STATE-OF-THE-ART REVIEW
Autophagy in the test tube: In vitro reconstitution
of aspects of autophagosome biogenesis
Yijian Rao, Nena Matscheko and Thomas Wollert
Molecular Membrane and Organelle Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
Keywords
autophagy; cellular recycling; in vitro
reconstitution; membrane biology; trafficking
Correspondence
T. Wollert, Molecular Membrane and
Organelle Biology, Max Planck Institute of
Biochemistry, Am Klopferspitz 18, 82152
Martinsried, Germany
Fax: +498985783430
Tel: +498985783420
E-mail: wollert@biochem.mpg.de
(Received 19 November 2015, revised 30
December 2015, accepted 14 January 2016)
Autophagy is a versatile recycling pathway that delivers cytoplasmic contents to lysosomal compartments for degradation. It involves the formation
of a cup-shaped membrane that expands to capture cargo. After the cargo
has been entirely enclosed, the membrane is sealed to generate a doublemembrane-enclosed compartment, termed the autophagosome. Depending
on the physiological state of the cell, the cargo is selected either specifically
or non-specifically. The process involves a highly conserved set of autophagy-related proteins. Reconstitution of their action on model membranes
in vitro has contributed tremendously to our understanding of autophagosome biogenesis. This review will focus on various in vitro techniques that
have been employed to decipher the function of the autophagic core
machinery.
doi:10.1111/febs.13661
It’s all about membranes –
the biogenesis of autophagosomes
Macroautophagy, here referred to as autophagy,
involves the formation of a cup-shaped membrane
sack, termed the phagophore or isolation membrane
(IM), which captures cytoplasmic material and delivers it to lysosomes (to the vacuole in yeast) [1,2].
During normal physiological conditions, superfluous
or damaged cytoplasmic material is the major cargo
for autophagy. The pathway thus allows cells to
maintain their homeostasis by preventing accumulation of harmful components such as damaged mitochondria or aggregated proteins. During starvation
and in response to cytotoxic stresses, autophagy is
strongly induced and largely recycles bulk cytoplasm
non-selectively [3].
The pathway is best characterized in yeast where 41
autophagy-related (Atg – AuTophaGy) proteins
coordinate the biogenesis of autophagosomes. Human
orthologs for many yeast Atg proteins have been identified, emphasizing the high degree of conservation
among eukaryotes. Most of them belong to the autophagic core machinery that is required for all autophagy-related processes [4].
The morphological hallmark of autophagy is the
appearance of a cup-shaped membrane that expands
Abbreviations
ALPS, amphipathic lipid packing sensor; Atg, autophagy related; BAR, Bin–amphiphysin–Rvs; ER, endoplasmic reticulum; GABARAP,
c-aminobutyric acid receptor associated protein; GATE-16, Golgi-associated ATPase enhancer of 16 kDa; HOPS, homotypic fusion and
protein sorting; IM, isolation membrane; LC3, microtubule-associated proteins 1A/1B light chain 3; MIM, MIT interacting motif; MIT,
microtubule interacting and trafficking; PAS, phagophore assembly site; PE, phosphatidylethanoleamine; PI3K, phosphatidylinsositol-3-kinase;
PI, phosphatidylinsositol; PROPPIN, β-propellers that bind polyphosphoinositides; S/L/GUV, small/large/giant unilamellar vesicle; SLB,
supported lipid bilayer; SNARE, soluble NSF attachment protein receptor; Ub, ubiquitin; WD, tryptophan–aspartic acid; WIPI, WD repeat
domain phosphoinositide-interacting.
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to successively enclose cytoplasmic cargo. In yeast, it
is widely accepted that such precursor membranes are
formed at a distinct location, the phagophore assembly
site (PAS), which is in close proximity to the vacuole
and endoplasmic reticulum (ER) exit sites [5,6]. By
contrast, IMs are formed at many sites simultaneously
in mammalian cells. Corresponding loci have been
characterized as specialized phosphatidylinositol
(PI)-3-phosphate enriched domains of the ER, termed
the omegasome [7].
Upon initiation of autophagy the Atg1-kinase
complex (ULK-complexes at the ER in humans),
which consists of the kinase subunit Atg1, Atg13,
Atg17, Atg29 and Atg31 in yeast, assembles at and
recruits Atg9 vesicles to the PAS [8–10]. For yeast, it
has been suggested that these vesicles undergo fusion
to initiate the formation of the phagophore (Fig. 1A)
A
B
[11]. In mammalian cells, mATG9 only transiently
interacts with IMs [12] to deliver membranes. Whether
a certain amount of mATG9 is incorporated into IMs
remains to be investigated [9]. Whereas Atg9 vesicles
are an essential membrane source for autophagy initiation in yeast, other sources including the endoplasmic
reticulum, the Golgi, the plasma membrane, recycling
endosomes as well as mitochondria have been
described in human cells [7,12–17].
The expansion of autophagic precursor membranes
requires the sequential recruitment of several critical
Atg complexes in a hierarchical and ordered fashion.
First, the autophagy-specific PI-3-kinase complex
localizes to IMs, enriching them in PI-3-phosphate
(Fig. 1B) [18,19]. In yeast, the complex comprises
the canonical PI-3-kinase subunits Vps34, Vps15 and
Vps30, which are also found in other PI-3-kinases,
C
D
Fig. 1. Schematic representation of autophagosome formation. (A) Atg16 might tether or organize Atg9 vesicles of the peripheral Atg9 pool.
The Atg1-kinase complex recruits Atg9 vesicles to the PAS to initiate the formation of autophagosomes. (B) Atg14, which forms part of the
PI-3-kinase complex, possesses a membrane curvature-sensing ALPS motif that is important to target the complex to early autophagic
precursor membranes or the edge of the IM. After enriching the IM in PI-3-phosphate (PI3P), downstream factors recruit the Ub-like
conjugation system to the PAS followed by conjugation of Atg8 to the lipid PE within IMs. (C) Atg8 might form together with Atg12–Atg5–
Atg16 a membrane scaffold at the convex face of the phagophore to regulate phagophore expansion. At the concave face, Atg8 functions
as a cargo adaptor and recognizes autophagic cargo through its interaction with cargo receptors. Expansion of IMs might involve fusion of
vesicles with their highly bent membrane edges. (D) Autophagic cargo is finally delivered to the vacuole (lysosomes in humans). This
process requires tethering of autophagosomes to vacuolar membranes by the membrane-tethering HOPS complex and the small RabGTPase Ypt7. Membrane fusion is catalyzed by SNARE proteins in a process that might be facilitated by the PI-3-kinase complex subunit
Atg14.
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Y. Rao et al.
and the autophagy-specific subunit Atg14. Beclin-1
and Atg14L/Barkor are the human homologs of Vps30
and Atg14, respectively.
Next, PI-3-phosphate effectors such as yeast b-propellers that bind polyphosphoinositides (PROPPINs)
and the human tryptophan–aspartic acid (WD) repeat
domain phosphoinositide-interacting (WIPI) proteins
associate with IMs and coordinate critical steps during
membrane expansion. This involves the recruitment of
the highly conserved autophagy-specific ubiquitin
(Ub)-like conjugation machinery to IMs by the yeast
PROPPIN Atg21 and human WIPI-2A, respectively
[20,21]. The coordinated action of two interconnected
Ub-like conjugation systems covalently attaches the
Ub-like protein Atg8 or its human orthologs LC3 (A,
B and C), c-aminobutyric acid receptor associated
protein (GABARAP), GABARAP-L1, and Golgiassociated ATPase enhancer of 16 kDa (GATE-16) to
phosphatidylethanolamine (PE) within autophagic
membranes (Fig. 1C) [22–24]. The amount of Atg8 at
the phagophore correlates with the size of autophagosomes [25,26], suggesting a direct role of Atg8 in
coordinating phagophore expansion. Interestingly,
small aberrantly shaped autophagosomes are still
formed in yeast atg8Δ cells, whereas extended IMs are
observed in human cells upon deletion of ATG5, indicating that Atg8 is important but not essential for
autophagy [26,27]. Atg8 and its human orthologs also
function as cargo adaptors by tethering specific cargo
to the phagophore through their interaction with
cargo-receptor proteins (Fig. 1C) [28–30]. The final
step in autophagosome biogenesis involves closure of
the phagophore, which requires the activity of the PI3-phosphate effector Atg18 [31].
The extraordinarily high demand for lipids that are
needed to expand autophagic membranes represents
without doubt a major challenge for cells, particularly
during starvation or stress conditions. Although many
organelles may supply lipids to generate autophagosomes, strong evidence for a direct contribution of the
ER–Golgi-intermediate compartment, ER exit sites,
lipid droplets and recycling endosomes is accumulating
[15,32–34].
After
autophagosome
completion,
Atg8/LC3
molecules that reside on the cytoplasmic face of
autophagosomes are recycled, i.e. proteolytically
cleaved off the membrane by Atg4 [35]. Human cells
express four Atg4 orthologs, with ATG4B being crucial for autophagy and able to cleave all human Atg8
orthologs [36,37]. Eventually, autophagosomes fuse
with lysosomal compartments in a process that
requires the membrane tethering complex homotypic
fusion and protein sorting (HOPS) and soluble NSF
2036
attachment protein receptor (SNARE) proteins
[38–40]. The latter family of membrane-trafficking proteins is not only required for this very last step in
autophagy, but also coordinates earlier events including initiation of autophagy [39,41–43].
Getting started – initiation of
autophagy
The most widely applied membrane model for reconstitution reactions, and presumably the handiest one,
utilizes small or large unilamellar vesicles (SUVs and
LUVs, respectively). These single membrane vesicles
are generated from mixtures of synthetic or natural
lipids by hydrating thin lipid films. SUVs are up to
100 nm in diameter and are often generated by
sonication, whereas LUVs are prepared by extruding
suspended lipid mixtures through filters with specific
pore sizes. LUVs thus have well-defined diameters of
100–800 nm [44]. Such vesicles have been used to
reconstitute the recruitment of autophagy-initiation
factors including yeast Atg1 and human Barkor/
ATG14L to sites of autophagosome formation in vitro
[45,46]. The pentameric Atg1-kinase complex plays a
central role in initiating autophagy [47,48]. Its kinase
subunit Atg1 harbors a tandem microtubule interacting and trafficking (MIT) domain that senses
membrane curvature [46,49]. The Atg1 MIT domain
binds small unilamellar vesicles with an average
diameter of 30 nm, possessing the physically smallest
possible size and consequently highest membrane curvature with respect to the lipid composition of such
vesicles (Fig. 2A) [46,50].
Interestingly, MIT domains do not belong to classical membrane curvature-sensing domains such as Bin–
amphiphysin–Rvs (BAR) domains or the amphipathic
lipid packing sensor (ALPS) motif [51,52]. Instead,
they are canonical protein–protein interacting domains
and recruit binding partners by recognizing a linear
peptide motif, termed the MIT interacting motif
(MIM). Correspondingly, the MIT domain of Atg1
binds Atg13 by recognizing its MIM [49]. Whether the
human Atg1 orthologs ULK1/2 also possess a tandem
MIT domain remains to be investigated. Their highly
conserved C termini, however, are essential for membrane localization and bind human ATG13 in vivo
[53], implying that membrane targeting of Atg1 and
ULK1/2 are structurally and functionally conserved.
Initiation of autophagy furthermore depends on the
recruitment of the autophagy-specific PI-3-kinase complex, which acts immediately downstream of the Atg1kinase or ULK1 complexes, respectively [48,54]. Atg14
and Barkor/ATG14L are integral components of yeast
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A
B
D
Ypt7
LUV
GDP
GUV
LUV
Ypt7
Ypt7
GTP
ATG14L
Atg1 MITdomain
Electron Micrograph
AlexaAtg16
Electron Micrograph
GUV
AlexaAtg8
HOPS
C
7,5 nm
LUV
E
Ub-like conjugation
system + ATP
Atg8
5,5
STX17 / SNAP29
3,5
1,5
Atg12–Atg5Atg16
Fluorescence
AFM
0
ATG14
VAMP8
ld on
scaffo ane
br support
mem
Fig. 2. In vitro reconstitutions of important steps in autophagy. (A) The Atg1-kinase and PI-3-kinase complexes are involved in nucleating
autophagy. The interaction of the MIT domain of Atg1 and ATG14L, members of the two respective complexes, with membranes has been
investigated using large unilamellar vesicles (LUVs). The cartoon shows the experimental set-up. Proteins of interest are incubated with
LUVs. Binding is analyzed by physically separating LUV from unbound protein and comparing bound and unbound protein fractions by SDS/
PAGE. The electron micrograph shows SUVs with an average diameter of 20 nm. Scale bar = 50 nm. (B) Giant unilamellar vesicles (GUVs)
can be visualized by incorporating fluorescently labeled lipids and employing confocal fluorescence microscopy. Fluorescent labeling of
proteins can be achieved by tagging with fluorescent proteins (green fluorescent protein (GFP) and spectral variants) or by chemically
attaching fluorescent dyes such as Alexa. Conjugation of Atg8 to GUV membranes was investigated using fluorescently labeled Atg8 and
Atg16. Both proteins co-localize to GUVs in confocal microscopy sections. Scale bar = 5 lm. (C) Supported lipid bilayers (SLBs) have been
used to structurally characterize the autophagic membrane scaffold, which comprises Atg8 and Atg12–Atg5–Atg16 complexes. The
fluorescence image shows a plain SLB, which contains fluorescent lipids. The height profile of an atomic force micrograph (AFM) shows the
membrane scaffold on an SLB. Colors indicate heights as defined by the calibration bar. Scale bar = 10 lm. (D) Membrane tethering by
proteins can be reconstituted using LUVs. The cartoon shows the experimental set-up of HOPS-mediated tethering of LUVs as described in
the text. The electron micrograph shows LUVs, which are tethered by proteins (not related to those shown in the cartoon [87]). (E) ATG14
facilitates SNARE-mediated fusion by stabilizing the STX17–SNAP29 subcomplex on autophagic membranes and by priming theses SNAREs
for fusion with VAMP8-containing lysosomal membranes. The process can be analyzed by content-mixing assays, using LUVs harboring the
respective SNAREs and containing fluorescent dyes as indicated by colors. Membrane fusion leads to mixing of the dyes, which can be
analyzed by measuring fluorescence-resonance energy transfer between the two fluorophores.
and human PI-3-kinase (PI3K) complexes I, targeting
them to autophagic membranes during initiation of
autophagy [18,55–57]. ATG14L possesses an ALPS
motif that has been shown to direct the human PI3K
complex to highly curved membranes [45]. Corresponding in vitro experiments using LUVs with various
diameters revealed that ATG14L senses curved membranes and specifically binds LUVs with diameters of
100 nm as opposed to 800 nm large vesicles (Fig. 2A).
Interestingly, a high concentration of PI-3-phosphate,
the product of the PI3K catalyzed reaction, outcompetes membrane curvature sensing by the ALPS motif
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Y. Rao et al.
of ATG14L [45]. The recruitment of ATG14L to flat
PI-3-phosphate-rich membranes appears to be essential
in later steps of autophagy [58].
In vitro reconstitution reactions using LUVs of
defined sizes thus uncovered a conserved property of
the two critical autophagy initiation complexes, the
Atg1-kinase (ULK1-kinase) and PI3K complex. Both
possess membrane curvature-sensing subunits that
apparently target them to highly curved membranes.
This binding preference is of physiological significance
as it seems to recruit these complexes to autophagic
precursor membranes, such as small Atg9 vesicles,
IMs, or membrane cradles of the ER.
Feeling hungry – maturation of
autophagosomes
The PI3K complex converts the autophagic precursor
into a PI-3-phosphate-rich membrane, allowing downstream effectors, including yeast PROPPINs and
human WIPI proteins, to be recruited. The members of
the two protein families possess conserved WD-repeats
that fold into a seven-bladed b-propeller domain and
bind PI-3-phosphate through their conserved FRRG
motif [59,60]. The yeast PROPPIN Atg21 and human
WIPI2 recruit the corresponding E3-like ligase complexes Atg12–Atg5–Atg16 (yeast) and ATG12–ATG5–
ATG16L1 (humans) to autophagic membranes [20,21].
Both ligase complexes catalyze the conjugation of Atg8
or its human orthologs to autophagic membranes to
promote phagophore expansion by a yet-to-be-identified molecular mechanism [61,62].
Biochemical reconstitutions of this Ub-like conjugation system on LUVs represent important steps to
understand autophagy at a molecular level. The earliest study recapitulated the conjugation of Atg12 to
Atg5 in cell lysates and demonstrated that the resulting
Atg12–Atg5 conjugate is essential for autophagy. The
reaction was found to be catalyzed by the E1- and E2like enzymes Atg7 and Atg10 [23]. The conjugation of
Atg8 to PE within LUV-membranes by a second Ublike conjugation system, involving the E1- and E2-like
enzymes Atg7 and Atg3, was first reconstituted in the
absence of Atg12–Atg5 [63]. Later, it was demonstrated that Atg12–Atg5 facilitates the Atg8-conjugation reaction and functions as a canonical E3-like
ligase complex by reconstituting the two Ub-like conjugation systems from purified components on LUVs
[61].
The reconstitution of the human Ub-like conjugation system is more challenging compared with the
yeast system because of its complexity, involving various homologs and isoforms, as well as limitations in
2038
producing all components in vitro. First reconstitutions
of human Atg8 orthologs on membranes thus
circumvented the need for producing components of
the Ub-like conjugation system by chemically tethering
LC3B and GATE-16 to LUVs [64].
The conjugation of LC3B, GABARAP-L1 and
GATE-16 to LUVs by the E1-like ATG7 and E2-like
ATG3 enzymes was only recently reconstituted.
Interestingly, the study revealed that ATG3 contains
an ALPS motif that targets the ATG3–ATG8 intermediate to highly bent membranes in vitro, thereby promoting conjugation of human ATG8 homologs to PE
within LUVs [65]. Thus, conjugation might primarily
take place at strongly bent edges of IMs. Since LC3B
and yeast Atg8 are homogeneously distributed on IMs
and phagophores in vivo [6], the function of the
E3-ligase complex Atg12–Atg5–Atg16L1, which was
not included in this reconstitution study, might be
required to recapitulate all aspects of the conjugation
reaction in vitro. The discrepancy between the suggested conjugation site and protein localization might
also be explained by diffusion of Atg8 or LC3B from
the edge to both faces of the phagophore. Thus, the
regulation of ATG8 conjugation and its distribution
on IMs remains an interesting avenue to follow for
future reconstitutions, provided that cup-shaped
phagophore membranes and the complete Ub-like conjugation machinery can be produced in vitro.
How Atg8 coordinates phagophore growth
remained, however, an open question. A recent series
of in vitro reconstitution studies set out to address this
issue. First insights into Atg8 function were provided
by a study utilizing once more LUVs. The authors of
the study observed clustering of Atg8-decorated LUVs.
Moreover, clustered LUVs underwent hemifusion, i.e.
only the outer leaflets of fusing bilayers merge. Tethering and hemifusion by Atg8 was suggested to play
important roles in phagophore expansion, presumably
by tethering donor vesicles to the growing phagophore
[66]. A later study demonstrated that SNARE-proteins, which promote membrane fusion reactions in all
other known trafficking pathways, are also essential
for autophagy and that extraordinarily high PE concentrations are the major driving force for SNAREindependent fusion by Atg8 [39].
More advanced in vitro reconstitution reactions
based on fluorescence microscopy were applied to further characterize the role of Atg8 during autophagy.
These systems utilized giant unilamellar vesicles
(GUVs), which are 10–100 lm in diameter. GUVs are
produced by hydrating a lipid film while applying an
electric AC field [67]. GUV membranes can be visualized by confocal fluorescence microscopy through
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Y. Rao et al.
incorporation of fluorescently labeled lipids and proteins, allowing observation of Atg8 conjugation to
GUVs in real time (Fig. 2B). One study showed that
Atg12–Atg5–Atg16 recruits the E2-like enzyme Atg3
to membranes in vitro to promote lipidation of Atg8.
Furthermore, the authors found that Atg12–Atg5–
Atg16 induced membrane tethering [68], comparable
to what had been observed for Atg8 [66]. Thus,
Atg12–Atg5–Atg16-mediated tethering of membranes
might be related to an early function of the complex
by promoting tethering of vesicles of the peripheral
Atg9 pool (Fig. 1A). Correspondingly, ATG16L1 vesicles were found to coalesce with ATG9-positive vesicles at recycling endosomes in a process that drives
autophagy initiation in human cells [69].
How Atg8 controls the size of autophagosomes was
addressed by another in vitro study in which the conjugation of fluorescently labeled Atg8 to GUVs and supported lipid bilayers (SLBs) was reconstituted. SLBs
are produced by depositing sonicated liposomes on
supports (e.g. silica or mica) and facilitating their
fusion to produce a bilayer by calcium addition. Fluorescent labeling of proteins by small chemical dyes is
essential to characterize protein topology and organization on membranes by coupled fluorescence confocal
and atomic force microscopy. Atg8 was found to associate with Atg12–Atg5 into well-defined particles, harboring multiple copies of each protein. Fluorescence
recovery after photobleaching demonstrated such particles to be mobile, exhibiting free lateral diffusion.
The presence of the dimeric coiled-coil protein Atg16,
however, induced the formation of a continuous protein lattice on the membrane with meshwork-like
appearance (Fig. 2C) [70]. Atg8 thus assembles
together with its E3-ligase Atg12–Atg5–Atg16 into a
membrane-scaffold that might structurally support
membrane expansion of phagophores. Membrane scaffold formation would thus explain how the amount of
Atg8 on phagophores defines the final size of
autophagosomes [71].
Selective types of autophagy are primarily operating
during periods of vegetative growth with sufficient
nutrient supply and in the absence of cytotoxic stress.
Selected cargo is tethered to IMs by direct interaction
with Atg8. Recently, the cargo-tethering function of
Atg8 and LC3B has been investigated by reconstituting the recruitment of the yeast cargo ApeI and
human ubiquitinated cargo by its corresponding cargo
receptors, yeast Atg19 and human p62, in vitro [72,73].
These studies revealed that tight interaction between
autophagic membranes and cargo is established by
multivalent binding of one Atg19 molecule to several
Atg8PE moieties or by coordinated clustering of p62,
Autophagy in vitro
respectively. As a consequence, cargo is selectively
enclosed by autophagic membranes while other cytoplasmic components are excluded.
Finishing up – fusion with lysosomal
compartments
In yeast, both Atg8–PE and Atg18 have directly been
implicated in promoting sealing of the membrane to
produce, double-membrane-enclosed autophagosomes
[31,66]. The major challenge for future in vitro experiments is the generation of membrane templates that
serve as substrate to correlate protein localization with
function to reveal the molecular processes that drive
autophagosome closure.
The last step in the life of an autophagosome is its
fusion with lysosomal compartments to degrade
sequestered material. This process is preceded by
deconjugation of Atg8 or its human orthologs from
the cytoplasmic leaflet of autophagosomes by Atg4
[74,75]. Moreover, fusion requires cooperation of
canonical membrane tethering and fusion machines
with Atg proteins. The initial contact between
autophagosomal and vacuolar/lysosomal membranes is
established by the canonical membrane tether HOPS,
which also facilitates tethering to and fusion of late
endosomes (multivesicular bodies) with vacuoles/lysosomes (Fig. 1D) [38,76,77]. The recruitment of the
HOPS complex to endosomal and vacuolar membranes is mediated by the Rab GTPase Ypt7 in its
GTP-associated form. By reconstituting this process
from purified components in vitro on LUVs, the
HOPS complex was found to cross-link two Ypt7molecules in trans, i.e. on two opposing membranes
(Fig. 2D) [78]. Although Ypt7 and its human homolog
Rab7 are required for autophagy in yeast [79] and
human cells [80], it is not clear whether they mediate
the contact of HOPS to autophagosomal membranes
as well.
SNAREs drive membrane fusion with limited selectivity [81] and therefore require tethering complexes to
provide specificity while establishing the first contact
between membranes [82]. In yeast, Ykt6, Vti1, Vam3
and Vam7 have been shown to mediate autophagosome–lysosome fusion [43,83,84]. The latter three are
Q-SNAREs, which are found on vacuolar membranes
and promote fusion by interacting with their cognate
R-SNARE. Ykt6 is such an R-SNARE and thus in
principle able to form a SNARE complex with these
vacuolar SNAREs. However, upon reconstitution of
Vti1/Vam3/Vam7 and Ykt6 in LUVs, fusion was
not observed [85]. Recently, the importance of
vacuolar lipids for SNARE-mediated fusion has been
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Y. Rao et al.
demonstrated, suggesting that regulatory factors are
required to promote fusion of Ykt6 vesicles to Vti1/
Vam3/Vam7 vesicles as well. Interestingly, a direct
interaction of the HOPS complex with Vam3 is
required for vacuolar fusion in yeast [86], suggesting
that HOPS facilitates both membrane tethering
through its interaction with Rab-GTPases and fusion
by binding specific SNAREs.
In human cells, the SNARE syntaxin-17 (STX17)
drives together with VAMP8 and SNAP29 the fusion
of autophagosomes with lysosomes in a process that
depends also on the interaction of STX17 with the
human HOPS complex [40]. This suggests that, similarly to yeast, human HOPS facilitates fusion of
autophagosomes with lysosomes by interacting with
SNAREs.
Unexpectedly, ATG14L was recently discovered to
promote fusion of autophagosomes with lysosomes in
a process that depends on its interaction with the two
autophagic SNAREs, STX17 and SNAP29 (Fig. 1E).
By reconstituting fusion of STX17/SNAP29 vesicles
with VAMP8 vesicles in vitro, ATG14L was found to
facilitate SNARE priming and thus membrane fusion
independently of its previously observed membrane
tethering function [58]. In summary, these data
indicate that concerted action of various autophagic
factors drives in cooperation with canonical membrane
trafficking complexes the last step in the life cycle of
autophagosomes.
Concluding remarks
In vitro reconstitutions identified precise molecular
mechanisms involved in the formation of autophagosomes in yeast and humans. The great advantage of
in vitro systems over in vivo experiments is the welldefined environment to study protein functions independently of other influencing factors and conditions.
In vitro reconstitutions thus not only characterize
biophysical properties of proteins and membranes,
they also reveal physical protein interactions. Findings
of such experiments might translate one-to-one to the
situation in vivo. This, however, needs to be substantialized using complementary in vivo assays.
Many questions concerning the molecular mechanism of autophagy remain to be answered in the
future, representing even greater challenges for in vitro
reconstitution reactions, mainly because more complex
systems involving many components and unusual
membrane templates such as artificial phagophores are
needed. The final goal of these experiments is to
recapitulate the biogenesis of autophagosomes from
purified components in the test tube. Once achieved,
2040
the detailed mechanistic understanding of the process
would facilitate drug development and might result in
generation of specific therapies to treat life-threatening
diseases such as cancer, neurodegeneration or autoimmune diseases.
Acknowledgements
Work in our laboratory was generously supported by
the Max Planck Society.
Authors contributions
T.W. wrote the manuscript with contributions of Y.R.
and N.M.
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