. F. Zheng, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: FRB, FKBP12-rapamycin-binding; NCR, nitrogen catabolite repression; UTR, untranslated region; ADH, alcohol dehydrogenase; YPD, yeast extract-peptone-dextrose.
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