Research Article
4963
TRAPPIII is responsible for vesicular transport from
early endosomes to Golgi, facilitating Atg9 cycling
in autophagy
Kanae Shirahama-Noda1, Shintaro Kira1, Tamotsu Yoshimori2,3 and Takeshi Noda1,2,*
1
Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka University, 565-0871 Osaka, Japan
Graduate School of Frontier Bioscience, Osaka University, 565-0871 Osaka, Japan
3
Department of Genetics, Graduate School of Medicine, Osaka University, 565-0871 Osaka, Japan
2
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 8 August 2013
Journal of Cell Science 126, 4963–4973
ß 2013. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.131318
Summary
Autophagy is a bulk protein-degradation process that is regulated by many factors. In this study, we quantitatively assessed the
contribution of each essential yeast gene to autophagy. Of the contributing factors that we identified, we focused on the TRAPPIII
complex, which was recently shown to act as a guanine-nucleotide exchange factor for the Rab small GTPase Ypt1. Autophagy is
defective in the TRAPPIII mutant under nutrient-rich conditions (Cvt pathway), but starvation-induced autophagy is only partially
affected. Here, we show that TRAPPIII functions at the Golgi complex to receive general retrograde vesicle traffic from early
endosomes. Cargo proteins in this TRAPPIII-dependent pathway include Atg9, a transmembrane protein that is essential for autophagy,
and Snc1, a SNARE unrelated to autophagy. When cells were starved, further disruption of vesicle movement from late endosomes to
the Golgi caused defects in Atg9 trafficking and autophagy. Thus, TRAPPIII-dependent sorting pathways provide Atg9 reservoirs for
pre-autophagosomal structure and phagophore assembly sites under nutrient-rich conditions, whereas the late endosome-to-Golgi
pathway is added to these reservoirs when nutrients are limited. This clarification of the role of TRAPPIII elucidates how general
membrane traffic contributes to autophagy.
Key words: Autophagy, TRAPP, Golgi-associated retrograde protein, Sorting nexin, Retromer, Atg9, Snc1
Introduction
Autophagy is an evolutionarily conserved intracellular degradation
system that contributes to a wide range of physiological phenomena
(Mizushima and Komatsu, 2011). However, the formation of the
autophagosome, a spherical membrane structure that sequesters the
degradation substrates, remains poorly understood (Yoshimori and
Noda, 2008). The autophagosome is surrounded by two membrane
layers, suggesting that it forms by membrane dynamics that differ
from the mechanisms generally responsible for vesicle budding.
Formation of the autophagosome is mediated by the Atg proteins,
which include two ubiquitin-like proteins (Nakatogawa et al., 2009).
In addition to the Atg proteins, numerous other proteins are involved,
either directly or indirectly, in autophagy. However, it remains
unclear how other membrane-trafficking pathways are related to
autophagy (Longatti and Tooze, 2009). Several yeast mutations that
cause defects in intracellular vesicular trafficking also affect
autophagy. For example, mutation of Tlg2, a t-SNARE localized
in endosomes and the Golgi, disrupts autophagy, but does not abolish
it (Abeliovich et al., 1999). Similarly, early and late sec mutants, as
well as mutants in the conserved oligomeric Golgi (COG) complex,
exhibit defective autophagy (Geng et al., 2010; Hamasaki et al.,
2003; Ishihara et al., 2001; Reggiori et al., 2004b; Yen et al., 2010).
Atg9 is a multiple-transmembrane protein that is essential for
autophagosome formation (Noda et al., 2000; Young et al., 2006);
it is recycled between pre-autophagosomal structure/phagophore
assembly sites (PAS), which are involved in autophagosome
formation, and other small structures called peripheral reservoirs
(Reggiori et al., 2004a). Mutation of Tlg2 or COG complexes
inhibits this recycling, suggesting that these factors are involved in
Atg9 dynamics (Ohashi and Munro, 2010; Yen et al., 2010). In
mammalian cells, Atg9 is transiently associated with some
endosomes (Kageyama et al., 2011; Orsi et al., 2012). A recent
study characterized Atg9-containing vesicles with respect to
physical characteristics such as their size and the number of
Atg9 molecules contained in each vesicle (Yamamoto et al., 2012);
however, the identity and significance of this vesicular-transport
pathway have not been clearly elucidated.
In this study, we screened a mutant collection containing
knockdowns of all essential genes in Saccharomyces cerevisiae, by
measuring the autophagic competency of each mutant. Among the
factors that we identified as contributing significantly to
autophagy, we focused on TRAPPIII, which has been reported to
be specifically involved in autophagy at the PAS, where
autophagosome formation takes place (Lynch-Day et al., 2010).
In contrast to the previous report, our results demonstrated that
TRAPPIII plays a role in general vesicular trafficking at the Golgi,
rather than in an autophagy-specific process. These findings
explain how general vesicular traffic contributes to autophagy.
Results
In the yeast Saccharomyces cerevisiae, an alkaline phosphatase assay
is widely used to quantitatively assess autophagy (Noda and
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Klionsky, 2008; Noda et al., 1995). To obtain a comprehensive
understanding of the mechanisms underlying regulation of autophagy,
we extended the alkaline phosphatase assay system by adapting the
procedure to 96-well microtiter plates, enabling large-scale
quantitative estimation of autophagic competency and the effects of
every non-essential gene mutation in yeast (S.K. et al., unpublished
results). Here, we applied this system to the essential gene knockdown
collection, including about 900 mutants, in which the mRNA for each
gene is destabilized by insertion of a marker gene into the 39untranslated region (DAmP method) (Schuldiner et al., 2005). From
this collection, we identified a group of genes, including TRS20 and
BET5, whose knockdown resulted in markedly low autophagic
activity (Fig. 1A,B). Trs20 and Bet5 are core subunits of TRAPP
complexes, which function at tethering step in several vesiculartransport pathways (Barrowman et al., 2010). The three TRAPP
complexes – TRAPPI, TRAPPII and TRAPPIII – have been reported
to function in endoplasmic reticulum (ER)-to-Golgi transport, intraGolgi transport and autophagy, respectively (Lynch-Day et al., 2010).
Each TRAPP complex serves as a guanine-nucleotide exchange
factor for the Rab GTPase Ypt1 (Lynch-Day et al., 2010), which was
also highly ranked as a candidate gene in our screen (Fig. 1A). The
only subunit that is specific to TRAPPIII, Trs85, was also highly
ranked in our screen of mutations of nonessential genes (S.K. et al.,
unpublished results). Therefore, based on the results of our unbiased
genome-wide screen, we focused on TRAPPIII in our subsequent
investigations.
Fig. 1. Genome-wide examination of autophagy in a collection of
knockdown mutants of essential genes. (A) Autophagic activities of a
collection of yeast mutants in which expression of essential genes was
knocked down. Cells were assayed for autophagic activity after 4 hours of
nitrogen starvation. Relative autophagic activities of each mutant compared
with the wild-type strain are plotted. Note that the efficiency of expression
knockdown differed between the strains. (B) Detailed results for the
representative TRAPP mutants indicated by arrows in A.
Atg9 is recycled between PAS and as-yet-unidentified
peripheral reservoirs, previously characterized as multiple
punctate compartments distributed throughout the cytosol (He
and Klionsky, 2007; Noda et al., 2000). Although Atg9 recycling
has previously been reported to be independent of TRAPPIII
(Lynch-Day et al., 2010), we re-examined this issue using a more
elaborate experimental system that exploits ubiquitylationmediated degradation of the IAA protein induced by addition
of auxin (NAA) to the medium (Nishimura et al., 2009). In the
presence of auxin, Trs85 tagged with IAA is not detected because
it is ubiquitylated and degraded, whereas in the absence of auxin,
TRS85-IAA is stably expressed from its own promoter and is not
degraded (Fig. 2A). Defect in API processing observed in the
absence of TRS85 due to degradation was recovered after
removal of auxin (Fig. 2B). We observed mCherry-Atg8 dot
formation in these conditions, and found that it is severely
defected in the presence of auxin, but recovered after removal of
auxin (Fig. 2C). Consistent with the results of a previous paper
(Lynch-Day et al., 2010), puncta of Atg9-36GFP were dispersed
through the cytoplasm even in the absence of Trs85, a pattern that
was indistinguishable from that observed in the presence of Trs85
(Fig. 2D, left top and bottom panels). When we knocked out
ATG1, a protein kinase required for movement of Atg9 from the
PAS to peripheral pools, Atg9-36GFP accumulated at the PAS,
which was observed as a bright punctate signal (Reggiori et al.,
2004a) (Fig. 2D, right bottom panel). By contrast, in the absence
of Trs85 in atg1 mutants, Atg9-36GFP was dispersed throughout
the cells (Fig. 2D, right top panel). Thus, Atg9 trafficking to the
PAS, and resultant PAS formation, is dependent on TRAPPIII.
Recently, TRAPPIII was reported to be associated with the
vesicle where Atg9 resides (Kakuta et al., 2012). Consistent with
this, subsets of Trs85-36mCherry-positive puncta and Atg936GFP-positive puncta were also double-positive (12.2% and
5.23%, respectively, in over 300 cells) (supplementary material
Fig. S1A). Furthermore, subsets of GFP-Ypt1 positive puncta and
a part of Atg9-36mCherry were also double positive (10.8% and
2.73%, respectively, in over 400 cells) (supplementary material
Fig. S1B). Therefore, at least some Atg9-positive puncta
correlate with the vesicles in which TRAPPIII and/or Ypt1
resides.
In contrast to the situation in nutrient-rich conditions,
TRAPPIII (trs85) and atg1 double-mutant cells did not exhibit
defective Atg9 transport to the PAS under starvation conditions
(Fig. 3A,B). On the basis of this observation, we hypothesized
that under starvation conditions, another pathway bypasses the
TRAPPIII-dependent pathway for transport to the PAS. The Cvt
pathway is a selective autophagic process in which API is
transported to the vacuole and processed to its mature form (Scott
et al., 1996). TRAPPIII (trs85) null mutants did not exhibit
defects in the Cvt pathway under starvation conditions, whereas
they were severely defective under nutrient-rich conditions,
consistent with the existence of a starvation-specific bypass
pathway (Meiling-Wesse et al., 2005; Nazarko et al., 2005).
In an effort to identify the bypass pathway, we first examined
Retromer, a protein complex that functions in retrograde
transport from the late endosomes to the Golgi (Seaman, 2005).
To date, Retromer has not been considered to be crucial for
autophagy (Kametaka et al., 1998). Atg9 trafficking is affected in
several double mutants in endosomal trafficking pathways,
although the precise role of these pathways is still ambiguous
(Ohashi and Munro, 2010). Although mutation in an essential
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TRAPPIII and autophagy
4965
Fig. 2. Atg9 movement depends on TRAPPIII. (A) Conditional expression of Trs85 was detected by immunoblotting for HA. In KNY74 cells, which
express the auxin receptor gene OsTIR1, the C-terminus of genomic TRS85 was tagged with IAA and 36HA, making the protein a target of ubiquitylation in the
presence of auxin (NAA). When NAA was removed from the medium, Trs85-IAA-36HA was expressed at normal levels. (B) Lysates of wild-type, trs85D, Trs85IAA-36HA cells before and after NAA wash-out were prepared by the alkaline lysis method and subjected to SDS-PAGE followed by immunoblotting for
API. Lysate equivalent to 0.2 OD600 units was loaded to each lane. Lane 1, wild-type; lane 2, trs85D; lane 3, Trs85-IAA-36HA (-NAA 2 h); lane 4, Trs85-IAA36HA (200 mM NAA). Upper and lower bands represent pro- and mature-form of API, respectively. (C) KNY74 cells harboring pRS314-26mCherry-Atg8
were incubated in the presence of 200 mM NAA to destabilize the Trs85-IAA-36HA for an overnight in SD containing 0.5% casamino acids (SCD) medium. The
cells were observed at the indicated time points after the NAA wash-out using Leica DIM6000B microscope. 26mCherry-Atg8-positive puncta were counted
(n.1000 cells) and the graph shows the number of cells for each condition (mean 6 s.d.). (D) TRS85-IAA-36HA (KNY74) and TRS85-IAA-36HA atg1D
(KNY76) cells expressing Atg9-36GFP from the native promoter were cultured in YPD in the presence of 200 mM NAA. Images were obtained 0 and 2 hours
after NAA was removed, using a Leica DIM6000B conventional fluorescence microscope.
subunit of Retromer, Vps17, did not affect the distribution of
Atg9-36GFP under nutrient-rich or starvation conditions (not
shown), a combination of vps17 and a TRAPPIII mutation (trs85)
increased the number of Atg9 puncta, even under starvation
conditions (Fig. 3A,B). This observation suggests that a
Retromer-dependent process bypasses the TRAPPIII-dependent
pathway when cells are starved.
On the basis of the possibility that the TRAPPIII-derived
defect is bypassed by a Retromer-dependent pathway, we
hypothesized that TRAPPIII has some unknown connection to
endosome-to-Golgi trafficking. Therefore, we asked whether
other retrograde endosome-to-Golgi transport processes play
bypass roles similar to that of the Retromer-dependent pathway.
Golgi-associated retrograde protein (GARP) is a complex that
functions as a tethering factor in endosome-to-Golgi transport,
and Vps51 is its essential subunit (Bonifacino and Hierro, 2010).
Similar to the case of Retromer, the vps51 trs85 double mutant
exhibited a severe defect in Atg9 trafficking under starvation
conditions in the atg1 background (Fig. 3A,B). In this vps51
trs85 atg1 triple mutant, we further observed that Atg9 failed to
reach the PAS: Atg9-36GFP was scarcely associated with API
puncta, which represent the PAS, in sharp contrast to the
significant overlap between Atg9-36GFP and API puncta in the
atg1 single mutant (Fig. 4). This increase was not due to
elevation of the amount of Atg9-36GFP protein because it was
not changed by these mutations (supplementary material Fig. S2).
Consistent with these results, we observed a marked reduction
in API processing after 1 hour of starvation in the vps17 trs85
double mutant, relative to each single mutant (Fig. 5A–C).
Likewise, the vps51 trs85 double mutant exhibited a severe
defect after 3 hours of starvation (Fig. 5A–C), although mutation
of the GARP subunit Vps51 was previously shown to only
slightly disrupt API processing (Reggiori et al., 2003). By
contrast, combination of the GARP mutation (vps51) with a
Retromer mutation (vps17) did not result in a marked defect even
under starvation conditions (Fig. 5B,C). These observations are
consistent with a model in which Retromer and GARP function
in the same bypass pathway from the late endosome to the Golgi.
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Fig. 3. Retromer- and GARP-dependent bypass pathways for Atg9 trafficking under starvation. (A) Wild-type (KNY93), atg1D (KNY94), atg1D
trs85D (KNY80), atg1D trs85D vps17D (KNY105) and atg1D trs85D vps51D (KNY280) cells expressing Atg9-36GFP were cultured at 26 ˚C in YPD and
subjected to nitrogen starvation for 2 hours. Images were obtained using an Olympus IX71 microscope equipped with a spinning-disk confocal system.
(B) Atg9-36GFP-positive puncta in each strain described in A were counted using G-count software; means 6 s.d. are shown (n.100). Representative results
from two independent experiments are shown.
To further understand the relationship between TRAPPIII and
the other endosomal pathways, we next examined the sorting
nexin complex, which functions in a retrograde pathway that is
distinct from the Retromer-mediated process (Hettema et al.,
2003). Mutation of the sorting nexin subunit Atg24 (also called
Snx4) disrupts API processing under nutrient-rich conditions
(Nice et al., 2002). When the sorting nexin mutation atg24 was
introduced into the TRAPPIII (trs85) mutant, the phenotype was
similar to that of each single mutant, unlike the combinations of
trs85 with mutations in GARP and Retromer subunits (Fig. 5A–
C). By contrast, combination of atg24 with a mutation in a GARP
(vps51) or Retromer subunit (vps17) exacerbated the API
processing defects relative to each single mutant (Fig. 5B,C).
This result indicates that TRAPPIII functions in the same
pathway as sorting nexin, and leads to the novel idea that
TRAPPIII is involved in endosome-to-Golgi retrograde
trafficking.
On the basis of this finding, we asked whether TRAPPIII
function might not be specific to autophagy, but rather associated
with general vesicular traffic. To answer this question, we
assessed the localization of a general cargo, GFP-tagged Snc1, a
v-SNARE protein involved in the fusion of secretory vesicles to
the plasma membrane. Snc1 recycles between the plasma
membrane, endosomes, and the Golgi complex by a
combination of endocytosis, retrograde transport and the
secretory pathway (Lewis et al., 2000). Compared with wildtype cells, the TRAPPIII (trs85) mutant exhibited altered
distribution of GFP-Snc1 (Fig. 6A,B). Furthermore, when the
TRAPPIII (trs85) mutation was combined with a GARP (vps51)
mutation, marked changes in the localization of GFP-Snc1 were
observed (Fig. 6A,B): no GFP signals were detected at the
plasma membrane, and most signals were dispersed throughout
the cytoplasm, implying that the GFP-Snc1 protein was packaged
in small transport vesicles (Fig. 6A,B). Ric1 is a guaninenucleotide exchange factor for Ypt6 that functions in a GARPdependent pathway (Siniossoglou et al., 2000). Consistent with
this, the phenotype of the ric1 trs85 double mutant was quite
similar to that of the vps51 trs85 mutant (supplementary material
Fig. S3A). We also noticed that GARP (vps51) and TRAPPIII
(trs85) double mutant, and ric1 and trs85 double mutant,
exhibited severe growth defects, growing only at lower
temperatures and at a slower overall rate (Fig. 6C and not
shown). Furthermore, combination of the TRAPPIII (trs85) and
Retromer (vps17) mutations resulted in a phenotype that was
milder overall than that of vps51 trs85, but still additive, whereas
the TRAPPIII (trs85) and sorting nexin (atg24) double mutant
did not exhibit an additive growth defect (Fig. 6C). Next, we
investigated another cargo of the endosome-to-Golgi pathway,
Vps10, which is a receptor for the vacuolar protease
carboxypeptidase Y. Vps10 mostly localized to the Golgi and
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TRAPPIII and autophagy
4967
Fig. 4. Atg9 movement to the PAS is severely affected in trs85 vps51 cells. atg1D (KNY310) and atg1D trs85D vps51D (KNY279) cells harboring genomic
Atg9-3xGFP and API-mStrawberry were cultured at 26 ˚C in YPD and shifted to nitrogen starvation for 2 hours. Images were obtained at the indicated time
points using an Olympus IX71 microscope equipped with a spinning-disk confocal system. The graph shows the quantification of Atg9-3xGFP and APImStrawberry colocalization. For each strain, 90–250 API-mStrawberry-positive puncta were examined. Scale bars: 5 mm.
endosome, and exhibited a typical patchy pattern (supplementary
material Fig. S3B) (Shi et al., 2011). However, the pattern of
Vps10 was more dispersed in the trs85 vps51 double mutant,
appearing as numerous small particles, and was also dispersed to
some extent in each single mutant (supplementary material Fig.
S3B). Taken together, these results indicate that TRAPPIII is
involved in general retrograde transport from the early
endosomes to the Golgi.
Because other TRAPP family members, TRAPPI and
TRAPPII, act in tethering step at the destinations of their
respective pathways (Barrowman et al., 2010), we predicted that
TRAPPIII would also function at its destination, the Golgi. To
investigate this idea, we determined whether Trs85-GFP
expressed at endogenous levels would colocalize with the
Golgi marker Sec7-mStrawberry in wild-type cells. In cells
under starvation conditions, 80.9% of Trs85-GFP signals were
also positive for Sec7-mStrawberry (Fig. 7A), whereas only
0.58% of Trs85-GFP signals overlapped with API-mStrawberry,
which represents the PAS in wild-type cells (Fig. 7B); more than
200 cells were counted for each quantification of colocalization.
Together, these results support a role for TRAPPIII in tethering to
the Golgi retrograde vesicles that originate from endosomes.
Accordingly, we investigated the localization of Ypt1 in relation
to the Golgi and Atg8 in wild-type cells (supplementary material
Fig. S4A,B). In a count of more than 60 cells, 77.7% of GFP-Ypt1positive dots were also positive for Sec7-mStrawberry, whereas
only 3.7% of GFP-Ypt1-positive dots were positive for
26mCherry-Atg8. Furthermore, a recent report showed that
Atg11 also acts together with Ypt1 and TRAPPIII in autophagy
(Lipatova et. al, 2012). However, in a count of more than 500 cells,
only 0.95% of GFP-Atg11-positive puncta were also positive for
Sec7-mStrawberry (supplementary material Fig. S4C). Together,
these results suggest that association of TRAPPIII and Ypt1 to
PAS is limited to a small population.
Discussion
In this study, we revealed an important novel feature of
membrane trafficking, namely, a role for TRAPPIII in
endosome-to-Golgi retrograde transport. The identity of the
tethering factor at this step has been a ‘missing piece’ in the study
of membrane traffic. We also reinterpreted the observation that
TRAPPIII-dependent Atg9 movement is crucial for autophagy, in
terms of the nutrient response. On the basis of these findings, we
propose a model (below) describing how general membrane
traffic is involved in the regulation of autophagy.
Combinations of mutations in TRAPPIII and GARP subunits
produced additive defects, whereas combinations of mutations in
TRAPPIII and sorting nexin subunits did not result in more
severe phenotypes. These findings indicate that TRAPPIII
receives vesicles from early endosomes at the Golgi (Fig. 8).
Our results showing that TRAPPIII functions in retrograde
transport from endosomes to the Golgi are consistent with the
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Fig. 5. API processing is disrupted by
combining mutations that affect
TRAPPIII and late endosome-to-Golgi
transport. (A) Summary of genes and
proteins investigated in these experiments.
(B) Each strain (SEY6210; NTY41; KNY92,
KNY125, KNY189, KNY191, KNY151,
KNY216, KNY217, KNY218, KNY222 or
KNY223) was grown at 26 ˚C in YPD,
transferred to medium lacking nitrogen, and
collected at the indicated time points. Cell
lysates were analyzed by immunoblotting
with anti-API antibody. White arrowhead,
pro-API; black arrowhead, mature-API.
(C) Band intensities were calculated for
mature API relative to total API (mature API
+ pro-API) in B. Representative results from
two independent experiments are shown.
previously reported roles of TRAPPI and TRAPPII in ER-toGolgi and intra-Golgi trafficking, respectively. Thus, all TRAPP
family members can now be considered to function at the Golgi.
Furthermore, our results suggest that Ypt1 functions at the Golgi
together with all members of the TRAPP family.
These findings provide important insights into the dynamics of
Atg9 (Fig. 8). Under nutrient-rich conditions, early endosome-toGolgi transport is crucial for the Cvt pathway. Because COGdependent Golgi function and exit from the Golgi are important
for autophagy (van der Vaart et al., 2010; Yen et al., 2010),
shuttling between the Golgi and early endosomes also appears to
be crucial. The essential cargo molecule for autophagy is Atg9,
although additional transmembrane cargo proteins, such as
Atg27, could also participate in this process. Atg9-GFP-positive
puncta in peripheral reservoirs exhibit marked movement
(Sekito et al., 2009), and only a small minority of these
structures (8–12%) colocalize with Golgi and endosomal
markers (Mari et al., 2010). The other labeled structures were
probably a mixture of vesicles providing anterograde and
retrograde transport between the Golgi and endosomes. During
autophagosome formation, some Atg9 will depart from these
pathways and arrive at the PAS. In TRAPPIII mutant cells, Atg9
was trapped in the vesicle and did not reach the PAS. As long as
autophagy proceeds normally, Atg9 is recycled back to the
peripheral pool via the PAS and the autophagosome and/or
vacuoles, but in mutant cells (e.g. atg1), it accumulates
abnormally in the PAS.
Another finding of this study is that the TRAPPIII-dependent
pathway is bypassed under starvation conditions (Fig. 8).
Previously, the existence of this alternative pathway might
have obscured the role of TRAPPIII in this process. Here, we
propose a model in which TRAPPIII and GARP function as two
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Fig. 6. Snc1 trafficking and growth are
disrupted by mutations affecting
TRAPPIII and late endosome-to-Golgi
transport. (A) Strains (KNY201, KNY204,
KNY205 and KNY225) expressing GFPSnc1 were cultured at 26 ˚C in YPD, and
GFP-derived signals were observed using an
Olympus IX71 microscope equipped with a
spinning-disk confocal system. (B) Based
on the distribution of GFP-Snc1 in A, the
cells were categorized into four groups, and
the relative size of each population was
determined (n.200 cells). (1) Diffuse: GFP
signals were diffusely located throughout
the cell. (2) Internal: GFP signals were
observed as internal puncta. (3) Polar: in
addition to internal puncta, GFP signals
were observed on the plasma membrane of a
bud. (4) Nonpolar: in addition to a bud, GFP
signals were observed on the plasma
membrane of the mother cell.
Representative results from two
independent experiments are shown.
(C) Serially diluted cells from each strain
(SEY6210; NTY41; KNY92, KNY125,
KNY189, KNY191, KNY151, KNY217 and
KNY218) were spotted onto YPD plates and
incubated for 3 days at the indicated
temperatures.
independent complexes at the Golgi. Although the vps51 mutant
exhibited defective API processing under nutrient-rich
conditions, this defect was not as pronounced as that of the
trs85 mutant, and autophagic activity was not affected under
starvation conditions. Therefore, the GARP-dependent pathway
must represent an alternative, rather than primary, pathway in
autophagy. However, Snc1 recycling mainly uses the GARPdependent pathway, and in this case the TRAPPIII-dependent
pathway appears to serve as the backup. In general, starvation
alters vesicular trafficking, and the early endosome-to-late
Fig. 7. Trs85 localization at the Golgi complex. (A) Wildtype cells (KNY306) harboring chromosomally integrated
TRS85-GFP and SEC7-mStrawberry were cultured in YPD
and shifted to medium lacking nitrogen for 3 hours. Images
were obtained at the indicated time points using an Olympus
IX71 microscope equipped with a spinning-disk confocal
system. (B) Wild-type cells (KNY305) harboring
chromosomally integrated Trs85-GFP and APImStrawberry were cultured in YPD and shifted to medium
lacking nitrogen for 3 hours. Images were obtained at the
indicated time points using an Olympus IX71 microscope
equipped with a spinning-disk confocal system. Scale bars:
5 mm.
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Journal of Cell Science 126 (21)
proposed in those studies. Future analysis of TRAPP-dependent
Atg9 trafficking in mammalian cells will determine whether our
model is universally applicable.
In preparing for sudden demand for autophagy, pooling the
transmembrane protein Atg9 as a cargo of vesicular transport
may be more favorable than statically storing the protein in a
specific organelle, such as the Golgi. How Atg9 is transferred
from these reservoirs to the PAS is still unclear. Our model and
those proposed in other studies predict that Atg9 is delivered to
the PAS via the Golgi through a canonical mechanism such as the
secretory pathway (Geng et al., 2010; Nair et al., 2011). This
important issue should be addressed in future studies.
Materials and Methods
Growth conditions and media
Journal of Cell Science
Cells were incubated at 30 ˚C unless otherwise indicated in YPD (1% Bacto yeast
extract, 2% Bacto peptone and 2% dextrose) or SD (0.17% yeast nitrogen base
without amino acids or ammonium sulfate, 0.5% ammonium sulfate, 2% dextrose,
and appropriate amino acids) until an OD600 value of 0.8–1.2 was reached. For
starvation experiments, cells were washed in deionized water, transferred to SD-N
(0.17% yeast nitrogen base without amino acids or ammonium sulfate, 2%
dextrose), and incubated at the indicated temperature. For auxin-based degradation
of Trs85, cells were grown overnight in YPD containing 200 mM 1naphthaleneacetic acid (NAA; Sigma N0640) and washed twice with fresh YPD
to remove the NAA. The cells were then resuspended in YPD at an OD600 value of
0.5, and incubated at 30 ˚C for the indicated times.
Fig. 8. Role of TRAPPIII in membrane trafficking and autophagy. Model
of membrane trafficking and Atg9 dynamics. TRAPPIII functions at the Golgi
in the same pathway as sorting nexin, but in a different pathway to GARP and
Retromer. Under nutrient-rich conditions, TRAPPIII plays a central role in
Atg9 movement. This pathway and Golgi-to-endosome anterograde transport
constitute a shuttling pathway. Under starvation conditions, the GARPdependent pathway acts as a bypass route. These pathways provide a reservoir
of Atg9, and some Atg9 is relocalized at the PAS, where it contributes to
autophagy. Solid lines represent pathways experimentally examined in this
study, and dashed lines reflect other published results.
endosome pathway is enhanced by starvation, possibly due to the
need for increased degradation in the vacuole (Hamasaki et al.,
2005; Jones et al., 2012). As a result, some Atg9 in early
endosomes will be relocated to late endosomes, and the late
endosome-to-Golgi pathway becomes another reservoir of Atg9.
Our model also explains why Tlg2, a t-SNARE localized on the
Golgi and early endosomes, is required only for the Cvt pathway
(Abeliovich et al., 1999). When mutations affecting vesicles at
their origin (Retromer, sorting nexin) and destination (TRAPPIII,
GARP) were combined, the phenotypes were milder (see
Fig. 5B,C; vps51 trs85 and atg24 vps51 double mutants) than
that of the trs85 vps51 double mutant. This implies that the
apparatuses at the origin and destination do not need to strictly
match. Also of note, even in the context of the strongest
phenotype (i.e. that of the trs85 vps51 mutant), some limited API
processing still occurred. This might reflect the existence of
another bypass pathway, such as the Fab1-dependent retrograde
pathway (Efe et al., 2007). Alternatively, even without the
reservoir sources, Atg9 expressed during starvation could support
a small amount of autophagic activity.
Our model might appear to conflict with previous reports that
TRAPPIII functions at the PAS in yeast and in forming
autophagosome in mammalian cells (Lynch-Day et al., 2010;
Kakuta et al., 2012; Lipatova et al., 2012; Huang et al., 2011;
Wang et al., 2013). Our model does not necessarily exclude these
possibilities, but our results suggest that the direct contribution of
TRAPPIII to autophagosome formation is not as large as
Genetic procedures
Yeast strains used in this study are listed in Table 1. PCR was used to create and
confirm all gene disruptions, tagged constructs and other modifications (Goldstein
et al., 1999; Janke et al., 2004; Longtine et al., 1998; Nakatogawa et al., 2012). The
PHO8 locus in the knockout collection and the DAmP haploid collection (Open Bio
System) (Schuldiner et al., 2005) was replaced with pho8D60 using a synthetic
genetic array method (Tong and Boone, 2007). Briefly, the TNY509 parental strain
harboring pho8D60 (Noda and Klionsky, 2008; Tong and Boone, 2007) was crossed
with each mutant strain on YPD in a rectangular plate (OmniTray, Nunc) with a 96pinned replicator (VP408, V&P Scientific). Haploid cells containing the pho8D60
allele and each mutant allele were selected using auxotrophic and drug-resistance
markers and sequential replica plating (Tong and Boone, 2007). The mStrawberry
sequence was PCR-amplified from pmStrawberry (Shu et al., 2006) and used to
replace the yeGFP sequence of pYM25 to generate pKN14; the resulting plasmid
was used for C-terminal tagging with mStrawberry. A partially digested PvuII
fragment of pGFP-Snc1 (306) (Lewis et al., 2000) was subcloned into pRS305
digested with PvuII to generate pKN24. The resulting plasmid was linearized with
EcoRV and integrated at the chromosomal LEU2 locus. A BamHI-SacI fragment
encoding 36GFP from the pAtg9-36GFP (306) plasmid (Geng et al., 2010) was
subcloned into pRS303 digested with the same enzymes to generate pKN15
(pRS303-36GFP). Sequence encoding the C-terminal region of ATG9 was PCR
amplified using primers 59-CGGGATCCCCTCTTCCGACGTCAGAC-39 and 59CCGCTCGAGGCACCATTTCTGGTCACATAC-39. The amplified fragment was
digested with BamHI and XhoI, and then subcloned into pKN15 digested with the
same enzymes to generate pKN17 (pRS303-Atg9-36GFP). pKN17 was linearized
with BglII and integrated at the ATG9 locus. pNHK53 and pMK43 (National
BioResource Project, Japan) harbored OsTIR1 and AtIAA17, respectively
(Nishimura et al., 2009). pNHK53 was cut with StuI and integrated at the URA3
locus of a wild-type strain. AtIAA17 in pMK43 was PCR-amplified, cut with HindIII
and XhoI, and subcloned into pYM24 digested with HindIII and SalI to add 36HA to
the C terminus of IAA17. The resultant plasmid, pKN19, was used as the template
for C-AID tagging of TRS85. pRS314-26mCherry-Atg8 was a gift from the Ohsumi
lab. pKN38 to express GFP-Ypt1 under the ADH1 promoter was constructed as
follows. The YPT1 gene was amplified from the wild-type genomic DNA by PCR
with the primer pair of 59-GGG GAT CCA ATA TGA ATA GCG AGT ACG-39GG
GA59GG GAT CCA ATA TGA ATA GCG AGT ACG-3G, and digested with
BamHI and XhoI. The resulting fragment was introduced into pBP73-A digested
with the same enzymes to generate pKN37, which has a GFP-YPT1 fusion gene
under the ADH1 promoter. The plasmid was then digested with SacI and XhoI and
the fragment containing GFP-YPT1 with the ADH-promoter was subcloned into
pRS304 digested with the same enzymes. The resulting plasmid pKN38 was
linearized at the unique EcoRI site within the YPT1 gene and integrated at the YPT1
locus of a wild-type strain harboring chromosomally tagged Sec7-mStrawberry.
Large-scale ALP assay
YPD plates were inoculated with cells from each pool using a 96-pin replicator and
incubated for 16–24 hours at 30 ˚C. Subsequently, the cells were suspended in
TRAPPIII and autophagy
4971
Table 1. Strains used in this study
Strain
Journal of Cell Science
Y3656
TNY509
SKY001
SEY6210
SEY6210.1
NTY41
KNY67
KNY74
KNY76
KNY93
KNY94
KNY95
KNY80
KNY103
KNY104
KNY105
KNY279
KNY92
KNY125
KNY189
KNY191
KNY151
KNY217
KNY218
KNY216
KNY222
KNY223
KNY201
KNY204
KNY205
KNY225
KNY305
KNY306
KNY310
KNY345
KNY351
KNY352
KNY353
KNY354
KNY355
KNY356
KNY357
KNY358
KNY359
KNY360
KNY361
Genotype
Reference
MATa can1D::MFA1pr-HIS3-MFa1pr-LEU2 ura3D leu2D his3D met15D lys2D
Y3656; pho8::pho8D60: natNT2
TNY509 pep4D::kanMX6
MATa ura3-52 leu2-3,112 his3D200 trp1-D901 lys2-801 suc2-D9
MATa ura3-52 leu2-3,112 his3D200 trp1-D901 lys2-801 suc2-D9
SEY6210; atg1D::kanMX6
SEY6210; OsTIR1-9xmyc:URA3 atg9::ATG9-3xGFP:HIS3
KNY67; trs85::TRS85-AtIAA17-36HA:hphNT1
KNY67; atg1D::kanMX6 trs85::TRS85-AtIAA17-36HA:hphNT1
SEY6210; atg9::ATG9-36GFP:HIS3
SEY6210; atg1D::kanMX6 atg9::ATG9-36GFP:HIS3
SEY6210; trs85D::URA3MX atg9::ATG9-36GFP:HIS3
SEY6210; atg1D::kanMX6MX6 trs85D:: CaURA3 atg9::ATG9-36GFP:HIS3
SEY6210; vps17D::TRP1 atg9::ATG9-36GFP:HIS3
SEY6210; trs85D::CaURA3 vps17D::TRP1 atg9::ATG9-36GFP:HIS3
SEY6210; atg1D::kanMX6 trs85D::CaURA3 vps17D::TRP1 atg9::ATG9-36GFP:HIS3
SEY6210; atg1D::kanMX6 trs85D::CaURA3 vps51D::natNT2 atg9::ATG9-36GFP:HIS3
ape1::APE1-mStrawberry:hphNT1
SEY6210; trs85D::URA3MX
SEY6210; vps17D::TRP1
SEY6210; atg24D::zeoNT3
SEY6210; vps51D::natNT2
SEY6210; trs85D::URA3MX vps17D::TRP1
SEY6210; atg24D::zeoNT3 trs85D::URA3MX
SEY6210; trs85D::URA3MX vps51D::natNT2
SEY6210; atg24D::zeoNT3 vps51D::natNT2
SEY6210; vps17D::TRP1 vps51D::natNT2
SEY6210; atg24D::zeoNT3 vps17D::TRP1
SEY6210; leu2::GFP-SNC1:LEU2
SEY6210; vps51D::natNT2 leu2::GFP-SNC1:LEU2
SEY6210; trs85D::CaURA3 leu2::GFP-SNC1:LEU2
SEY6210; trs85D::CaURA3 vps51D::natNT2 leu2::GFP-SNC1:LEU2
SEY6210; trs85::TRS85-GFP:HIS3MX6 sec7::SEC7-mStrawberry:hphNT1
SEY6210; trs85::TRS85-GFP:HIS3MX6 ape1::APE1-mStrawberry:hphNT1
SEY6210; atg1D::kanMX6 atg9::ATG9-3xGFP:HIS3 ape1::APE1-mStrawberry:hphNT1
SEY6210; atg9::ATG9-3xGFP:HIS3 sec7::SEC7-mStrawberry:hphNT1
SEY6210; ric1::kanMX6 snc1::GFP-SNC1:LEU2
SEY6210; ric1::kanMX6 trs85::CaURA3 snc1::GFP-SNC1:LEU2
SEY6210; vps10::VPS10-GFP:His3MX6
SEY6210; trs85::CaURA3 vps10::VPS10-GFP:His3MX6
SEY6210; vps51::natNT2 vps10::VPS10-GFP:His3MX6
SEY6210; trs85::CaURA3 vps51::natNTS vps10::VPS10-GFP:His3MX6
SEY6210; atg9::ATG9-3xmCherry:hphNT1 ypt1::PADH-GFP-YPT1:TRP1
SEY6210; atg9::ATG9-3xGFP:HisMX6 trs85::TRS85-3xmCherry:hphNT1
SEY6210; sec7::SEC7-mStrawberry:hphNT1 ypt1::PADH-GFP-YPT1:TRP1
SEY6210; ypt1::PADH-GFP-YPT1:TRP1 pRS314-2xmCherry-Atg8
SEY6210; atg11::PADH-GFP-Atg11:natNT2 sec7::SEC7-mStrawberry:hphNT1
200 ml of SD-N medium in 96-well plates and incubated for 4 hours at 30 ˚C. The
plates were centrifuged, and the supernatant was discarded. To each well was
added 50 ml of ice-cold lysis buffer (10 mM Tris-HCl at pH 9.0, 10 mM MgSO4
and 10 mM ZnSO4,) and ,10 ml of 0.6 mm zirconia/silica beads (Biomedical
Science). The plates were sealed with parafilm and mixed vigorously on a rotary
shaker at 2500 rpm for 10 minutes at 4 ˚C. After a brief centrifugation, 150 ml of
ice-cold lysis buffer was added, and the plates were centrifuged for 15 minutes at
490 g at 4 ˚C. Protein levels were determined from 50 ml aliquots of the
supernatants using the bicinchoninic acid method, and enzymatic activity was
measured in 50 ml aliquots as described in a previous report (Noda and Klionsky,
2008) with slight modifications.
Microscopy
Cells were observed Leica AF6500 fluorescent imaging system mounted on a
DIM6000 B microscope (HCX PL APO 636 /1.40–0.60 oil-immersion objective
lens, mercury lamp) under the control of LAS-AF software (Leica
Microsystems) or on a Yokogawa CSU-X spinning-disk confocal system
(Yokogawa Electric Corp., Japan) mounted on an Olympus IX71 microscope
(1006 NA 1.4 PlanApo objective lens) equipped with an Andor iXon CCD
camera (Andor Technology, UK) under the control of the Andor IQ software.
Images were processed using Adobe Photoshop. Atg9 puncta were counted using
the G-Count software (G-Angstrom, Japan). Leica TCS SP8 confocal system
mounted on a DMI 6000 CS microscope (HCX PL APO 1006 /1.4 Oil STED
(Tong and Boone, 2007)
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objective lens, Leica Microsystems) was used to observe the cells in
supplementary material Fig. S2.
Protein extraction and immunoblotting
Yeast cells were harvested, suspended in 100 ml of 0.2 M NaOH and 1% (v/v) 2mercaptoethanol, and incubated on ice for 10 minutes. One milliliter of chilled
acetone was added to the suspension, and the samples were incubated for
10 minutes on ice. After centrifugation at 16,000 g for 10 minutes, cell pellets
were washed once with chilled acetone and dissolved in Laemmli sample buffer
using a water-bath sonicator and boiled for 5 minutes. Aliquots (0.2 OD600 units)
were resolved using SDS-PAGE, and proteins were detected with the appropriate
antibodies [anti-aminopeptidase I (Noda et al., 2000), anti-HA (12CA5), anti-Atg9
(a gift from the Ohsumi lab)]. Band intensities were measured using ImageJ
software. Lysates to detect Atg9 were prepared by glass bead lysis. Cells were
suspended in lysis buffer (20 mM PIPES, pH 6.8, 200 mM sorbitol, 5 mM EDTA)
containing Complete protease inhibitor cocktail (Roche) and 4 mM PMSF, and
disrupted by six rounds of vortexing for 30 seconds with 30 second intervals on
ice. Triton X-100 was added to the lysate at the final concentration of 0.5%. The
lysates were incubated on ice for 5 minutes and centrifuged at 5000 g for
10 minutes at 4 ˚C. The supernatants were incubated at 65 ˚C for 10 minutes in
Laemmli sample buffer before performing SDS-PAGE followed by
immunoblotting with anti-Atg9 (a gift from the Ohsumi lab) and anti-PGK (A6457, Molecular Probes).
4972
Journal of Cell Science 126 (21)
Acknowledgements
The authors would like to thank Dr Daniel J. Klionsky (University of
Michigan), Dr Scott D. Emr (Cornell University), Dr Charles Boone
(University of Toronto), Dr Yoshinori Ohsumi, Dr Hayashi
Yamamoto (Tokyo Institute of Technology), and Dr Roger Tsien
(University of California, San Diego) for various strains and
plasmids; the National BioResource Project Yeast (Japan) for
plasmids; and Mr Yuuma Ito for technical assistance.
Author contributions
K.S.N. did most of the experiments. S.K. established the novel
autophagy screening method and performed the genome-wide
screening. T.Y. contributed valuable discussion. T.N. designed the
study and wrote the manuscript.
Funding
This work was supported in part by the Special Coordination Funds
for Promoting Science and Technology from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of
Japan; and by a grant from the Cell Science Research Foundation.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.131318/-/DC1
Journal of Cell Science
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