Molecular Biology of the Cell
Vol. 21, 3998–4008, November 15, 2010
Membrane Delivery to the Yeast Autophagosome from the
Yohei Ohashi and Sean Munro
Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK
Submitted May 24, 2010; Revised August 13, 2010; Accepted September 14, 2010
Monitoring Editor: Benjamin S. Glick
While many of the proteins required for autophagy have been identified, the source of the membrane of the autopha-
gosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked.
The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-
to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of
Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9
cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and
endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells.
However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only
required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears
that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from
the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for
discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.
Autophagy allows eukaryotic cells to recycle proteins and
organelles by enveloping then in a double membrane to
form autophagosomes which then fuse with lytic organelles
for digestion. Initially identified as a response to starvation,
autophagy has been found to be involved in many impor-
tant biological processes including the clearance of patho-
gens and cytoplasmic aggregates, mobilizing lipid stores
and remodeling of tissues (Rubinsztein et al., 2007; Xie and
Klionsky, 2007; Farre ´ et al., 2009). Proteins required for au-
tophagosome formation have been identified by screening
for yeast mutations that affect starvation-induced autophagy
or the related Cvt pathway that delivers oligomerized en-
zymes to the vacuole during normal growth (Tsukada and
Ohsumi, 1993; Thumm et al., 1994; Harding et al., 1995;
Nakatogawa et al., 2009). However, the origin of the double-
membrane of the autophagosome is less well understood
(Juhasz and Neufeld, 2006; Longatti and Tooze, 2009). It has
been suggested that membrane could extend out from the
endoplasmic reticulum (ER) to wrap the target for digestion,
or alternatively that autophagosomes could grow by the
delivery of vesicular carriers that bud from other organelles
such as mitochondria or endosomes (Reggiori et al., 2004b;
Axe et al., 2008; Hayashi-Nishino et al., 2009; Yla ¨-Anttila et
al., 2009; Hailey et al., 2010).
One integral membrane protein, Atg9, has been found on
the forming autophagosome in both yeast and mammals
(Lang et al., 2000; Noda et al., 2000; Young et al., 2006), and
because it has six transmembrane domains its delivery must
be accompanied by the delivery of membrane. In yeast Atg9
is found at the forming autophagosome and in peripheral
sites that have been suggested to be mitochondria (Reggiori
et al., 2004a; He et al., 2006; Reggiori and Klionsky, 2006).
Yeast Atg9 appears to cycle between the forming autopha-
gosome and the peripheral sites, however the precise nature
of these sites and the membrane trafficking routes taken by
Atg9 remain unclear. The genetic screens for proteins re-
quired for autophagy have found only a few known com-
ponents of membrane traffic, and these are typically proteins
such the SNARE Vam3 which act on the vacuole to mediate
fusion with the completed autophagosome (Darsow et al.,
1997). The related Cvt pathway has been found to require
several proteins involved in traffic between the Golgi
apparatus and endosomes, including the SNARE Tlg2, the
GARP/VFT tethering complex, the sorting nexins Atg20 and
Atg24, and the TRAPP subunit Gsg1/Trs85 (Abeliovich et
al., 1999; Nice et al., 2002; Reggiori et al., 2003; Meiling-Wesse
et al., 2005). However, these proteins are not required for
bulk autophagy and so do not appear relevant to the deliv-
ery of membrane to the autophagosome during starvation
(Juhasz and Neufeld, 2006). In mammalian cells Atg9 is
found in the TGN and endosomal system and has been
proposed to be delivered to the forming autophagosome
from one or more of the compartments that constitute this
system (Young et al., 2006; Razi et al., 2009). However, such
a source for autophagosomal membranes would appear to
lack support from yeast studies where the components that
mediate traffic in the Golgi-endosomal system are only re-
quired for the Cvt pathway.
This article was published online ahead of print in MBoC in Press
on September 22, 2010.
Address correspondence to: Sean Munro (email@example.com.
© 2010 Y. Ohashi and S. Munro. This article is distributed by The
American Society for Cell Biology under license from the au-
thor(s). Two months after publication it is available to the public
under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License(http://creativecommons.org/li-
We have examined the trafficking of Atg9 as a means of
understanding more about the routes by which membrane
gets to the forming autophagosome during starvation. Re-
straining Atg9 to the ER and early Golgi reveals that it must
move to later parts of the endomembrane system to be able
to reach the autophagosome and exert its function. Exami-
nation of a range of membrane traffic components reveals
the SNARE Gos1 to be a further component of Golgi-endo-
some traffic that is required for the Cvt pathway but, as with
those found previously, it does not appear to be required for
starvation-induced autophagy. However, we find that com-
bining mutations that appear only to affect the Cvt pathway
results in profound synergistic defects in autophagy in
starved cells. This suggests that in yeast the Golgi-endosome
system, rather that the ER or mitochondria, provides Atg9-
containing membrane to the forming autophagosome.
MATERIALS AND METHODS
Yeast Strains and Plasmids
Yeast strains were based on BY4741 (MATa his3?1 leu2?0 met15?0 ura3?0),
and all single deletion strains were in the BY4741 background and obtained
from Open Biosystems (Huntsville, AL). Double mutants were created using
the PCR method with KanMX or S. pombe HIS5 markers, and fluorescent
markers RFP-APE1, ATG9-GFP, RFP-SCS2, Cherry-TLG1, and TOM20-RFP
were introduced by homologous recombination [RFP was TagRFP (Evrogen,
Moscow, Russia) or tdTomato (Campbell et al., 2002) as indicated, Supple-
mental Table S1]. An integration plasmid to express Atg9-EGFP under the
TPI1 promoter was constructed follows: ATG9 promoter-TPI1 promoter-
ATG9-EGFP-NatMX in pBluescriptII(KS-) (Supplemental Table S2). Homolo-
gous recombination was performed using a unique XbaI site in the ATG9
promoter region. Strains carrying this construct showed a similar distribution
of Atg9-EGFP to that described previously (Sekito et al., 2009). For the GFP-
Atg8 and ALP assays integration plasmids carrying EGFP-ATG9-MYC,
KKXX, and RxR, were created by inserting the coding region between the
TPI1 promoter and NatMX of pYOintF8, and introduced into ?atg9 genome
by homologous recombination in the ATG9 promoter.
Yeast Growth and Protein Extraction
YEPD (1.1% yeast extract, 2.2% bactopeptone, 2% glucose, and 0.0055% ade-
nine sulfate) or -URA medium was used for vegetative phase growth. For
nitrogen starvation, overnight cultures in YEPD or -URA were diluted 1/50
and grown at 30°C to early log phase in YEPD or -URA medium, then
resuspended in nitrogen starvation medium (SD-N, 0.17% yeast nitrogen base
lacking ammonium sulfate and amino acids with 2% glucose) and incubated
for 4 h. For protein extraction, 5 OD600of cells were collected and resus-
pended in 100 ml of sample buffer, disrupted with an equal volume of glass
beads using a homogenizer (FastPrep-24, MP Biomedicals, Aurora, OH, 45 s
at intensity 6.5). Immunoblots were with anti-Ape1 (Dan Klionsky, University
of Michigan), anti-GFP raised in rabbits, or anti-FLAG (Sigma-Aldrich, St.
Louis, MO, A-8592). CPY pulse-chase was performed as described previously
(Sapperstein et al., 1996), using anti-CPY (Rockland, Gilbertsville, PA 100-401-135).
Alkaline Phosphatase Assay
Phosphatase assays for autophagy using a cytosolic form of Pho8 (Pho8?60)
were as described previously (Noda and Ohsumi, 1998). Briefly, 1–2 ? 107
cells were collected by centrifugation, washed with water, and resuspended
in 0.2 ml assay buffer (250 mM Tris-HCl (pH 9.0), 10 mM MgSO4, 10 ?M
ZnSO4) and vortexed with glass beads. After centrifugation, 50 ?l of super-
natant was transferred to 0.5 ml of assay buffer. Fifty microliters of 55 mM
?-naphthyl phosphate (Sigma) was added to start the reaction and, after
incubation at 30°C, was stopped by addition of 0.5 ml of 2 M glycine-NaOH
(pH 11.0). Fluorescent emission at 472 nm after excitation at 345 nm was
measured with a fluorometer (Perkin-Elmer, Foster City, CA). One unit is the
release of 1 ?mol ?-naphthol/min/mg protein. Protein concentration was
measured using by the Bradford method (Bio-Rad, Hercules, CA).
Microscopy of Yeast Strains
Cells expressing fluorescent fusion proteins were photographed with an
100 ? 1.49 NA objective on a Nikon Eclipse TE2000 epifluorescent microscope
using a CCD camera (CoolSNAP-HQ2, Roper Scientific, Tucson, AZ) and RFP
and GFP filters (Chroma Technology, Rockingham, UT). Images were ac-
quired and analyzed using MetaMorph and ImageJ, and levels adjusted with
Adobe Photoshop. For FM4-64 labeling cells were incubated in 8 ?M FM4-64
(Molecular Probes, Eugene, OR) at 30°C, washed twice and incubated in
medium lacking FM4-64 at 30°C, timing being indicated in the figure legends.
Atg9 Can Be Retained in the ER and Early Golgi
To be able to investigate whether Atg9 can travel to the auto-
phagosome by direct delivery of membrane from the ER we
attempted to create a version of the protein whose localization
membrane proteins to accumulate in the ER by directing re-
trieval from the cis-Golgi in COPI-coated vesicles. The C-ter-
minal cytoplasmic tail of Atg9 is relatively long (?250 resi-
dues), and although recognition of the KKXX signal by the
COPI coat is reduced by increased separation from the mem-
brane, recognition of RXR is apparently independent of dis-
tance (Shikano and Li, 2003). Atg9 is normally found in the
forming autophagosome and in a peripheral pool of small
structures (He et al., 2006; Reggiori and Klionsky, 2006). When
Atg9 was expressed with an RXR motif at its C terminus it
instead accumulated in the ER as well as in some additional
puncta, which presumably reflect cycling through the cis Golgi
(Figure 1A). To examine delivery to the autophagosome we
compared GFP-Atg9 to aminopeptidase1 (Ape1). Ape1 forms
oligomers in the cytoplasm and is delivered directly to the
vacuole by the Cvt pathway in normal growth conditions and
by bulk autophagy during starvation (Harding et al., 1995).
the forming autophagosome (Shintani et al., 2002). Comparison
with RFP-Ape1 revealed that the addition of RXR inhibited the
delivery of Atg9-GFP to Ape1 aggregates (Figure 1B; 95% of
RFP-Ape1 structures were labeled with GFP-Atg9 vs. 8% with
GFP-Atg9-RXR, n ? 100). As expected, the Atg9 cytoplasmic
tail is too long to allow recognition of a C-terminal KKXX
motif. However, Atg9 retains some functionality when the
tail is truncated to just 38 residues (He et al., 2008), and
this truncated form was found to be relocated to the ER
when a KKXX motif was attached to the C terminus (Sup-
plemental Figure S1A).
The Cvt Pathway and Autophagy Depend on Atg9 Being
Able to Leave the ER/Early Golgi
We next examined whether the relocation of Atg9 to the ER
results in a defect in autophagy. We initially assayed the con-
version of Ape1 from the precursor (proApe1) to the mature
form which occurs when autophagosomes fuse to the vacuole.
The rescue of Ape1 maturation in an Atg9 null strain by GFP-
Atg9 was blocked by attachment of the RXR signal, and hence
ER retention, in both rich medium and during nitrogen star-
vation (Figure 1C). Although the activity of full-length Atg9
was, as expected, unaffected by the KKXX motif, the version
with a 38 residue tail which could be relocated to the ER by
KKXX also showed a loss of activity when this motif was
attached (Figure 1B and Supplemental Figure S1A). It is pos-
sible that the lack of processing of proApe1 during autophagy
could reflect a cargo-specific defect. We thus examined two
other widely used reporters for starvation-induced autophagy.
The first is the release of free GFP from a fusion to the protein
Atg8 that is covalently attached to lipids in the autophagosome
and is digested after fusion to the vacuole (Shintani and Klion-
sky, 2004). The second is the vacuolar delivery and hence
activation of a cytosolic form of the proenzyme alkaline phos-
phatase (Noda and Ohsumi, 1998). With both assays we again
found that the RXR motif blocked the ability of GFP-Atg9 to
allow autophagy during starvation conditions (Figure 1, C and
D). Taken together these results indicate that Atg9 normally
has access to the ER and early Golgi but that it has to move
beyond the early Golgi to be able reach the autophagosome.
Atg9 Delivery to the Autophagosome
Vol. 21, November 15, 20103999
Intracellular Distribution of Atg9
Direct delivery from the ER is not the only route that has
been proposed for delivery of membrane to the forming
autophagosome. In yeast Atg9 is located in peripheral
puncta as well as the forming autophagosome, and it has
been proposed that these puncta correspond to mitochon-
dria (Reggiori et al., 2004a; He et al., 2006; Reggiori and
Klionsky, 2006; Yen et al., 2007). When we examined the
distribution of Atg9-GFP we were able to detect some la-
beled puncta close to mitochondria, particularly under star-
vation conditions (Figure 2A). However, when mitochon-
drial morphology was altered genetically, there was not a
concomitant large-scale relocalization of Atg9-GFP (Figure
2A). In addition, time-lapse imaging revealed that the Atg9-
GFP puncta were much more mobile than the mitochondria
and appeared to be moving past them and also through
other parts of the cell were there are no mitochondria (Sup-
plemental Video 1). Similar observations have been recently
reported by Sekito and coworkers (Sekito et al., 2009), and
the apparent transient colocalization of Atg9-GFP puncta
and mitochondria may be linked to mitophagy or could
simply reflect the fact that both structures can move along
actin cables (Frederick and Shaw, 2007; Monastyrska et al.,
2008). When we compared Atg9-GFP to other organelle
markers the only substantial colocalization we observed was
with endocytic compartments as labeled by the late endoso-
mal sodium/proton exchanger Nhx1 (Bowers et al., 2000),
the endocytosed fluorescent dye FM4-64 (Vida and Emr,
ER to the forming autophagosome. (A) Fluores-
cence micrographs of yeast expressing GFP-
Atg9 with a C-terminal myc-tag followed by
nothing or an RXR motif [KLRRRRI (Michelsen
et al., 2007)]. The fusions were expressed with a
TPI1 promoter from a centromeric vector and
protein RFP-Scs2. The RXR motif increases the
amount of Atg9 in the ER and reduces that
present at aggregates of mRFP-Ape1. Scale
bars ? 2 ?m. (B) Anti-Ape1 immunoblots of
lysates from wild-type cells, or from an ATG9
deletion strain transformed with either an
empty plasmid, or the same GFP-Atg9 plas-
mids shown in A, and also one with the C-
terminal myc-tag followed by a KKXX motif
(SKKSL). Cells were harvested after growth in
rich medium or after four hours of nitrogen
starvation to induce autophagy. Attachment of
the RXR motif to Atg9 blocks its activity under
both conditions. (C) Anti-GFP immunoblots of
ATG8 promoter on a CEN plasmid. The yeast
strains are as in A and B but with the ATG9
fusions integrated into the genome. The cells
were grown to midlog phase and either har-
vested (0) or starved for nitrogen for 4 h. Dele-
tion of Atg9 prevents delivery of GFP-Atg8 to
the vacuole and release of free GFP. (D) Alka-
line phosphatase activity in strains expressing a
cytosolic form of Pho8 (Pho8?60) that lacks a
transmembrane domain and so only becomes
active upon delivery to the vacuole by bulk
autophagy (Noda and Ohsumi, 1998). Strains
are as in C but with PHO13 deleted and PHO8
truncated to PHO8?60 by integration of TDH3
promoter. Cells were grown and starved for 4 h
as in C (error bars indicate SD of three indepen-
Atg9 cannot get directly from the
Y. Ohashi and S. Munro
Molecular Biology of the Cell4000
1995), or the SNARE Tlg1 whose steady state distribution is
primarily in endosomes as it cycles between endosomes and
the Golgi (Holthuis et al., 1998) (Figure 2, B and C). This
colocalization was maintained in the absence of Atg11 which
is required for the formation of autophagosomal, but not the
peripheral, pool of Atg9 (Figure 2C), and was also seen in
the enlarged endosomes that are induced by deletion of the
multivesicular body forming protein Vps4 (Figure 2C).
These results indicate that Atg9 traffics through the Golgi
and then resides in both the forming autophagosome and, at
least in part, in endosomal compartments rather than being
Analysis of SNARE Requirements for Cvt Pathway and
To obtain further insight into the route by which Atg9-contain-
ing membrane reaches the autophagosome we looked for traf-
ficking components that are required for autophagy. We ini-
tially examined the SNARE proteins as these act in all known
trafficking steps within the endomembrane system. To ob-
tain a comprehensive picture of the involvement of SNARE
proteins in the Cvt pathway and autophagy, Ape1 matura-
tion was examined in individual mutants for all eleven of
the SNAREs that are not essential for viability (Figure 3A).
As reported previously the vacuolar SNAREs Vam3 and
Vam7 that mediate fusion of the autophagosome with the
vacuole are required for both autophagy and the Cvt path-
way (Figure 3A; Darsow et al., 1997). In addition, we found
that deletion of the Golgi SNARE Gos1 caused an accumu-
lation of proApe1 in rich medium comparable to that seen
with loss of the SNARE Tlg2 that has been previously re-
ported to act in the Cvt pathway (Abeliovich et al., 1999). The
stability of mature Ape1 means that the mature protein
generated during starvation in stationary phase persists in
the vacuole long after initiation of log-phase growth, but
imaging of GFP-Apel in strains lacking either Gos1 or Tlg2
confirmed the accumulation of unprocessed Ape1 aggre-
gates, and hence a defect in the Cvt pathway, during log-
phase growth in rich medium (Figure 3B). However, neither
Gos1 nor Tlg2 were required for Ape1 processing when
autophagy was induced by starvation (Figure 2A).
COG Complex Subunits Contribute to Efficient
Functioning of the Cvt Pathway
The SNAREs Tlg2 and Gos1 act in distinct pathways for
recycling of membrane back from the endosomes to the
Golgi, or within the Golgi itself, and are believed to partic-
ipate in distinct SNARE complexes (Holthuis et al., 1998;
McNew et al., 1998; Parlati et al., 2002). Gos1 has been pro-
posed to interact with the octameric COG complex which
comprises two lobes, A and B, with the latter lobe containing
four nonessential subunits (Cog5-8) that appear to be in-
volved in recycling back to the Golgi from endosomes
(Whyte and Munro, 2001; Ungar et al., 2006). Thus we asked
whether mutants in lobe B subunits show defects in the Cvt
pathway and autophagy. Figure 3C shows that in mutants
lacking any one of Cog5-8 there was a small but reproduc-
ible accumulation of unprocessed Ape1 in rich (i.e., nonstar-
vation) medium. In these COG subunit mutants there was
also an increase in the number of cells that where aggregates
GFP. (A) Fluorescent micrographs comparing
Atg9-GFP with the mitochondrial outer mem-
brane marker Tom20-RFP in wild-type cells or
cells lacking the mitochondrial fusion protein
Ugo1 and grown in YEPD or starved for nitro-
gen for four hours [SD(-N)] as indicated. The
loss of Ugo1 causes the mitochondria to form
clumps of fragments (Sesaki and Jensen, 2001),
but Atg9-GFP remains scattered, with colocal-
ization only observed occasionally, usually in
forming buds. (B) Fluorescence micrographs
comparing Atg9-GFP to the late Golgi protein
Sec7-RFP or the late endosomal protein Nhx1-
RFP in wild-type cells in rich medium. (C)
Fluorescence micrographs comparing Atg9-
GFP to RFP-Ape1, the endocytic tracer FM4-64
(10 min pulse, 10 min chase), or mCherry-Tlg1
(Chry-Tlg1) in wild-type cells in rich medium.
(D) As for C except that the cells are either
lacking the autophagosome scaffold protein
Atg11 which is required for membrane to ac-
cumulate at the autophagosome (Shintani and
Klionsky, 2004), or lacking the endosomal pro-
tein Vps4, in which the endocytic compart-
ment expands (Babst et al., 1997).
Intracellular distribution of Atg9-
Atg9 Delivery to the Autophagosome
Vol. 21, November 15, 20104001
of GFP-Ape1 had accumulated in the cytoplasm (Figure 3B).
As with the ?gos1 mutant, accumulation of proApe1 in the
COG mutants was lost when bulk autophagy was induced
by starvation (Figure 3, B and C). Thus Cog5-8 appear to be
only required for autophagosome formation under nonstar-
vation conditions, and while this article was in preparation
similar effects of cog5-8 mutants on Ape1 processing in the
Cvt pathway but not autophagy were reported by Klionsky
and coworkers (Yen et al., 2010).
Synthetic Genetic Interactions Reveal Role of Cvt
Pathway Components in Autophagy
The above results reveal that Atg9 needs to travel past the
early Golgi to reach the autophagosome in starved cells and
that it does not appear to accumulate in mitochondria but
rather it is, at least in part, localized to endosomes. However,
the only endosome/Golgi SNAREs that are required for the
Cvt pathway, Gos1 and Tlg2, do not appear to be required
for autophagy during starvation. Nonetheless, previous
studies on recycling between the Golgi and endosomes have
revealed a remarkable degree of redundancy with many
double mutants showing synergistic interactions (Tong et al.,
2004; Sciorra et al., 2005; Burston et al., 2009). This appears to
be due to there being multiple routes between endosomes
and Golgi, and also to the ability of some cargo proteins to
use a different route when their normal route is missing
(Hettema et al., 2003; Quenneville et al., 2006). This led us to
wonder whether the Golgi and endosomal proteins previ-
ously thought to contribute only to the Cvt pathway are in
fact also contributing to autophagy but in a manner that is
redundant and hence only visible when more than one
protein is missing. We first analyzed strains lacking one or
other of the SNAREs Gos1 and Tlg2, and the COG subunit
Cog8. Not only were the Cvt defects observed in the single
mutants enhanced in the double mutations, but now defects
could be clearly seen under starvation conditions in the case
of the ?cog8?gos1 double mutant (Figure 4A).
We next examined the sorting nexins Atg20 and Atg24
(Snx4). These proteins form a heterodimer and act in traffic
in one of the routes from endosomes to the Golgi and are
required for the Cvt pathway but not bulk autophagy (Nice
et al., 2002; Hettema et al., 2003). Atg24 also forms het-
erodimers with a relative of Atg20, called Snx41, but the
Atg24/Snx41 complex appears to be exclusively involved in
endosome to Golgi traffic and not the Cvt pathway (Hettema
et al., 2003). Figure 4B shows that the ?cog8?atg24 double
mutant has a clear defect in autophagy despite there being
no detectable defect with either single mutation. Likewise,
combining mutants in Atg20 or Atg24 with those in the
SNAREs Gos1 or Tlg2 results in defects in Ape1 maturation
in starved cells, with a near complete block in ?gos1?atg24
and ?tlg2?atg24, despite the individual mutations showing
little defect (Figure 4C). These strong phenotypes are due to
the loss of the deleted genes as they could be rescued by the
relevant genes on plasmids (Supplemental Figure S2).
To test whether these findings were generally relevant
to Cvt-specific proteins we examined double mutants in a
range of other genes encoding components of endosome-
Golgi traffic that appear to contribute to the Cvt pathway but
not to autophagy. These were the GTPase Ypt6, its effector
the GARP/VFT subunit Vps51, and the TRAPP subunit
Gsg1 (Trs85) (Siniossoglou and Pelham, 2001; Reggiori et al.,
2003; Meiling-Wesse et al., 2005; Lynch-Day et al., 2010). We
also examined the PtdIns(3)P binding protein Atg21 that is
required for the Cvt pathway but has no reported role in
traffic (Krick et al., 2008). In every case combining mutations
in these proteins with mutants in sorting nexin subunits
again resulted in synergistic defects in autophagy (Figure 5).
The Effects of Double Mutants of Cvt Pathway
Components on Other Assays for Autophagy
To confirm that the above synergistic effects on starvation-
induced autophagy seen with combinations of Cvt path-
way components were not confined to Ape1 processing we
tested some mutant combinations with other assays for auto-
phagy. The strongly synergistic combinations ?gos1?atg24
and ?tlg2?atg24 showed blocks in both the GFP-Atg8 process-
ing assay and the Pho8?60 vacuolar activation assay that were
are involved in the Cvt pathway. (A) Ape1
grown in rich medium (YEPD) for three hours
or under conditions of nitrogen starvation for
four hours [SD(-N)]. Vam3 and Vam7 are in-
volved in the fusion of vacuolar membranes
and so are required for maturation of Ape1 in
vegetative and starvation conditions. Deletion
of Pep12 inhibits Ape1 maturation, which at
least in part appears to be because of its known
role in the delivery of vacuolar hydrolases
(Becherer et al., 1996). In starved cells some
GFP-Ape1 could be seen in undigested auto-
phagosomes inside the vacuole (Supplemental
Figure 1B), but in rich medium we also ob-
served an accumulation of GFP-Ape1 aggre-
gates adjacent to the vacuole and so there may
also be defects in autophagosome formation in
the Cvt pathway. (B) Localization of GFP-
Ape1 by fluorescence microscopy in the indi-
cated strains. Cells were grown in YEPD or in
SD(-N) and labeled with FM4-64. In YEPD a
puncta of GFP-Ape1 was observed in 4% of
wild-type cells, but this rose in ?atg1 (31%),
?cog8 (29%), ?gos1 (12%), and ?tlg2 (24%). In
SD(-N), GFP signals were mainly observed in-
Golgi SNAREs and COG subunits
side the vacuole in all the strains except ?atg1. (C) Ape1 immunoblot analysis in wild-type (WT) or the indicated deletion mutants. Mutants
lacking COG lobe B subunits, or YPT6 or its exchange factor RIC1 show reduced Ape1 maturation only in vegetative phase (YEPD).
Y. Ohashi and S. Munro
Molecular Biology of the Cell4002
not seen with the single mutants (Figure 6, A and B). In addi-
tion, GFP-Atg8 accumulated in more than one puncta in the
cytosol of the double mutants (Figure 6, C and D), a phenotype
associated with defects in autophagosome formation (Reggiori
et al., 2004b; Meiling-Wesse et al., 2005; Yen et al., 2010).
The Ape1 processing assay, the GFP-Atg8 cleavage assay,
and the Pho8 activation assay all depend on the SNARE Vam3
and active hydrolases being present in the vacuole (Darsow et
al., 1997). However, pulse-chase analysis showed that the mat-
uration of the vacuolar hydrolase carboxypeptidase Y is not
SD(-N) by immunoblotting in the indicated mutants. (A) Analysis of ?cog8 and two SNARE mutants, ?gos1 and ?tlg2. (B) Analysis of ?cog8
and single mutants for three related sorting nexins, Atg20, Atg24, and Snx41. (C) Analysis of two SNARE mutants (?gos1 and ?tlg2) and two
sorting nexin mutants.
Combinations of Golgi-endosomal mutations cause defects in autophagy. Ape1 maturation was analyzed both in YEPD and
by immunoblotting in the indicated mutants. (A) Analysis of mutants lacking GARP/VFT subunit Vps51, or Ypt6 that recruits GARP/VFT,
and the sorting nexins (?atg20 and ?atg24). (B) Analysis of mutants in Cvt component Atg21 and sorting nexins (?atg20 and ?atg24). (C)
Analysis of mutants in TRAPP subunit Gsg1 and sorting nexin Atg24.
Combinations of Cvt pathway mutations cause defects in autophagy. Ape1 maturation was analyzed both in YEPD and SD(-N)
Atg9 Delivery to the Autophagosome
Vol. 21, November 15, 20104003
affected in the double mutants that show strong Ape1 process-
ing defects (e.g., ?gos1?atg24 and ?tlg2?atg24), whereas it is,
as expected, blocked in the ?vam3 control (Supplemental Fig-
ure S3A). Likewise the double mutations did not show severe
vacuole fragmentation defects, or indeed perturbation of
growth rate (Supplemental Figure S3, B and C). Defects in
endosomal components can also result in the increased turn-
over of membrane proteins by vacuolar degradation, but there
Atg9 in the double mutants (Supplemental Figure S3D). Taken
together these data indicate that the vacuole in these double
mutants is fusion competent and contains active hydrolases,
and therefore the defects in autophagy seen with double mu-
tants of Cvt pathway components must be upstream of vacu-
olar processing and hence reflect defects in autophagosome
Accumulation of Atg9-GFP in the Autophagosome Is
Impaired in Double Mutants of Cvt Pathway Components
Because Atg9-GFP appears to be at least in part located in
endosomal compartments, it seemed possible that the above
combinations of mutations in components of Golgi-endosomal
traffic that affect autophagy were affecting delivery of Atg9 to
the autophagosome. To investigate the effects of the various
double mutants on the delivery of Atg9 to the forming auto-
phagosome, we compared the distribution of Atg9-GFP to that
of RFP-Ape1. In the single mutants there was no substantial
change in the number of cells that showed labeling of the Ape1
aggregate with Atg9-GFP (Figure 7, A and B). However, in
double mutants with a severe defect in Ape1 processing there
was in general a large reduction in the number of cells where
starvation conditions. This suggests that combining mutations
in Cvt pathway can result in the delivery of Atg9 to the form-
ing autophagosome being impaired, even in starvation condi-
tions, which would provide an explanation for the observed
defects in autophagy. The only exceptions to this general trend
were those mutants containing Atg20, where Atg9 could still
reach the forming autophagosome in rich medium even
though Ape1 processing was defective. This could reflect a role
for Atg20 in retrieval of Atg9 to peripheral sites, with elevated
Atg9 being detrimental to autophagosome maturation. This
associated with Ape1 aggregates (Figure 7A).
The SNAREs Gos1 and Tlg2 Are Required for Different
Steps in Atg9 Recycling
After Atg9 is delivered to the autophagosome it appears to
be retrieved back to the peripheral pool, presumably allow-
ing it to participate in multiple rounds of autophagosome
formation (Reggiori et al., 2004a). Thus there must be mem-
brane trafficking routes to deliver Atg9 to the forming auto-
phagosome and also to remove it and deliver it back to the
to starvation conditions for four hours (SD-N). The positions of GFP-Atg8 and the free GFP that is released after autophagic delivery to the vacuole
are indicated. (B) Alkaline phosphatase activity in the indicated strains expressing Pho8?60 that lacks a transmembrane domain and so is only
independent experiments. (C) Quantitation of the distribution of GFP-Atg8 in cells grown as in A. For each strain ?150 cells were counted. The
values show the proportion of the population with one, or more than one puncta of GFP-Atg8, and are based on a projections of six focal planes.
The increases in frequency of cells with multiple GFP-Atg8 puncta in all the double mutants are statistically significant (?2test, two-tailed values
p ? 0.0001). (D) Representative single focal plane images of the cells quantified in C. Combination of SNARE and Atg24 mutations results in the
appearance of multiple puncta of GFP-Atg8 consistent with defects in autophagosome formation.
Combined mutants in Golgi-endosomal components show synergistic defects in starvation-induced autophagy. (A) Anti-GFP immu-
Y. Ohashi and S. Munro
Molecular Biology of the Cell4004
peripheral sites, at least some of which we suggest are
endosomes. Thus if a mutant combination reduces the accu-
mulation of Atg9 in the forming autophagosome it could
reflect a direct defect in delivery, or it could be that delivery
is normal but there is a defect in the retrieval route that
occurs after Atg9 has exited the autophagosome. These pos-
sibilities can be distinguished using an epistasis analysis
based on the Atg1 kinase, the so called “Take Atg1 kinase
away (TAKA)” assay (Shintani and Klionsky, 2004). Atg1 is
required for exit of Atg9 from the autophagosome at the
start of the retrieval route (Reggiori et al., 2004a). Thus if a
mutant, or mutant combination, affects delivery, when Atg1
is removed from the mutant background the accumulation
of Atg9 at the autophagosome should remain inhibited.
However, if the defect is in the retrieval route then removal
of Atg1 should result in accumulation of Atg9 in the auto-
phagosome as it cannot enter the defective retrieval route.
We therefore examined the effect of deleting Atg1 in combi-
nations of Cvt mutants that show strong autophagy defects.
Figure 8 shows that when Atg1 activity is removed from the
?tlg2?atg24 strain then delivery of Atg9 remains substantially
inhibited. In contrast, when the SNARE Gos1 is absent in
combination with ?atg24, then removal of Atg1 results in Atg9
accumulating at the forming autophagosome. This indicates
that when cells are sensitized by removal of Atg24, then the
route that Atg9 must travel to the forming autophagosome is
dependent upon Tlg2, but not Gos1. In contrast the retrieval
not Tlg2, that appears to occur after the Atg1-dependent exit of
participate in distinct SNARE complexes and hence different
trafficking processes (McNew et al., 1998; Parlati et al., 2002;
Burri and Lithgow, 2004). These results suggest that during
starvation-induced autophagy, Cvt components are making a
redundant contribution to both the delivery of Atg9-containing
membrane to the forming autophagosome and also to its re-
The results reported here demonstrate that in yeast Atg9 has to
move beyond the ER and early Golgi to be able to function in
autophagy. This finding suggests that substantial amounts of
bilayer membrane are not delivered directly from the ER to the
forming autophagosome, although we cannot exclude the pos-
sibility that there is a process that transports some bilayer
directly from the ER to the autophagosome in carriers that
exclude Atg9. The conclusion that at least Atg9 must traffic out
of the ER via the Golgi is consistent with previous reports that
Fluorescence micrographs comparing the distribution of Atg9-GFP and RFP-Ape1 in the indicated representative strains in YEPD or SD(-N).
Arrows indicate RFP-Ape1 dots which are not colocalized with Atg9-GFP. (B) Quantification of Atg9-GFP and RFP-Ape1 colocalization in
both YEPD (rich) and SD(-N). For each strain 50–100 RFP-Ape1 containing structures were examined.
Atg9 does not reach Ape1-containing autophagosomes in double mutants for Golgi-endosomal trafficking components. (A)
Atg9 Delivery to the Autophagosome
Vol. 21, November 15, 2010 4005
conditional mutations that block ER to Golgi traffic affect au-
tophagy (Ishihara et al., 2001; Reggiori et al., 2004b), although it
could have been argued in this case that the general, and
ultimately lethal, block in secretion used in these experiments
could have affected other processes apart from Atg9 delivery,
or could reflect indirect effects mediated via the stress path-
ways known to be induced when secretion is blocked (Mizuta
and Warner, 1994; Nanduri and Tartakoff, 2001; Geng et al.,
2010). In addition, we do not find evidence to support previous
suggestions that Atg9 is localized to mitochondria and hence
bilayer membrane can be delivered from mitochondria to the
forming autophagosome (Reggiori et al., 2004a; He et al., 2006;
Reggiori and Klionsky, 2006; Yen et al., 2007), a route that
would have required unprecedented membrane trafficking
processes and machinery.
Instead, our results demonstrate that during starvation
the delivery of Atg9, and hence membrane, to the autopha-
gosome is perturbed by removal of combinations of compo-
nents of the membrane traffic routes that link the Golgi and
endosomal systems. These components had previously been
shown to be required for efficient functioning of the Cvt
pathway, but did not appear to be required for starvation-
induced autophagy (Abeliovich et al., 1999; Nice et al., 2002;
Reggiori et al., 2003; Meiling-Wesse et al., 2005). The ability of
the yeast Golgi-endosomal system to compensate for many
single gene deletions under laboratory conditions would
explain why some of these genes need to be deleted in
combination before defects in autophagy become apparent
and why these genes were not found in the seminal genetic
screens that identified the proteins that regulate and initiate
autophagy (Tsukada and Ohsumi, 1993; Thumm et al., 1994;
Harding et al., 1995; Nakatogawa et al., 2009). The fact that
single mutants show defects only in the Cvt pathway and
not in starvation conditions might reflect that fact that dur-
ing starvation it could become important to deliver mem-
brane from more sources in the Golgi-endosome system so
that no one aspect of endocytic traffic becomes depleted
when there is a larger flux through the autophagic pathway.
This would also explain why a block in the flow of mem-
brane from the ER into the secretory pathway rapidly affects
autophagy but has a slower effect on the Cvt pathway (Ishi-
hara et al., 2001; Reggiori et al., 2004b).
Our results imply that the Cvt-pathway is more similar to
starvation-induced autophagy than previously appreciated,
and hence the two processes differ more in rate than in
mechanism. The majority of cellular components thought to
be specific to the Cvt-pathway are components of Golgi-
endosomal traffic which we show here are also important for
autophagy. Of the remainder, Atg19 is a receptor for a
specific cargo (Ape1) that needs to efficiently directed into
forming autophagosomes in nonstarved cells, and the Cvt-
specific scaffold protein Atg11 appears to be also required
for autophagy if Atg17 is absent (Suzuki et al., 2007). More-
over, Atg11 is distantly related to FIP200 (S.M., unpublished
observation), a mammalian protein which is required for
starvation-induced autophagy (Hara et al., 2008).
The link between Golgi-endosome components and starva-
tion-induced autophagy is also consistent with the fact that the
forming autophagosome has many of the properties of an
endocytic compartment including enrichment in PtdIns(3)P
and its effector proteins (Hettema et al., 2003; Krick et al., 2008;
Obara et al., 2008), the apparent exchange of membrane with
compartments in the Golgi-endosome system, and the ability
to mature to fuse with the vacuole. Indeed we note that Atg2,
a protein found on the autophagosome and required for auto-
phagy, is distantly related to the endosomal trafficking compo-
nent Vps13 (S.M., unpublished observation). Delivery of mem-
affects different parts of the Atg9 recycling itinerary. (A) Fluores-
cence micrographs comparing the distribution of Atg9-GFP and
RFP-Ape1 in the indicated strains with or without Atg1 deleted.
Cells were grown in YEPD to midlog phase and shifted to starvation
conditions for four hours (SD-N). Deletion of Atg1 blocks exit of
Atg9 from the autophagosome that forms around Ape1 aggregates
(Reggiori et al., 2004a). The autophagosome does not form in the
absence of Atg11, and so this serves as a negative control. In
?gos1?atg24 the majority of forming autophagosomes show an
accumulation of Atg9 in the absence of Atg1 (arrows), whereas in
?tlg2?atg24, the forming autophagosomes tend to lack Atg9 (ar-
rows), indicating that delivery rather than retrieval is defective. (B)
Quantitation of the experiments in A in which the presence of Atg9
in forming autophagosomes was quantified by counting how many
Ape1 aggregates had associated Atg9-GFP. 100–200 mRFP-Ape1
puncta were analyzed for each condition.
Combining loss of Atg24 with loss of different SNAREs
Y. Ohashi and S. Munro
Molecular Biology of the Cell 4006
brane and Atg9 from compartments of the endocytic system
would also be consistent with recent findings that conditional
inactivation of either of the essential Golgi GTPase activities
Arf1/2 or Ypt31/32 inhibits autophagy, as these activities are
likely to be required directly and indirectly for both traffic
through the Golgi and also recycling from endocytic compart-
ments (Hicke et al., 1997; Geng et al., 2010; van der Vaart et al.,
2010). This requirement does not appear to reflect a need to
traffic Atg9 to the cell surface, and indeed we do not see Atg9
accumulating at the surface in endocytic mutants such as
?end3 (YO and SM, unpublished observations). Our finding
that a RXR motif is recognized when attached to Atg9 indicates
that the protein must move through the cis Golgi where COPI
will be needed to determine where Atg9 leaves the Golgi, as
recent studies indicate that inactivation of the late Golgi Arf
exchange factor Sec7 does not result in accumulation of Atg9 in
the late Golgi (van der Vaart et al., 2010). Given the complexi-
ties of the yeast Golgi-endosome system, further work will also
be required to map which precise routes Atg9 can use and
when, and indeed it is possible that some components have a
role on the autophagosome itself as well as being required for
the traffic of Atg9 into a post-Golgi compartment and on to the
autophagosome. This has recently been suggested for the es-
sential (lobe A) subunits of the COG complex (Yen et al., 2010),
but because these subunits are also required for traffic of mem-
brane proteins through the Golgi then further work will be
required to determine the point at which these particular mu-
tations are blocking Atg9 delivery (VanRheenen et al., 1999;
Ram et al., 2002).
Overall, our results suggest that Atg9-containing bilayer is
recycling between the forming autophagosome and multiple
compartments in the Golgi-endosome system rather than the
ER or mitochondria. This provides a possible resolution of
the discrepancy between previous work in yeast and recent
studies in mammalian cells where the two orthologues of
Atg9 are found to be localized to TGN and endosomes
(Young et al., 2006; Razi et al., 2009). It should be added that
while our results suggest that bilayer may not be delivered
directly from the ER to the autophagosome, this need not
preclude some phospholipids being transferred from the ER
or mitochondria by cytosolic carrier proteins (Voelker, 2009).
Our demonstration that the Golgi–endosome system rather
than the ER or mitochondria is apparently involved in Atg9
delivery to the forming yeast autophagosome will hopefully
allow the yeast system to guide study of these and other
issues in both yeast and mammalian cells.
We thank Alison Gillingham and Maki Ohashi for comments on the manu-
script and Daniel Klionsky for reagents. This work was supported by a grant
from the Medical Research Council (to S.M.), and by postdoctoral fellowships
(to Y.O.) from the European Molecular Biology Organization, the Human
Frontiers Scientific Program, and the Japanese Society for the Promotion of
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