The Tor and PKA signaling pathways independently
target the Atg1/Atg13 protein kinase complex
to control autophagy
Joseph S. Stephana,b, Yuh-Ying Yeha,b, Vidhya Ramachandrana,b, Stephen J. Deminoffa, and Paul K. Hermana,b,1
aDepartment of Molecular Genetics andbProgram in Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, OH 43210
Edited by Scott D. Emr, Cornell University, Ithaca, NY, and approved August 6, 2009 (received for review March 25, 2009)
that has been implicated in a number of biological processes, includ-
ing organismal aging, innate immunity, and the progression of
human cancers. This pathway was initially identified as a cellular
response to nutrient deprivation and is essential for cell survival
Tor pathway, that coordinate cell growth with nutrient availability.
These pathways appear to target a complex of proteins that contains
the Atg1 protein kinase. The data here show that autophagy in
Saccharomyces cerevisiae is also controlled by the cAMP-dependent
protein kinase (PKA) pathway. Elevated levels of PKA activity inhib-
ited autophagy and inactivation of the PKA pathway was sufficient
to induce a robust autophagy response. We show that in addition to
Atg1, PKA directly phosphorylates Atg13, a conserved regulator of
Atg1 kinase activity. This phosphorylation regulates Atg13 localiza-
tion to the preautophagosomal structure, the nucleation site from
which autophagy pathway transport intermediates are formed.
Atg13 is also phosphorylated in a Tor-dependent manner, but these
modifications appear to occur at positions distinct from the PKA
PKA and Tor pathways function independently to control autophagy
in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site
of signal integration within this degradative pathway.
cAMP-dependent protein kinase ? macroautophagy ? stationary phase ?
Tor protein kinase
sible for the turnover of bulk cytoplasmic protein and organelles
(1, 2). This pathway was initially identified as a cellular response
to nutrient deprivation (3, 4). However, recent studies indicate
that autophagy is involved in a wide variety of physiological
processes, including tissue remodeling during development, the
removal of protein aggregates, and innate immune responses (5,
6). During autophagy, an isolation membrane emanates from a
(PAS) in Saccharomyces cerevisiae and the phagophore assembly
site in mammals (7, 8). This double membrane encapsulates
nearby cytoplasm and ultimately targets it to the vacuole/
lysosome for degradation. The breakdown products are then
recycled to allow for the synthesis of the macromolecules needed
for survival during the period of starvation (9). The cellular
components mediating autophagy were initially described in S.
cerevisiae, and orthologs of many of these Atg proteins have since
been identified in other eukaryotes (10, 11).
The flux through the autophagy pathway is tightly controlled
by multiple signaling pathways, including the Tor pathway, that
are responsible for coordinating cell growth with nutrient avail-
ability. One of the key targets of this control appears to be a
complex of proteins that contains the Atg1 protein kinase
(12–15). Atg1 is specifically recruited to the PAS, and activated,
in response to conditions that induce autophagy (7, 16). In
contrast, most Atg proteins are constitutively localized to this
acroautophagy (hereafter autophagy) is a highly-
conserved membrane trafficking pathway that is respon-
nucleation structure (17). Recent work suggests that the mam-
malian Atg1 proteins, ULK1 and ULK2, are also recruited to the
phagophore assembly site, and activated, upon nutrient depri-
vation (18, 19). A key question that remains is how do these
signaling pathways work together through Atg1 to ensure the
appropriate autophagic response.
In S. cerevisiae, the cAMP-dependent protein kinase (PKA)
signaling pathway has also been implicated in the control of
how it is integrated with that of the Tor pathway, is still somewhat
is critical for the appropriate control of autophagy in S. cerevisiae.
activity, and this control appears to occur independently, or in
parallel to, that exerted by the Tor pathway. For example, both of
pathways are important, but independent, regulators of autophagy,
and that the Atg1 protein kinase complex is a key site of signal
integration within this pathway.
Inactivation of the PKA Pathway Is Sufficient to Induce Autophagy.
Elevated levels of PKA activity have been shown to inhibit the
autophagy process (13). Here, we tested whether the inactivation
of this signaling pathway was also sufficient to induce autophagy.
To shutdown PKA signaling, we used an inducible form of a
dominant negative allele of RAS2, known as RAS2ala22(22). In
S. cerevisiae, the Ras proteins, Ras1 and Ras2, regulate cAMP
production, and thus PKA activity, by directly stimulating ad-
enylyl cyclase (23, 24). The RAS2ala22allele used here was under
the control of the promoter from the MET3 gene, a locus that is
repressed when methionine is in the growth medium (25). We
found that autophagy was efficiently induced upon expression of
the RAS2ala22protein and that the kinetics of induction were
similar to that observed with rapamycin treatment (Fig. 1A).
Moreover, this RAS2ala22-mediated induction was dependent
upon the presence of Atg1 (Fig. 1B). To directly compare the
effects of inhibiting the Tor and PKA pathways, we used a
concentration of rapamycin that produced a growth arrest
similar to that observed in the MET3-RAS2ala22strain. [Note that
rapamycin specifically inhibits the TORC1 complex and that we
will be referring to this complex when we discuss Tor signaling
V.R., and S.J.D. performed research; J.S.S., Y.-Y.Y., V.R., and S.J.D. contributed new re-
agents/analytic tools; J.S.S., Y.-Y.Y., V.R., S.J.D., and P.K.H. analyzed data; and J.S.S. and
P.K.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
October 6, 2009 ?
vol. 106 ?
no. 40 ?
in this report (26).] Therefore, a decrease in PKA function that
was sufficient to arrest S. cerevisiae growth resulted in a con-
comitant increase in autophagy activity. These results are con-
sistent with a previous study that used a galactose-inducible form
of this RAS2ala22allele (13).
The second method to inactivate PKA activity made use of an
‘‘analog-sensitive’’ version of this enzyme. This approach for
inactivating protein kinases was pioneered by Kevan Shokat and
his colleagues, and involves altering a specific residue within the
kinase active site (27, 28). Particular substitutions at this ‘‘gate-
keeper’’ position render these enzymes sensitive to membrane-
soluble inhibitors, like 1NM-PP1 (29). Here, we used a yeast
strain that has an ‘‘analog-sensitive’’ allele of TPK1, tpk1-as, as
the sole source of PKA activity (27, 28). S. cerevisiae has three
functionally redundant PKA catalytic subunits that are encoded
by the TPK1–3 genes (30). The tpk1-as strain used here, gener-
ously provided by Dr. James Broach, lacks TPK2 and TPK3, and
contains an allele of TPK1, tpk1-M164G, that is sensitive to the
drug, 1NM-PP1. Interestingly, we found that 1NM-PP1 concen-
trations that inhibited cell growth in the tpk1-as strain also
resulted in a robust induction of the autophagy pathway (Fig. 1
C–E and Figs. S1 and S2). For these experiments, autophagy
activity was assessed with three different assays that are de-
scribed in the Materials and Methods. Therefore, the inhibition
of PKA signaling by two different means resulted in an induction
of autophagy activity similar to that observed upon inactivation
of the Tor pathway. These results are significant because they
contradict a recent study that suggested that the loss of PKA
activity was not sufficient to induce autophagy (21). This latter
study also used a yeast strain that contained analog-sensitive
versions of PKA; this strain is in the same genetic background as
the tpk1-as strain used here. However, this previous study used
a 1NM-PP1 concentration of only 0.1 ?M, a concentration that
did not have a significant effect upon the growth rate of the
tpk1-as strain (Fig. 1C). Therefore, the low level of autophagy
analog-sensitive cells. In all, our data here indicate that the
inactivation of the PKA pathway is sufficient to induce autoph-
agy activity in S. cerevisiae cells.
Atg13 Is a Substrate for PKA in Vitro and in Vivo. The Atg13 protein
physically interacts with Atg1 and is required for full Atg1
protein kinase activity in vitro (14, 31–35). Although the mech-
anistic basis of this activation is not yet understood, the Atg1-
Atg13 interaction in S. cerevisiae appears to be regulated by Tor
signaling activity. A recent study also identified Atg13 as a
candidate substrate for PKA in this budding yeast (20). This
identification was based on the presence of evolutionarily-
conserved matches in Atg13 to the PKA consensus phosphory-
lation site. Two sites were very similar to the consensus of
R-R-x-S/T-B, where x refers to any amino acid and B to a
hydrophobic residue (Sites 2 and 3; Fig. 2A) (36). A third
conserved site that deviates more from the consensus was also
identified (Site 1). We found that each of these sites was
phosphorylated by PKA in vitro, and alteration of all three sites
resulted in a greater than 95% decrease in Atg13 phosphoryla-
tion (Atg13-AAA; Fig. 2 B and C and Fig. S3). Similar results
that specifically recognizes phosphorylated PKA sites (Fig. 2D)
(37, 38). For these experiments, Atg13 was precipitated from cell
extracts and the level of PKA phosphorylation was assessed by
Western blotting with this ?-substrate antibody. This Atg13
signal was lost upon phosphatase treatment, and was restored by
a subsequent incubation with PKA and ATP (Fig. 2D). In
addition, this in vivo signal was elevated in cells that possessed
elevated levels of PKA activity (Fig. 2E). Finally, Atg13 recog-
nition by this ?-substrate antibody was lost following the inac-
Atg13 was a direct substrate for PKA in S. cerevisiae.
The Loss of PKA Phosphorylation on Atg13 Was Correlated with the
Induction of Autophagy. Atg13 has been shown to be required for
Atg1 kinase activity in vitro (14). Here, we tested whether this
protein was also required for Atg1 activity in vivo by taking
advantage of a previous observation concerning the mobility of
this protein in SDS-polyacrylamide gels (14, 20). In particular,
autophosphorylation was found to retard the mobility of Atg1 in
these gels, and the presence of this slower-migrating band can
induce autophagy. (A) Autophagy induction upon rapamycin treatment
and/or expression of the dominant-negative RAS2ala22allele. Wild-type cells
(TN125) were grown to mid-log phase and treated with 20 ng/mL rapamycin
SC minimal medium lacking methionine for 4 h at 30 °C to induce expression
from the MET3 promoter. Autophagy levels were assessed with an alkaline
white bars indicate the relative levels of ALP activity in the untreated controls
and the black bars the activity following the indicated treatments. (B) The
autophagy activity induced upon inactivation of the Ras/PKA pathway was
dependent upon the presence of the Atg1 protein. Autophagy levels were
assessed with the ALP-based assay in isogenic wild-type and atg1? cells after
4 h of rapamycin treatment (R) or exposure to the RAS2ala22protein (A). (C)
Growth curves for isogenic TPK1 and tpk1-as strains in YPAD at 30 °C are
shown. The drug 1NM-PP1 was present at the indicated concentrations. (D)
Inactivation of PKA signaling in the tpk1-as strain resulted in the induction of
autophagy. Autophagy levels were assessed with the ALP-based assay in
tpk1-as cells (PHY4710) that were treated for 5 h with either 200 ng/mL
rapamycin or the indicated concentrations of 1NM-PP1. (E) Cells over-
expressing Ape1 were treated for 4 h with either 200 ng/ml rapamycin or 25
?M 1NM-PP1, and the relative level of Ape1 processing was assessed by
Western blotting. Cont, untreated cells.
Inactivation of the Ras/PKA signaling pathway was sufficient to
www.pnas.org?cgi?doi?10.1073?pnas.0903316106Stephan et al.
serve as an indicator of Atg1 kinase activity in vivo (Fig. 3A). We
found that the proportion of Atg1 in the slower migrating form
increased upon rapamycin treatment (Fig. 3A). This activation of
Atg1 did not occur in cells that lacked either Atg13 or Atg17;
these mutants were the only atg strains tested that exhibited a
significant defect in this Atg1 autophosphorylation. Therefore,
Atg13 was required in vivo for Atg1 protein kinase activity.
We used the tpk1-as strain described above to examine the
temporal relationship between the PKA phosphorylation of
Atg13, and the induction of both Atg1 kinase activity and
autophagy. We found that the PKA-dependent phosphorylation
of Atg13 was largely gone after 30 min of treatment with the
drug, 1NM-PP1 (Fig. 3B). The first signs of Atg1 kinase activity
were apparent at this time, but the fraction of Atg1 in the
slower-migrating form continued to increase at later time points
(Fig. 3B). Autophagy activity was found to increase in a similar
manner (Fig. 3C). Finally, we found that this inactivation of the
PKA pathway also resulted in the re-localization of Atg1 from
the cytoplasm to the PAS (Fig. 3D). These data therefore
indicated that the loss of Atg13 phosphorylation preceded the
activation of Atg1 and the induction of the autophagy pathway.
As observed previously, we found that Atg13 was largely cyto-
plasmic in growing cells and was associated with the PAS upon
nutrient limitation (Fig. 4A). Here, we tested whether this
localization to the PAS was regulated by PKA phosphorylation.
A clear precedent exists as PKA phosphorylation has been
shown to inhibit the PAS association of Atg1 (20). We found that
and nitrogen-starved cells (Fig. 4A and Fig. S4). Moreover, the
inhibited by the presence of the RAS2val19allele that results in
constitutively-elevated levels of PKA activity (39). This inhibi-
tion was dependent upon the PKA sites as the Atg13-AAA
protein was still constitutively localized to the PAS in RAS2val19
cells (Fig. 4A and Fig. S4). Finally, we found that the wild-type
Atg13 protein was recruited to the PAS following the inactiva-
tion of the Ras/PKA pathway (Fig. 4B). These data are therefore
consistent with PKA phosphorylation regulating the association
of Atg13 with the PAS.
Previous studies have shown that Atg13 interacts with a
number of proteins, most notably Atg1 and Atg17 (40–42). Since
Atg17 has been suggested to play a role in organizing the PAS,
we tested whether the PAS association of Atg13-AAA was
dependent upon the presence of Atg17 (43–45). Indeed, we
found that the Atg13-AAA variant was less efficiently targeted
to the PAS in cells lacking Atg17 (Fig. 4 C and D). In addition,
wild-type Atg13 protein and this interaction was no longer
influenced by rapamycin treatment (Fig. 4E) (41). Instead,
Atg13-AAA appeared to be constitutively associated with
sites of PKA phosphorylation in Atg13 are shown. (B and C) The in vitro
phosphorylation of Atg13 was dependent upon the presence of the above
three PKA sites. The indicated Atg13 variants were precipitated from yeast
S. cerevisiae Tpk1 (C). S, serine; A, alanine. (D) The three PKA sites were
required for the in vivo phosphorylation of Atg13. The indicated Atg13
proteins were immunoprecipitated from yeast cell extracts with an ?-HA
antibody and treated with ? phosphatase and then incubated with bPKA and
3 mM ATP, as indicated. The level of PKA phosphorylation was assessed by
Western blotting with an ?-substrate antibody that recognizes phosphory-
lated PKA sites. (E) The in vivo level of PKA phosphorylation on Atg13 was
elevated in a strain over-expressing Tpk1. (F) Recognition by the ?-substrate
antibody was lost following inactivation of PKA. The tpk1-as strain was
incubated with 10 ?M 1NM-PP1 for 4 h and the PKA phosphorylation level of
Atg13 was assessed by Western blotting with the ?-substrate antibody.
The Atg13 protein was a substrate for PKA. (A) The three conserved
Atg1 and the induction of autophagy. (A) Atg13 and Atg17 were required for
Atg1 autophosphorylation in vivo. Cell extracts were prepared from the
indicated yeast strains and the levels of autophosphorylated Atg1 were
assessed by Western blotting. The cells were treated with 200 ng/mL rapamy-
atg1-K54A. In the bottom panel, Atg1 was immunoprecipitated from yeast
levels of Atg13 phosphorylation by PKA (top panel) and of the ‘‘activated’’
form of Atg1 (bottom) were assessed by Western blotting at the indicated
times after the addition of 25 ?M 1NM-PP1 to a culture of tpk1-as cells. (C)
Autophagy activity was assessed with the ALP-based assay at the indicated
of an Atg1-YFP protein was assessed by fluorescence microscopy 4 h after the
addition of 25 ?M 1NM-PP1 to a culture of tpk1-as cells.
Stephan et al.PNAS ?
October 6, 2009 ?
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Atg17. In contrast, alteration of the PKA sites did not signifi-
cantly influence the Atg1-Atg13 interaction (Fig. S5A). Finally,
Atg17 was found to be associated with the PAS in both growing
and nitrogen-starved cells, and this localization was not inhibited
by the presence of RAS2val19(Fig. S5B) (40, 45). Therefore, PKA
was apparently regulating the PAS association of Atg13, at least
in part, by interfering with its interaction with Atg17.
The PKA and Tor Pathways Independently Target the Atg13 Protein.
The simultaneous inactivation of both the PKA and Tor path-
ways produced a more rapid and greater induction of autophagy
and 5A). This result is consistent with these pathways working
independently of each other to control autophagy. To examine
this possibility, we tested how shutting down one of these
pathways would influence the activity of the other. For these
experiments, we used the PKA- and Tor-dependent phospho-
rylations of Atg13 as reporters for the activity of the respective
signaling pathways. We found that the in vivo level of Atg13
phosphorylation by PKA was not diminished upon rapamycin
treatment (Fig. 5B). In addition, the inactivation of the Ras/PKA
pathway did not result in a loss of the Tor-dependent phosphor-
ylation present on Atg13 (Fig. 5C). This latter phosphorylation
causes Atg13 to run as a broad smear on SDS-polyacrylamide
gels. This smear rapidly collapses into a tight, faster-migrating
band upon rapamycin treatment (Fig. 5C) (14, 46). Finally, the
in vitro phosphorylation of Atg13 by PKA did not alter the
mobility of this protein in SDS-polyacrylamide gels (see Fig. 2).
Therefore, although the precise locations of the Tor-dependent
phosphorylation sites on Atg13 have yet to be identified, these
positions appear to be distinct from the PKA sites described
here. In all, these data are consistent with the Tor and PKA
pathways working independently to control autophagy.
Autophagy is a nonspecific degradative process that must be
tightly controlled to prevent the inappropriate turnover of
material needed for cell growth. The work here adds to our
current understanding of this process by demonstrating that the
(A) Fluorescence microscopy was performed with cells that contained the
indicated YFP-Atg13 fusion proteins. The Atg13 proteins had either wild-type
(Atg13-SSS) or nonphosphorylatable versions (Atg13-AAA) of the three PKA
sites. The CFP-Atg11 fusion protein was present in all cells and served as a
marker for the PAS. The RAS2val19allele was present in the indicated strains.
Nitrogen starvation was achieved by transferring the cells from SC glucose
minimal medium to the SD-N medium for 1 h at 30 °C. (B) The localization of
a wild-type Atg13-YFP protein in the indicated cells was assessed by fluores-
cence microscopy. A22, RAS2ala22. (C) Atg17 was required for the efficient
localization of Atg13-AAA to the PAS in log phase cells. The fraction of cells
with Atg1-AA or Atg13-AAA present in a perivacuolar punctate spot in the
strain. (D) Representative images for the indicated strains in C. (E) Alteration
of the PKA phosphorylation sites in Atg13 influenced the interaction with
extracts and the relative levels of the associated Atg17 were assessed by
Western blotting. The cell extracts were prepared from either mid-log phase
cultures (Log) or from cells that were treated with rapamycin (Rap). S, Atg13-
SSS; A, Atg13-AAA.
autophagy activity. (A) Simultaneous inactivation of the PKA and Tor path-
ways resulted in an elevated autophagy response relative to the loss of either
pathway alone. Autophagy levels were assessed with the ALP-based assay in
tpk1-as cells that had been treated for the indicated time with either 25 ?M
1NM-PP1, 200 ng/mL rapamycin, or both reagents. The data shown are from
a single experiment that was representative of at least three independent
replicates. (B) The PKA phosphorylation of Atg13 was not diminished upon
inactivation of the Tor pathway. Atg13 was immunoprecipitated from cells
level of PKA phosphorylation was subsequently assessed by Western blotting
with the ?-PKA substrate antibody. (C) Inactivation of the Ras/PKA pathway
did not influence the Tor-dependent phosphorylation of Atg13. Cell extracts
were prepared from the indicated cells and the level of Tor-dependent
phosphorylation of Atg13 was assessed by Western blotting. In the left-hand
lanes, cells containing either MET3-RAS2ala22(A22) or a control plasmid (-)
were incubated in methionine-free medium for 6 h to allow for expression
incubated for 4 h with 0 or 5 ?M 1NM-PP1. The bottom panel in the middle
blotting with the ?-PKA substrate antibody. Note that the relative spread of
the Atg13 ‘‘smear’’ is dependent upon the running conditions of the gel. (D)
A model depicting the proposed roles of the PKA and Tor pathways in the
control of autophagy. The dashed lines indicate the Atg1 and Atg13 interac-
tions with each other or the PAS (and perhaps Atg17), and the solid lines
indicate the regulatory effects of the PKA and/or Tor pathways on these
interactions. See the text for additional details.
The PKA and Tor pathways independently target Atg13 to control
www.pnas.org?cgi?doi?10.1073?pnas.0903316106Stephan et al.
Ras/PKA pathway is an important regulator of autophagy in S.
cerevisiae. Inactivation of this signaling pathway by two different
methods was sufficient for a robust induction of autophagy
activity. This control appears to be exerted through the Atg1
complex as both Atg1 itself and Atg13, a key regulator of this
kinase, were shown to be direct substrates for PKA (20) (Fig. 2).
For both proteins, this phosphorylation appears to regulate the
association with the PAS. Interestingly, the data here indicate
that this control by PKA is independent of that exerted by the
Tor pathway. For example, although both pathways regulate
Atg13 phosphorylation, each appears to control a distinct set of
phosphorylation events. In all, this report suggests that the
Atg1/Atg13 complex serves as an important point of signal
integration within the autophagy pathway. The relevance of this
complex as a regulatory control point may be evolutionarily
conserved as both Atg1 and Atg13 have been found to be targets
for the Tor pathway in other eukaryotes (32, 33, 35, 47).
The autophagy pathway in S. cerevisiae therefore appears to be
under the control of at least two independent regulatory inputs.
A model that attempts to summarize our current understanding
of this regulation is presented in Fig. 5D. In this model, PKA
activity specifically inhibits the PAS association of both Atg1 and
Atg13. In contrast, the Tor pathway appears to influence both
this localization and the Atg1-Atg13 interaction that is necessary
for the activation of Atg1 kinase activity (14). Therefore, inac-
tivation of the Tor pathway would result in the formation of an
active Atg1-Atg13 complex at the PAS and the ensuing induction
of autophagy (1). Upon the loss of PKA activity, however, it is
less clear how the Atg1 enzyme is activated. One possibility is
that the increased local concentrations of Atg1 and Atg13 at the
PAS are sufficient to override the inhibitory effects of Tor
activity on the Atg1-Atg13 interaction. Alternatively, Atg13
could be selectively dephosphorylated at the PAS. Although
further experimentation is needed to distinguish between these
possibilities, the work here shows that the loss of PKA signaling
results in increased Atg1 activity and the induction of autophagy.
An additional question that arises is why more than one signaling
pathway might be needed for the regulation of autophagy. An
interesting possibility is that these pathways are responding to
distinct nutritional cues and that different types of starvations
might elicit distinct autophagy responses in the cell (48). The
existence of these multiple inputs would therefore provide the
cell with greater flexibility in its response to changing environ-
mental conditions. Determining how these pathways influence
this degradative process, and how these signaling activities are
coordinated, are therefore important questions for future work.
Materials and Methods
construction, are included in SI Text.
Yeast Strain Construction and Growth Conditions. The yeast strains used in this
study were PHY1220 (MAT? his3-?200 leu2–3,112 lys2–801 trp1–101 ura3–52
suc2-?9), PHY1942 (PHY1220 prc1::HIS3 pep4?::LEU2 prb1?::hisG), TN125
(MATa ade2 his3 leu2 lys2 trp1 ura3 pho8::pho8?60), YYK126 (TN125
atg1?::LEU2), YYK130 (TN125 atg13?::TRP1), PHY3687 (TN125 atg1?::LEU2
atg13?::kanMX), Y3175 (ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1
ura3–1 tpk2::KAN tpk3::TRP1 tpk1-M164G), and PHY4710 (Y3175
pho8::pho8?60). This latter strain was generated by integration of a previ-
ously described plasmid, PTN9, into Y3175 (49). Strains carrying the MET3-
of 100 ?M CuSO4to the growth medium. The drug, 1NM-PP1, was generously
provided by Kevan Shokat.
Western Blotting and Immunoprecipitations. Protein extracts for Western
transferred to nitrocellulose membranes (Hybond ECL, Amersham Bio-
sciences) at 4 °C. The membranes were probed with the appropriate primary
and secondary antibodies and the Supersignal chemiluminescent substrate
(Pierce) was used to illuminate the reactive bands. The immunoprecipitation
experiments were performed as described (37, 38).
Autophagy Assays. The alkaline phosphatase (ALP) assay for autophagy activ-
ity was performed as described (13, 49). This assay measures the delivery and
subsequent activation of an altered form of the Pho8 phosphatase by the
autophagy pathway. Two additional assays assessed the processing of ami-
nopeptidase I (Ape1) and the accumulation of autophagic bodies, and are
discussed in SI Text.
PKA Phosphorylation Assays. In general, the in vitro phosphorylation assays
were performed with HA epitope-tagged proteins that were under the con-
trol of the yeast CUP1 promoter in the yeast strain, PHY1942. The strains were
grown to mid-log phase in selective SC minimal medium containing 2%
glucose, and induced with 100 ?M copper sulfate for 90 mins. The HA
epitope-tagged Atg13 variants were isolated on an ?-HA antibody resin
(Roche), and incubated with [?-32P] ATP (PerkinElmer) and 5 U bovine PKA
catalytic subunit (Sigma), as described previously (20, 38). A Western immu-
noblot control was performed with an ?-HA antibody (Sigma) to assess the
relative amount of Atg13 present in each sample.
The in vivo level of PKA phosphorylation was assessed with an ?-PKA
substrate antibody (Cell Signaling) as described (37, 38). Briefly, the substrate
proteins were immunoprecipitated from yeast cell extracts, separated on
SDS-polyacrylamide gels and the relative level of occupancy at the PKA sites
was assessed by Western blotting with the ?-PKA substrate antibody used at
a concentration of 1:2,000.
Fluorescence Microscopy. The CFP-Atg11, YFP-Atg13, and YFP-Atg17 fusions
were under the control of the inducible promoter from the yeast CUP1 gene.
Expression of these fusion proteins was induced by the addition of 100 ?M
CuSO4for 1 h at 30 °C. The samples were imaged as described (20).
ACKNOWLEGEMENTS. We thank James Broach, Daniel Klionsky, Takeshi Noda,
study, and members of the Herman lab for helpful discussions and comments
on the manuscript. J.S.S. was supported, in part, by a graduate student
fellowship from the Jeffrey Seilheimer Lung Cancer Foundation. This
work was supported by a grant from the National Institutes of Health
(GM65227) to P.K.H.
1. Noda T, Suzuki K, Ohsumi Y (2002) Yeast autophagosomes: De novo formation of a
membrane structure. Trends Cell Biol 12:231–235.
Nat Cell Biol 9:1102–1109.
3. Kopitz J, Kisen GO, Gordon PB, Bohley P, Seglen PO (1990) Nonselective autophagy of
cytosolic enzymes by isolated rat hepatocytes. J Cell Biol 111:941–953.
4. Schworer CM, Mortimore GE (1979) Glucagon-induced autophagy and proteolysis in
Sci USA 76:3169–3173.
5. Levine B, Klionsky DJ (2004) Development by self-digestion: Molecular mechanisms
and biological functions of autophagy. Dev Cell 6:463–477.
6. Klionsky DJ (2007) Autophagy: From phenomenology to molecular understanding in
less than a decade. Nat Rev Mol Cell Biol 8:931–937.
7. Suzuki K, et al. (2001) The pre-autophagosomal structure organized by concerted
functions of APG genes is essential for autophagosome formation. EMBO J 20:5971–
8. Seglen PO, Gordon PB, Holen I (1990) Non-selective autophagy. Semin Cell Biol
9. Mizushima N, Klionsky DJ (2007) Protein turnover via autophagy: Implications for
metabolism. Annu Rev Nutr 27:19–40.
10. Klionsky DJ, et al. (2003) A unified nomenclature for yeast autophagy-related genes.
Dev Cell 5:539–545.
11. Meijer WH, van der Klei IJ, Veenhuis M, Kiel JA (2007) ATG genes involved in non-
selective autophagy are conserved from yeast to man, but the selective Cvt and
pexophagy pathways also require organism-specific genes. Autophagy 3:106–116.
12. Noda T, Ohsumi Y (1998) Tor, a phosphatidylinositol kinase homologue, controls
autophagy in yeast. J Biol Chem 273:3963–3966.
13. Budovskaya YV, Stephan JS, Reggiori F, Klionsky DJ, Herman PK (2004) The Ras/cAMP-
process in Saccharomyces cerevisiae. J Biol Chem 279:20663–20671.
14. Kamada Y, et al. (2000) Tor-mediated induction of autophagy via an Apg1 protein
kinase complex. J Cell Biol 150:1507–1513.
Stephan et al.PNAS ?
October 6, 2009 ?
vol. 106 ?
no. 40 ?
15. Stephan JS, Herman PK (2006) The regulation of autophagy in eukaryotic cells: Do all Download full-text
roads pass through Atg1? Autophagy 2:146–148.
16. Kim J, Huang WP, Stromhaug PE, Klionsky DJ (2002) Convergence of multiple auto-
phagy and cytoplasm to vacuole targeting components to a perivacuolar membrane
compartment prior to de novo vesicle formation. J Biol Chem 277:763–773.
Saccharomyces cerevisiae. FEBS Lett 581:2156–2161.
18. Chan EY, Kir S, Tooze SA (2007) siRNA screening of the kinome identifies ULK1 as a
multidomain modulator of autophagy. J Biol Chem 282:25464–25474.
19. Hara T, et al. (2008) FIP200, a ULK-interacting protein, is required for autophagosome
formation in mammalian cells. J Cell Biol 181:497–510.
ics approach identifies substrates of the cAMP-dependent protein kinase. Proc Natl
Acad Sci USA 102:13933–13938.
21. Yorimitsu T, Zaman S, Broach JR, Klionsky DJ (2007) Protein kinase A and Sch9
cooperatively regulate induction of autophagy in Saccharomyces cerevisiae. Mol Biol
interfere with the CDC25- dependent activation of wild-type RAS in Saccharomyces
cerevisiae. Mol Cell Biol 9:390–395.
23. Field J, et al. (1990) Mutations of the adenylyl cyclase gene that block RAS function in
Saccharomyces cerevisiae. Science 247:464–467.
24. Suzuki N, et al. (1990) Leucine-rich repeats and carboxyl terminus are required for
interaction of yeast adenylate cyclase with RAS proteins. Proc Natl Acad Sci USA
25. Mountain HA, Bystrom AS, Larsen JT, Korch C (1991) Four major transcriptional
responses in the methionine/threonine biosynthetic pathway of Saccharomyces cer-
evisiae. Yeast 7:781–803.
26. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism.
27. Bishop AC, Buzko O, Shokat KM (2001) Magic bullets for protein kinases. Trends Cell
28. Bishop AC, et al. (1998) Design of allele-specific inhibitors to probe protein kinase
signaling. Curr Biol 8:257–266.
29. Bishop AC, et al. (2000) A chemical switch for inhibitor-sensitive alleles of any protein
kinase. Nature 407:395–401.
30. Toda T, Cameron S, Sass P, Zoller M, Wigler M (1987) Three different genes in S.
cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell
31. Funakoshi T, Matsuura A, Noda T, Ohsumi Y (1997) Analyses of APG13 gene involved
in autophagy in yeast, Saccharomyces cerevisiae. Gene 192:207–213.
32. Jung CH, et al. (2009) ULK-Atg13-FIP200 complexes mediate mTOR signaling to the
autophagy machinery. Mol Biol Cell 20:1992–2003.
33. Ganley IG, et al. (2009) ULK1.ATG13.FIP200 complex mediates mTOR signaling and is
essential for autophagy. J Biol Chem 284:12297–12305.
34. Chan EY, Longatti A, McKnight NC, Tooze SA (2009) Kinase-inactivated ULK proteins
inhibit autophagy via their conserved C-terminal domains using an Atg13-
independent mechanism. Mol Cell Biol 29:157–171.
35. Hosokawa N, et al. (2009) Nutrient-dependent mTORC1 association with the ULK1-
Atg13-FIP200 complex required for autophagy. Mol Biol Cell 20:1981–1991.
36. Denis CL, Kemp BE, Zoller MJ (1991) Substrate specificities for yeast and mammalian cAMP-
dependent protein kinases are similar but not identical. J Biol Chem 266:17932–17935.
37. Chang YW, Howard SC, Herman PK (2004) The Ras/PKA signaling pathway directly
targets the Srb9 protein, a component of the general RNA polymerase II transcription
apparatus. Mol Cell 15:107–116.
38. Deminoff SJ, Howard SC, Hester A, Warner S, Herman PK (2006) Using substrate-
binding variants of the cAMP-dependent protein kinase to identify novel targets and
a kinase domain important for substrate interactions in Saccharomyces cerevisiae.
39. Toda T, et al. (1985) In yeast, RAS proteins are controlling elements of adenylate
cyclase. Cell 40:27–36.
40. Cheong H, et al. (2005) Atg17 regulates the magnitude of the autophagic response.
Mol Biol Cell 16:3438–3453.
41. Kabeya Y, et al. (2005) Atg17 functions in cooperation with atg1 and atg13 in yeast
autophagy. Mol Biol Cell 16:2544–2553.
42. Nair U, Klionsky DJ (2005) Molecular mechanisms and regulation of specific and
nonspecific autophagy pathways in yeast. J Biol Chem 280:41785–41788.
regulation of protein recruitment to initiate sequestering vesicle formation for non-
specific autophagy in Saccharomyces cerevisiae. Mol Biol Cell 19:668–681.
44. Suzuki K, Kubota Y, Sekito T, Ohsumi Y (2007) Hierarchy of Atg proteins in pre-
autophagosomal structure organization. Genes Cells 12:209–218.
45. Kawamata T, Kamada Y, Kabeya Y, Sekito T, Ohsumi Y (2008) Organization of the
pre-autophagosomal structure responsible for autophagosome formation. Mol Biol
46. Scott SV, et al. (2000) Apg13p and Vac8p are part of a complex of phosphoproteins
that are required for cytoplasm to vacuole targeting. J Biol Chem 275:25840–
47. Chang YY, Neufeld TP (2009) An Atg1/Atg13 complex with multiple roles in TOR-
mediated autophagy regulation. Mol Biol Cell 20:2004–2014.
48. Dechant R, Peter M (2008) Nutrient signals driving cell growth. Curr Opin Cell Biol
in the yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 210:126–132.
www.pnas.org?cgi?doi?10.1073?pnas.0903316106 Stephan et al.