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We acknowledge support from the Global Climate Energy Project
at Stanford University. H. W. acknowledges support from the
Stanford Interdisciplinary Graduate Fellowship. S.X. and F.B.P.
were supported by the Center on Nanostructuring for Efficient
Energy Conversion (CNEEC) at Stanford University, an Energy
Frontier Research Center funded by the U.S. Department of
Energy (DOE), Office of Science, Basic Energy Sciences, under
award DE-SC0001060. F.A.-P. and J.K.N acknowledge financial
support from the DOE, Office of Basic Energy Sciences, to the
SUNCAT Center for Interface Science and Catalysis. C. T.
acknowledges support from the National Science Foundation
Graduate Research Fellowship Program (GRFP) grant DGE-114747.
Y.C. acknowledges support to initiate the catalysis research from
the DOE, Basic Energy Sciences, Materials Sciences and
Engineering Division, under contract DE-AC02-76SF00515. The
authors acknowledge the help of aberration-corrected
STEM-HAADF from P. Yang and Y. Yu at the National Center for
Electron Microscopy at the Molecular Foundry. Work at the
Molecular Foundry was supported by the DOE Office of Science,
Office of Basic Energy Sciences, under contract DE-AC02-
05CH11231. The authors also acknowledge helpful discussions with
M. Logar. All of the data are available in the main paper and
Materials and Methods
Figs. S1 to S30
28 March 2016; accepted 17 October 2016
The ATG conjugation systems are
important for degradation of the
inner autophagosomal membrane
Kotaro Tsuboyama,1 Ikuko Koyama-Honda,1 Yuriko Sakamaki,2 Masato Koike,3
Hideaki Morishita,1 Noboru Mizushima1†
In macroautophagy, cytoplasmic contents are sequestered into the double-membrane
autophagosome, which fuses with the lysosome to become the autolysosome. It has been
thought that the autophagy-related (ATG) conjugation systems are required for autophagosome
formation. Here, we found that autophagosomal soluble N-ethylmaleimide–sensitive factor
attachment protein receptor (SNARE) syntaxin 17–positive autophagosome-like structures
could be generated even in the absence of the ATG conjugation systems, although at a
reduced rate. These syntaxin 17–positive structures could further fuse with lysosomes,
but degradation of the inner autophagosomal membrane was significantly delayed. Accordingly,
autophagic activity in ATG conjugation–deficient cells was strongly suppressed. We suggest
that the ATG conjugation systems, which are likely required for the closure (i.e., fission) of
the autophagosomal edge, are not absolutely essential for autolysosome formation but are
important for efficient degradation of the inner autophagosomal membrane.
Macroautophagy (hereafter, autophagy) is a highly inducible intracellular degrada- tion system (1–3). First, a flat membrane sac, termed the isolation membrane or the phagophore, elongates, bends, and
sequesters a part of the cytoplasm. Then, its
edge is closed by membrane fission to form the
double-membrane structure, the autophagosome
(4). The autophagosome fuses with lysosomes
and becomes the autolysosome. Lysosomal enzymes
selectively degrade the inner autophagosomal
membrane (IAM), but not the outer autopha-
gosomal membrane (OAM), and then finally de-
grade the enclosed cytoplasmic contents.
To characterize autophagosome maturation
in mammalian cells, we used the autophagosomal
soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) syntaxin 17 (STX17)
as an autophagosome marker (5). Elongating isolation membranes were microtubule-associated
protein light chain 3 (LC3) positive but STX17
negative (Fig. 1A, red arrows, and movie S1) (5).
Later, STX17 was recruited to the whole cir-
cumference of ring-shaped structures (Fig. 1A,
green arrows). The shape of elongating isola-
tion membranes was elliptical but became al-
most completely spherical when STX17 was
recruited (Fig. 1B). We hypothesize that fission
between the OAM and IAM during the closure
of the autophagosomal edge causes a morpho-
logical change into stable spherical bodies that
occurs immediately before or after the recruit-
ment of STX17.
After STX17 recruitment, several small LAMP1-
positive lysosomes (or late endosomes) associated with the autophagosomes (Fig. 1C, 2 to
8 min, blue arrows). Then, the autophagosomal
membrane became LAMP1-positive (Fig. 1C, 10
to 16 min, blue arrows). At almost the same time,
these structures became positive for Lyso Tracker
Red (LTR), a weak-base probe for acidic compartments. The LTR signals appeared also as
ring-shaped structures on the STX17-positive
structures (Fig. 1C, 4 to 10 min, red arrows),
which suggests that the space between the OAM
and IAM is acidified. Next, the ring-shaped LTR
signal collapsed, and the lumen of the autolysosomes filled with LTR, which indicated the
IAM degradation (Fig. 1C, 12 to 16 min). The
STX17 signal gradually disappeared after the collapse of the LTR ring structure (Fig. 1C, 12 to
14 min). This STX17 dissociation was independent of luminal acidification because it was not
affected by bafilomycin A1 treatment (fig. S1).
Thus, we detected four steps during autophagosome maturation: STX17 recruitment (step a),
lysosomal fusion (step b), IAM degradation (step c),
and STX17 release (step d) (Fig. 1C). The total
lifetime of STX17 (steps a to d) and the durations (means ± SEM) between each step (a to b,
b to c, and c to d) were 11.0 ± 0.6, 2.1 ± 0.3, 6.6 ±
0.6, and 2.2 ± 0.2 min, respectively (Fig. 1C).
Autophagosome formation requires the two
ubiquitin-like systems: the ATG12 conjugation
system and the ATG8 (LC3s and g-aminobutyric
acid receptor–associated proteins in mammals)
conjugation system (6, 7). Ubiquitin-like ATG12
and ATG8 are covalently conjugated to ATG5
and phosphatidylethanolamine (PE), which are
catalyzed by the common E1-like protein ATG7
and the specific E2-like proteins ATG10 and ATG3,
respectively. Conjugation of ATG8 or LC3 with
1036 25 NOVEMBER 2016 • VOL 354 ISSUE 6315 sciencemag.org SCIENCE
1Department of Biochemistry and Molecular Biology,
Graduate School and Faculty of Medicine, The University of
Tokyo, Tokyo 113-0033, Japan. 2Research Center for Medical
and Dental Sciences, Tokyo Medical and Dental University,
Tokyo 113-8510, Japan. 3Departments of Cell Biology and
Neuroscience, Juntendo University Graduate School of
Medicine, Bunkyo-Ku, Tokyo 113-8421, Japan.
*These authors contributed equally to this work.
†Corresponding author. Email: firstname.lastname@example.org
13 December 2015; resubmitted 28 March 2016
Accepted 17 October 2016