COMMENTARY
New insights into autophagosome– lysosome fusion
Shuhei Nakamura
1,2
and Tamotsu Yoshimori
1,2,
*
ABSTRACT
Macroautophagy (autophagy) is a highly conserved intracellular
degradation system that is essential for homeostasis in eukaryotic
cells. Due to the wide variety of the cytoplasmic targets of autophagy,
its dysregulation is associated with many diseases in humans, such
as neurodegenerative diseases, heart disease and cancer. During
autophagy, cytoplasmic materials are sequestered by the
autophagosome – a double-membraned structure – and transported
to the lysosome for digestion. The specific stages of autophagy are
induction, formation of the isolation membrane (phagophore),
formation and maturation of the autophagosome and, finally, fusion
with a late endosome or lysosome. Although there are significant
insights into each of these steps, the mechanisms of autophagosome–
lysosome fusion are least understood, although there have been
several recent advances. In this Commentary, we will summarize the
current knowledge regarding autophagosome–lysosome fusion,
focusing on mammals, and discuss the remaining questions and
future directions of the field.
KEY WORDS: Phosphoinositides, Autophagy, Fusion, Lysosome
Introduction
Macroautophagy, hereafter referred to as autophagy, is a catabolic
process that targets a wide var iety of cellu lar components including
proteins, lipids, damaged organelles a nd pathogens. Autophagy
nor mally occurs at a basal level, but it is accelerated by a
variety of stresses such as starvation, accumu lation of abnormal
proteins, organelle damage and pathogen infection. Autophagy
was origin ally consid ered to be a bulk, non -selec tive degradation
system; however, it is now known that autophagy selectively
degrades tar gets and contributes to intracellular h ome ostasis
(Kawabata and Yoshimori, 2016). During autophagy, a small
cisterna, called the isolation membrane (phagophore), elongates
and surrounds a part of the cytop lasm to form a double-membraned
structure, called the autophagosome. Autophagosomes either
fuse with late endosomes to form amphisomes, which then fuse
with lysosomes, or they fuse directly wit h lysosomes ( Berg et al.,
1998; Fader et al., 2008). After fusion with the lysosome, they are
called autolysosomes and the sequestered contents are d igested
(Fig. 1 ).
Since the identification of autophagy-related genes (ATGs) in
yeast (Tsukada and Ohsumi, 1993), the functions of their homologs
have been identified and extensively studied, especially in mice and
mammalian cells (Mizushima et al., 2011). Briefly, activation of the
unc-51-like kinase 1 (ULK1; Atg1 in yeast) complex is crucial for
the initiation of autophagy. Then, activation of the class III
phosphatidylinositol 3-kinase complex, which comprises PI3K
(Vps34 in yeast), beclin 1, VPS15 (PIK3R4) and ATG14L
(ATG14), triggers vesicle nucleation. The subsequent elongation
and closure of the isolation membrane are mediated by two ubiquitin-
like ATG conjugation pathways, ATG5-ATG12 and LC3/Atg8. In
mammals, there are seven Atg8 orthologues; MAP1LC3A/B/C,
GABARAP and GABARAPL1/2/3 (all of which are hereafter
referred to as LC3). LC3 is widely used as a marker for the
microscopic detection of isolation membranes and autophagosomes.
After synthesis, LC3 is processed at its C terminus by Atg4 and
becomes LC3-I, which has a glycine residue at the C-terminal end.
LC3-I is subsequently conjugated with phosphatidylethanolamine
(PE) to become LC3-II by a ubiquitination-like enzymatic reaction. In
contrast to the cytoplasmic localization of LC3-I, LC3-II associates
with both the outer and inner membranes of the autophagosome.
PE-conjugated LC3 (LC3-II) and unconjugated LC3 (LC3-I) can be
detected separately by immunoblot analysis, and the amount of
LC3-II is also widely used for the quantification of autophagic
activity (Kabeya et al., 2000).
Although the extensive characterization of ATG genes has
yielded insights into the mechanisms of autophagy activa tion and
autophagosome formation, how the fusion of autophagosomes with
endosomes and/or ly sosomes is contr o lled remains poorly unders tood.
Nevertheless, recent studies hav e started to uncover the molecular
mechanisms that regulate the fusion steps. Several experimental
approa ches have contributed to identifying the conditions that
are necessary for the autophagosome–lysosome fusion step to
occur. For instance, the V-ATPase inhibitor bafilomycin A1
(BafA1), a macrolide antibiotic derived from Streptomyces
griseus, which blocks degradation in autolysosomes and/or
autophagosome–lysosome fusion (Klionsky et al., 2008;
Yamamoto et al., 1998; Yoshimori et al., 1991), and LC3 tandem
tagged with RFP and GFP (RFP-GFP–LC3), which loses its GFP
fluorescence after fusion with the lysosome, have facilitated to
detect the blockage of autophagosomal fusion (Kimura et al., 2007).
In this Commentary, we survey recent findings with regard to the
molecular mechanisms underlying the autophagosome–lysosome
fusion step, with a focus on mammalian studies, and also discuss
future perspecti ves for the field.
Completion of autophagosomes
The timing of autophagosome–lysosome fusion is very important
and only the closed autophagosomes can fuse with lysosomes. This
raises the question how the closure of autophagosomes is regulated.
In mammals, a defect in the ATG-conjugation system results in
accumulation of unclosed autophagosomes (Fujita et al., 2008a;
Kishi-Itakura et al., 2014; Mizushima et al., 2001; Sou et al., 2008),
implying that it is likely to function in elongation and closure of
autophagosomes, and is important for transition of the isolation
membrane into the autophagosome. In addition to its role in
autophagosome maturation, the ATG conjugation system
(consisting of ATG3, ATG5 and ATG7) has been recently shown
1
Department of Genetics, Graduate School of Medicine, Osaka University,
565-0871 Osaka, Japan.
2
Laboratory of Intracellular Membrane Dynamics,
Graduate School of Frontier Biosciences, 565-0871 Osaka University, Osaka,
Japan.
*Author for correspondence (tamyoshi@fbs.osaka-u.ac.jp)
T.Y., 0000-0001-9787-3788
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© 2017. Published by The Company of Biologists Ltd
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Journal of Cell Science (2017) 130, 1209-1216 doi:10.1242/jcs.196352
Journal of Cell Science
to be required for efficient degradation of the inner autophagic
membrane; however, it is not required for autophagosome–
lysosome fusion, although the rate of autophagosome formation is
reduced to ∼30% in ATG conjugation-deficient cells (Tsuboyama
et al., 2016). By contrast, another study that was using cell lines in
which the entire ATG8 protein family had been knocked out
revealed that LC3 and GABARAP proteins are not required for
autophagosome formation, but are crucial for autophagosome–
lysosome fusion (Nguyen et al., 2016). The lack of fusion is
probably due to the impaired recruitment of the adaptor protein
PLEKHM1 (McEwan et al., 2015) (see also below) to
autophagosomes. The inconsistency between these two studies
might reflect the non-overlapping function of the ATG conjugation
system and LC3 and GABARAP. Why only the closed
autophagosomes are recognized by several fusion factors
(discussed below) is currently unclear.
Movement of autophagosomes and lysosomes
The cytoskeleton has many functions, including the structural
maintenance of cells, cell division and movement. Although
microtubules are dispensable for autophagy in yeast (Kirisako
et al., 1999), they are essential for the fusion step in mammals
(Aplin et al., 1992; Kochl et al., 2006; Monastyrska et al., 2009).
Autophagosomes are thought to form randomly throughout the
cytoplasm, whereas late endosomes and lysosomes are
predominantly found in the perinuclear region. Therefore, once
complete and closed autophagosomes have been generated, they
need to be delivered to the perinuclear region. The minus-end-
directed dynein−dynactin motor complex moves cargo to the
perinuclear region, whereas most kinesins are plus-end-directed
motor proteins that drive their cargo towards the cell periphery
(Gross et al., 2007). Given that lysosomes localize to the perinuclear
regions, the minus-end-directed transport of autophagosomes
appears reasonable and, indeed, live-imaging shows that mature
autophagosomes move along microtubule tracks towards the
lysosomes (Kimura et al., 2008). The efficient movement of
autophagosomes is inhibited by microinjection of antibodies against
LC3, suggesting a role for LC3 during this process (Kimura et al.,
2008). In addition, kinesin-dependent plus-end-directed transport is
essential for the correct positioning of autophagosomes because the
depletion of the kinesin KIF5B blocks autophagy and results in
perinuclear clustering of autophagosomes (Cardoso et al., 2009).
Similarly to mammals, in Drosophila, the PX-domain-containing
kinesin Klp98A controls the formation, fusion and intracellular
positioning of autophagic vesicles (Mauvezin et al., 2016).
Interestingly, the localization of lysosomes determines the rate of
autophagosomal fusion. Increasing the perinuclear localization of
lysosomes by depletion of kinesins KIF1B-β and KIF2A leads to
increased autophagosomal fusion, whereas disper sion of lysosomes
to the periphery by overexpressing the motors reduces fusion rates
(Korolchuk et al., 2011). Thus, the coordinated transport of both
autophagosomes and lysosomes is essential for fusion; but how are
autophagosomes and lysosomes connected to microtubules? The
small GTPase Rab7, which acts as a molecular switch and,
presumably, is recruited to late autophagosomes ( Gutierrez et al.,
2004), links autophagosomes to microtubule motors through
FYCO1 (FYVE and coiled-coil domain-containing 1), thereby
mediating kinesin-driven movement towards the cell periphery
(see below) (Pankiv et al., 2010). Rab7 also works in the reverse
direction by interacting with Rab-interacting lysosomal protein
(RILP), the cholesterol sensor ORP1L (also known as OSBPL1A)
and dynein, in order to facilitate transport of autophagosomes,
autolysosomes and lysosomes to the perinuclear region (Jordens
et al., 2001; Wijdeven et al., 2016) (Fig. 2).
Similarly to microtubules, actin filaments form tracks to move
various intracellular cargos by using the myosin family of motor
proteins. Several pieces of evidence suggest that actin is involved in
autophagosome–lysosome fusion. For instance, histone deacetylase
6 (HDAC6) recruits the cortactin-dependent actin remodeling
machinery, which in turn assembles the actin network that
stimulates autophagosome–lysosome fusion (Lee et al., 2010).
Interestingly, HDAC6 and actin assembly are dispensable for
Lysosome
Amphisome
Autolysosome
Late endosome
Autophagosome
Isolation membrane
Degradation
Fusion
Elongation
Nucleation
Recycling of molecules
Maturation
Fig. 1. Overview of autophagy. Upon induction of
autophagy by stress, cytoplasmic materials are
sequestered by a double-membraned structure, called
autophagosome. These autophagosomes fuse with
late endosomes (termed amphisomes) or lysosomes to
become autolysosomes, in which the sequestered
cargos are degraded and recycled for the maintenance
of cellular homeostasis. Autophagy can be divided into
several steps: formation of the isolation membrane
(nucleation), elongation of the isolation membrane
(elongation), completion and transport of the
autophagosome (maturation), docking and fusion
between autophagosome and lysosome (fusion), and
degradation of the cargo s inside the autolysosome
(degradation).
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Journal of Cell Science
starvation-induced autoph agy, but are required for the selective
degradation of aggregated proteins (Lee et al., 2010). Moreover, the
actin motor myosin VI and Tom1, a component of the ESCRT
machinery and binding partner of myosin V1 on endosomes, are
involved in fusion, as the loss of both factors reduces
autophagosomal delivery of endocytic cargo and blocks
autophagosome–lysosome fusion (Tumbarello et al., 2012).
Fusion of autophagosome and lysosome – Rabs, SNAREs and
tethering factors
After autophagosomes arrive at their destination, they fuse with the
endocytic system. However, it is difficult to distinguish the movement
from the actual fusion, most likely because these two processes seem
to occur almost simultaneously. Currently, our knowledge of the
machinery involved in this process is based on the general
understanding of intracellular membrane trafficking, particularly
with regard to three sets of protein families: Rab GTPases,
membrane-tethering complexes and soluble N-ethylmaleimide-
sensitive factor attachment protein receptors (SNAREs). Rab
proteins localize to specific membranes and recruit tethering
complexes that act as bridges to bring the compartments intended
for fusion together. These tethering complexes, in turn, help SNARE
proteins to physically drive the fusion of opposing lipid bilayers.
Rab proteins
The small GTPases of the Ras-related protein in brain (Rab) family
are evolutionally conserved, crucial regulators of membrane
trafficking in eukaryotic cells. They recruit specific effector
proteins, such as cargo adaptors to form transport vesicles, motor
proteins to move the vesicle to its target membrane, as well as
tethering proteins to aid the fusion machinery when the vesicular
cargo reaches its destination (Stenmark, 2009; Zhen and Stenmark,
2015). Each Rab protein localizes to a distinct membrane
compartment and, by doing so, Rabs are thought to provide
specificity to membrane trafficking. Membrane-associated Rabs are
activated by specific guanine nucleotide exchange factors (GEFs)
that drive GTP binding. Upon binding GTP, Rabs conformationally
change to interact with their effector proteins. Subsequently, Rabs
are inactivated by specific GTPase-activating proteins (GAPs) that
hydrolyze the bound GTP to GDP, causing loss of effector binding
and extraction from the membranes.
It has been suggested that some members of the Rab family
regulate autophagy. Rab7, which is localized on late endosomes and
lysosomes, and is essential for subsequent endocytic membrane
trafficking from late endosome to lysosome, is also important for
autophagosome–lysosome fusion and the subsequent degradation
of autophagosomal contents (Gutierrez et al., 2004; Jager et al.,
2004; Kirisako et al., 1999). Rab7 might be also recruited to late
autophagosomes. Gutierrez et al. showed that, upon induction of
autophagy, an increase in the labeling intensity of Rab7 is observed
on the autophagic vacuole (notice that the term autophagic vacuole
refers to nascent autophagosomes and autophagosomes that have
fused with late endosomes and lysosomes) because Rab7 staining
on late autoph agic vacuole is stronger than that on early autophagic
vacuoles (based on by immunofluorescence microscopy and
immunoelectron microscopy) (Gutierrez et al., 2004). The group
also claimed that Rab7 delivery to autophagosomes is detected
before fusion with a LAMP-1-positive compartment. Knockdown
of Rab7 causes accumulation of late autophagic vacuoles, indicating
that Rab7 function is only needed for the final maturation of late
autophagic vacuoles, probably the fusion with lysosomes (Gutierrez
et al., 2004; Jager et al., 2004). Interestingly, Rab7 is involved in
the formation of GAS-containing autophagosome-like structures
(GcAVs) that sequester invading group A streptococcus,
(Yamaguchi et al., 2009). Rab7 also functions in isolation
membrane expansion during mitophagy (Yamano et al., 2014).
These results indicate that Rab7 functions during the early phase of
selective types of autophagy. Owing to such multiple and
overlapping roles, it has proven difficult to determine the specific
role of Rabs during autophagosome–lysosome fusion.
Nevertheless, thapsigargin, an ER stressor widely used to induce
autophagy, blocks the recruitment of Rab7 to mature
autophagosomes and inhibits their fusion with endocytic vesicles
without affecting endocytosis, highlighting the vital role of Rab7 in
the fusion step (Ganley et al., 2011).
In mammals, a GEF that activates Rab7 for fusion has not been
identified. In Drosophila fat cells, the guanosine exchange complex
of Ccz1 and Mon1 (Ccz1−Mon1) recruits Rab7 to PI3P-positive
autophagosomes (Hegedus et al., 2016), and loss of the Ccz1–
Mon1–Rab7 complex impairs autophagosome–lysosome fusion.
Here, Rab5 recruits Ccz1−Mon1 to endosomes in order to activate
Rab7, which facilitates endosome maturation and fusion with
the lysosome. However, Rab5-null mutants exhibit normal
autophagosome–lysosome fusion, and Rab5 is dispensable for
Ccz1–Mon1-dependent recruitment of Rab7 (Hegedus et al., 2016).
Two compo nents of the PI3K complex, UV radiation resistance-
associated (UVRAG) and Rubicon (RUBCN) are involved in
endocytic transport, autophagosome maturation and/or
autophagosome–lysosome fusion through Rab7 (Liang et al.,
2008; Matsunaga et al., 2009; Tabata et al., 2010; Zhong et al.,
2009), although they have opposite effects. UVRAG promotes
autophagosome–lysosome fusion, whereas Rubicon inhibits it.
UVRAG, which localizes to the endoplasmic reticulum and
endosomes, binds to VPS16, a subunit of the homotypic fusion
and protein sorting (HOPS) complex (Liang et al., 2008) to
stimulate Rab7 GTPase activity and autophagosome–lysosome
fusion, whereas Rubicon binds to UVRAG and negatively regulates
Microtubules
–+
Perinuclea
r
Peripheral
Lysosome
Late autophagosome
FYCO1
Dynein
Dynactin
RILP
Kinesin
Rab7
ORP1L
Rab7
LC3-II
PI3P
Key
Fig. 2. Transport of autophagosomes. Rab7 GTPase links
autophagosomes to a microtubule motor through FYCO1 to facilitate kinesin-
driven movement towards the cell periphery. FYCO1 has been identified to
bind to LC3, but it also binds to the phospholipid PI3P, a component of the
autophagosome membrane. Rab7 also binds to RILP and ORP1L in order to
mediate dynein and/or dynactin-driven movement towards the perinuclear
region under normal cholesterol conditions. When levels of cholesterol are low,
ORP1L forms a contact site with VAP-A, which prevents dynactin recruitment
and blocks minus-end transport (not shown in this figure). Lysosomal
positioning also determines the rate of autophagosome–lysosome fusion.
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VPS34 activity (Sun et al., 2011). Under nutrient-rich conditions,
UVRAG is phosphorylated by mechanistic target of rapamcycin
complex 1 (mTORC1) (Kim et al., 2015), which enhances the
interaction with Rubicon and impairs VPS34 kinase activity, as well
as the interaction between UVRAG and the HOPS complex, thus
affecting autophagosome maturation. Prevention of UVRAG
phosphorylation increases the rate of autophagosome maturation
and lysosomal degradation, indicating that mTORC1 not only
regulates the induction of autophagy but also facilitates fusion
through UVRAG. However, the function of UVRAG during fusion
is controversial because another study found that UVRAG neither
interacts with HOPS nor regulates autophagosome–lysosome
fusion (Jiang et al., 2014). Similarly, UVRAG is dispensable for
autophagosome–lysosome fusion in Drosophila (Takats et al.,
2014). Further studies are needed to clarify the function of UVRAG
during the fusion.
An active, GTP-bound Rab protein binds to various effectors that
usually regulate vesicle motility and fusion with the correct membrane
compartment. Re cent findings suggest that PLEKHM1, which has
originally been identified as a Rubicon homolog, functions as a Rab7
effector and is involv e d in autophagosome–ly sosome fusion through
Rab7, the HOPS comple x, and LC3 and/or GABARAP (McEw an
et al., 2015; T aba ta et al., 2010) (Fig. 3). Sever al other Rab7 effectors
ha v e been charac terized, such as RILP and FYC O1, both of which
function in autophagosome tr ansport (see abov e and belo w).
In addition to Rab7, Rab33b is known to regulate the fusion step.
Rab33b is a Rab protein localized at the Golgi complex that plays a
role in autophagosome formation through interaction with ATG16
(Itoh et al., 2011). Its GAP ornithine aminotransferase-like 1 (OATL1,
also known as TBC1D25) is recruited onto autophagosomes through
direct interaction with ATG8, and OATL1 overexpression has been
shown to inhibit autophagosome–lysosome fusion (Itoh et al., 2011).
It has been recently shown that during T-tubule remodeling in
Drosophila, Rab2 localizes to completed autophagosomes and
interacts with the HOPS complex to promote autophagosome–
lysosome fusion (Fujita et al., 2017). These results suggest that,
although Rab7 plays a pivotal role during autophagosome–lysosome
fusion, further small GTPases are involved in this process and other –
so-far unappreciated – functions of members of this protein family
might be discovered in future.
SNAREs
More than 60 SNAREs determine membrane fusion specificity and
drive the fusion processes of mammalian cells. Functionally,
SNAREs are grouped into v-SNAREs on donor vesicles and t-
SNAREs on target vesicles. Structurally, SNAREs are divided into
Q-SNAREs (which have a Q amino acid residue) and R-SNAREs
(which have an R amino acid residue). Q-SNAREs are further
divided into Qa-, Qb- and Qc-SNAREs based on the amino acid
sequence of the SNARE domain. These SNAREs form a parallel
four-helix bundle composed of Qa-, Qb-, Qc- and R-SNAREs to
bridge the two fusing membranes.
Upon starvation in mammals, the Qa-SNARE syntaxin 17
(STX17) is recruited, presumably from the cytosol to closed
autophagosomes, and mediates autophagosome–lysosome fusi on
by binding to its partners, the Qbc-SNARE SNAP29 and the
lysosomal R-SNARE VAMP8 (Itakura et al., 2012) (Fig. 3).
Accordingly, depletion of STX17 causes accumulation of
autophagosomes. Similar machineries and mechanisms are also
used for autophagosome–lysosome fusion in Drosophila (Takats
et al., 2013). Furthermore, the O-linked N-acetylglucosamine
(O-GlcNAc) modification of SNAP29 negatively regulates
SNARE-dependent fusion between autophagosomes and
lysosomes (Guo et al., 2014). Consequently, knockdown of O-
GlcNAc transferase or mutating SNAP29 O-GlcNAc sites promotes
formation of the SNAP29-containing SNARE complex and
increases fusion between autophagosomes and lysosomes (Fig. 3).
It has recently been shown that the isolation membrane forms at
the ER-mito chondria contact site and STX17 is also involved in this
process (Hamasaki et al., 2013). STX17 localizes to the ER under
feeding conditions but, upon starvation, it relocalizes to ER-
mitochondria contact sites (Hamasaki et al., 2013). Here, STX17
binds to, and so recruits, ATG14L to ER-mitochondria contacts to
initiate formation of the isolation membrane. By contrast, STX17,
which is involved in autophagosome–lysosome fusion, is
presumably recruited from the cytosol to closed autophagosomes
(Itakura et al., 2012). Further studies are required to understand how
cells utilize different pools of STX17 depending on the context (ER
or cytoplasm). In addition to STX17, ATG14L binds to a binary
complex formed between STX17 and SNAP29 (STX17–SNAP29)
and facilitates its interaction with VAMP8 to promote
autophagosome–lysosome fusion (Diao et al., 2015) (Fig. 3),
indicating the interaction between STX17 and ATG14L both in the
early and late steps of autophagy.
Tethering factors
Membrane tethers are thought to provide another level of
specificity, and to facilitate docking and fusion by bridging
opposing membranes and by stimulating SNARE complex
formation. In addition to its well-known role in the endocytic
pathway, the HOPS complex functions as a tethering factor for
autophagosomal fusion (Jiang et al., 2014) (Fig. 3). STX17 interacts
with the HOPS complex, including VPS33A, VPS16, VPS11,
Rab7
Late endosome/lysosome
Autophagosome
ATG14L
SNAP29
STX17
VAMP8
Rab7
EPG5
SNAP29
PLEKHM1
HOPS
O-GlcNAcylated SNAP29
LC3-II
OGT
Starvation
Fig. 3. Role of SNAREs in mediating autophagosome–lysosome fusion.
EPG5 is recruited to late endosomes/lysosomes together with Rab7 and VAMP-
8, where it tethers late endosomes/lysosomes by binding to LC3 and STX17–
SNAP29; this facilitates the assembly of the trans-SNARE complex for fusion. In
contrast to EPG5, ATG14L binds to STX17 and STX17–SNAP29 Qabc
intermediate SNARE complexes on autophagosomes, but not to STX17–
SNAP29-VAMP8 RQabc SNARE complexes, suggesting that ATG14L acts
earlier than EPG5. PLEKHM1 is an adaptor protein that intera cts with Rab7,
HOPS–SNARE comple xes and LC3 and/or GABARAP proteins to facilita te
autophagosome–lysosome fusion. EPG5, ATG14L and the HOPS complex
function as tethering fa ctors, but their exact r ela tionship still needs to be clarified.
O-GlcNAcylated SNAP-29 which is generated by O-linked β-N-
acetylglucosamine (O-GlcNAc) tr ansfer ase (OGT), has a reduced binding affinity
for its partner SNAREs. This modification is suppressed by starvation, and a
reduce in the levels of O-GlcNAcylated SNAP-29 actsas a signal for the assembly
of SNAP-29-containing trans-SNARE complexes, thus stimulating autophagy.
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VPS18, VPS39 and VPS41. Consistent with this, these HOPS
subunits are recruited to STX17-positive autophagosomes upon
starvation. Importantly, knockdown of VPS33A, VPS16 or VPS39
blocks the autophagic flux and causes accumulation of STX17- and
LC3-positive autophagosomes, suggesting that HOPS promotes
autophagosome–lysosome fusion with STX17 (Jiang et al., 2014).
Most of these findings are also observed in Drosophila (Takats
et al., 2014). A recent structural study of Vps33 in the thermophilic
fungus Chaetomium thermophilum suggest that Vps33 promotes
SNARE assembly by precisely positioning and aligning SNAREs
(Baker et al., 2015). This finding provides us with a novel insight
how the HOPS complex promotes SNARE assembly.
How are HOPS complexes recruited to autophagosomes and
lysosomes? On late endosomes and lysosomes, Rab7 recruits its
effectors PLEKHM1 and RILP that bind to the HOPS components
VPS39 and VPS41, respectively (Wijdeven et al., 2016). These
effectors, thus, jointly recruit the HOPS complex for fusion. This is
similar in yeast, where the Rab7 homologue Ypt7 acquires HOPS
by binding to both Vps39 and Vps41 (Ostrowicz et al., 2010; Plemel
et al., 2011). In addition to PLEKHM1 and RILP, Rab7 recruits
another effector, the cholesterol sensor ORP1L, that binds to Rab7
in the presence of RILP and negatively regulates fusion (Johansson
et al., 2007; Wijdeven et al., 2016). At low levels of cholesterol,
ORP1L on late endosomes or lysosomes interacts with the ER
protein VAP-A to form the ER-autophagosome contact site. This
contact site prevents the recruitment of PLEKHM1 to Rab7, and
consequently, that of the HOPS complex, which results in a defect
in autophagosome-late endosome/lysosome fusion. Interestingly,
ORP1L and the presence of this contact site prevent the recruitment
of dynein and/or dynactin by RILP and, thus, minus-end-directed
transport of late autophagosomes (Wijdeven et al., 2016). These
observations suggest that the fusion of autophagosomes to late
endosomes and lysosomes and the transport of late autophagosomes
are regulated by cho lesterol. However, the in vivo function of
cholesterol during the fusion is still unclear and needs to be
addressed in future studies.
Ectopic P granules protein 5 (EPG5) was originally identified in a
C. elegans genetic screen and is another Rab7 effector and tethering
factor that determines the fusion specificity of autophagosomes with
endosomes/lysosomes (Tian et al., 2010; Wang et al., 2016) (Fig. 3).
EPG5 is recruited to late endosomes/lysosomes through direct
interaction with Rab7 and the late endosomal/lysosomal R- SNARE
VAMP7/VAMP8. EPG5 also binds to LC3/LGG-1 (LGG-1 is the C.
elegans Atg8 homol og) through its LC3-interacting region (LIR)
motif and to assembled STX17–SNAP29 complexes (Qabc-
SNARE) on autophagosomes. EPG5 stabilizes and facilitates the
assembly of STX17–SNAP29–VAMP8 trans-SNARE complexes
and, thus, is most likely to promote the fusion between
autophagosomes and lysosomes. Loss of EPG5 causes abnormal
fusion of autophagosomes with various endocytic vesicles, partly due
to increased assembly of the STX17–SNAP25–VAMP8 complex.
Consistent with this, SNAP25 knockdown partially suppresses the
effect on autophagy because of EPG5 depletion (Wang et al., 2016).
These findings have, thus, begun to answer the question as to
why autophagosomes specifically fuse with late endosomes and
lysosomes. In addition to HOPS and EPG5, as mentioned previously,
ATG14L directly binds to the STX17–SNAP29 binary complex on
autophagosomes and promotes STX17–SNAP29–VAMP8-mediated
autophagosome fusion with lysosomes, thus functioning as a
tethering factor (Diao et al., 2015) (Fig. 3). However the precise
relationship between HOPS, EPG5 and ATG14L is still unclear, and
needs to be clarified in future studies. Tectonin beta-propeller repeat-
containing 1 (TECPR1) also functions as a tethering factor and we
discuss its role further on in the text (Chen et al., 2012).
Phosphoino sitides in autophagosome–lysosome fusion
Phosphoinositides (PIs) function in intracellular membrane
trafficking. Phosphorylation of the third, fourth, and fifth position
of the PI inositol ring produces different variants, in particular PI3P
[phosphatidylinositol (3)-phosphate] that has a well-characterized
role in autophagosome formation. Although most studies focus on
the proteins that are involved in the autophagy process, several
recent studies have revealed roles for PIs during the late stage of
autophagy, including the transport of autophagosomes and the
autophagosomal fusion with the lysosome.
Studies in yeast provide hints on how autophagosomes become
competent to fuse with ly sosomes. Dephosphorylation and clearance of
PI3P by the PI3P phospha tase Ymr1 on the completed autophagosome
are essential for fusion of autophagosome and vacuole (Cebollero et al.,
2012). Clearance of PI3P triggers the dissocia tion of the A TG
machinery fr om the outer autophagosomal membrane, which makes
the autophagosome competent for fusion. By contrast, mammalian
forms of PI3P phosphatases, such as myotubularin-rela ted pr otein 3
(MTMR3) and MTMR14 (also known as Jumpy), appear to have
different functions during autophagy (Taguchi-Atarashi et al., 2010;
Vergne et al., 2009). Knockdown of MTMR3 and MTMR14 incr eases
autophagosome formation, indicating that these phosphatases are
negativ e regula tors of autophagy and tha t they function during the early
stages of autophagosome formation. It is, thus, worthwhile to further
investigate whether other PI phosphatases exert similar functions in
mammals.
As mentioned above, FYCO1 binds to LC3, PI3P and Rab7, and is
involv ed in the mov ement of autophagosomes (Fig. 2). Endogenous
FYCO1 localizes on perinuclear cytosolic vesicles but, upon
sta rvation, it is also dis tribut ed in the cell periphery in a
microtu bule-dependent manner (Pankiv et al., 2010). FYCO1
functions as an adaptor protein between autophagosomes and
microtu bule plus-end-directed molecular motors – as evidenced by
FYCO1 -depleted cells tha t a ccu mula te autophagosomes in perinuclear
clusters (Pankiv et al., 2010). Recently it has been shown that FYCO1
on endosomes intera cts directly with the KLC2 light chain of kinesin1
during the transloca tion of endosomes to the cell periphery (Raiborg
et al., 2015). Thus, it seems worthwhile to examine whether the same
kinesins ar e used during the transport of autophagosomes.
In addition to the HOPS complex, the PI3P-binding
proteinTECPR1 has been suggested to function as a tethering
factor in autophagosomal fusion (Chen et al., 2012) (Fig. 4).
TECPR1-depleted cells have impaired autophagic flux, and
accumulate autophagic vacuoles and substrates, including p62
(officially known as SQSTM1) and lipidated LC3 (LC3-II) (Chen
et al., 2012). TECPR1 was originally identified through its interaction
with ATG5 (Behrends et al., 2010). Whereas phagophore (isolation
membrane) localization of the Atg12–Atg5–Atg16 complex is
dependent on the presence of PI3P, Chen et al. show that TECPR1
and ATG16 form mutually exclusive complexes with the ATG12–
ATG5 conjugate, and TECPR1 binds PI3P upon association with the
Atg12–Atg5 conjugate (Chen et al., 2012; Fujita et al., 2008b).
Furthermore, TECPR1 localizes to lysosomes/autolysosomes and
recruits the ATG12–ATG5 conjugate, which enables its binding to
PI3P, thereby possibly facilitating autophagosome maturation and
autophagosome–lysosome fusion. However, another study found that
TECPR1 also functions in phagophore biogenesis and maturation
during selective autophagy (Ogawa et al., 2011). These discrepancies
might either reflect dual roles of TECPR1 or distinct functions in
1213
COMMENTARY Journal of Cell Science (2017) 130, 1209-1216 doi:10.1242/jcs. 196352
Journal of Cell Science