Results The C-terminus of Atg8L is cleaved in HEK293 cells, and the Gly116 of Atg8L is essential for cleavage The C-termini of yeast Atg8, mammalian LC3, GABA-RAP and GATE-16 are post-tr
Trang 1mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3
Isei Tanida, Yu-shin Sou, Naoko Minematsu-Ikeguchi, Takashi Ueno and Eiki Kominami
Molecular Cell Biology, Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan
Ubiquitylation and ubiquitylation-like reactions are
post-translational modifications that play indispensable
roles in many cellular events Atg8⁄ Apg8 ⁄ Aut7 is a
ubiquitin-like (Ubl) protein essential for autophagy in
the yeast Saccharomyces cerevisiae [1] Genetic analyses
of yeast ATG gene mutants have suggested that the
C-terminus of Atg8 is cleaved by Atg4 or a protease
activated by Atg4⁄ Apg4 to expose the C-terminal Gly
of Atg8 [2–4] Subsequently, Atg8 is activated by
Atg7⁄ Apg7 ⁄ Gsa7 ⁄ Cvt2, an E1-like enzyme [5–8],
transferred to Atg3⁄ Apg3 ⁄ Aut1, an E2-like enzyme [3],
and finally conjugated to phosphatidylethanolamine
[3] This conjugation reaction is essential for
auto-phagy under conditions of starvation and in the
cyto-plasm for the vacuole-targeting (Cvt) pathway under
nutrient-rich conditions
To date, three Atg8 homologs have been
character-ized in mammals: LC3 (microtubule-associated protein 1
light chain 3, MAP1-LC3) [9,10], 4-aminobutyrateA -receptor associated protein (GABARAP) [11–13], and Golgi-associated ATPase enhancer of 16 kDa (GATE-16) [12–14] Following cleavage by human Atg4B⁄ autophagin 1, the C-terminal Gly of each of these Atg8 homologs is exposed, as is also observed for yeast Atg8 [13,15] Human Atg4A⁄ autophagin 2 also cleaves the C-terminus of GATE-16 [16] Following C-terminal cleavage, each Atg8 homolog is activated by human Atg7, an E1-like enzyme, to form a transient E1-sub-strate intermediate [12,17,18] Each Atg8 homolog is subsequently transferred to human Atg3, an E2-like enzyme, to form a transient E2-substrate intermediate, and modified to the membrane-bound forms, LC3-II, GABARAP-PL (GABARAP-II), and GATE-16-II [12,19] Recently, it has been shown that LC3-II and GABARAP-PL are protein–phospholipid conjugates [15], with phosphatidylethanolamine thought to be the
Keywords
autophagy; GABARAP; GATE-16; LC3;
ubiquitylation-like modification
Correspondence
E Kominami, Department of Biochemistry,
Juntendo University School of Medicine,
2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421,
Japan
Fax: +81 3 5802 5889
Tel: +81 3 5802 1031
E-mail: kominami@med.juntendo.ac.jp
(Received 23 December 2005, revised
4 April 2006, accepted 5 April 2006)
doi:10.1111/j.1742-4658.2006.05260.x
Murine Atg8L⁄ Apg8L has significant homology with the other known mammalian Atg8 homologs, LC3, GABARAP and GATE-16 However, it
is unclear whether murine Atg8L modification is mediated by human Atg4B, Atg7 and Atg3 Expression of Atg8L in HEK293 cells led to clea-vage of its C-terminus In vitro, the C-terminus of Atg8L was cleaved by human Atg4B, but not human Atg4A or Atg4C Atg8L-I formed an E1-substrate intermediate with Atg7C572S, and an E2-substrate intermediate with Atg3C264S A modified form of Atg8L was detected in the pelletable fraction in the presence of lysosomal protease inhibitors under nutrient-rich conditions Cyan fluorescent protein (CFP)–Atg8L colocalized with yellow fluorescent protein (YFP)–LC3 in HeLa cells in the presence of the inhibi-tors However, little accumulation of the modified form of Atg8L was observed under conditions of starvation These results indicate that Atg8L
is the fourth modifier of mammalian Atg8 conjugation
Abbreviations
CFP, cyan fluorescent protein; GABARAP, 4-aminobutyrate A receptor-associated protein; GABARAP-PL, GABARAP–phospholipid conjugate; GFP, green fluorescent protein; LC3, human microtubule-associated protein 1 light chain 3; LC3-I, soluble unmodified form of LC3; LC3-II, LC3–phospholipid conjugate; PL, phospholipid; PVDF, poly(vinylidene difluoride); TRX, thioredoxin; YFP, yellow fluorescent protein.
Trang 2phospholipid bound to LC3 [13,20] LC3-II and
GABARAP-PL are both deconjugated by human
Atg4B [15] It is unclear whether the target of
GATE-16 is a phospholipid
Recently, a fourth mammalian Atg8 homolog has
been reported, mouse Atg8L⁄ Apg8L [21] The amino
acid sequence of mouse Atg8L⁄ Apg8L shows 100%
and 54% identity to those of human Atg8L and yeast
Atg8, respectively, and 33%, 60% and 86% identity to
the amino acid sequences of LC3, GATE-16 and
GABARAP, respectively In addition, Atg8L shares a
conserved Gly at its C-terminus Following chemical
modification of Atg8L with a C-terminal vinyl sulfone,
mouse Atg8L–vinyl sulfone was shown to react with
Atg4B, suggesting that mouse Atg8L is a substrate of
Atg4B [21] However, no direct evidence that mouse
Atg8L is cleaved by Atg4B has yet been reported
Fur-thermore, both the homology between Atg8L and the
three other mammalian Atg8 homologs and the
con-served C-terminal Gly in all four proteins suggest that
Atg8L may also be a substrate of mammalian Atg7
and Atg3 Therefore, we examined whether Atg8L is a
substrate of these three enzymes involved in
mamma-lian Atg8 conjugation
Results
The C-terminus of Atg8L is cleaved in HEK293
cells, and the Gly116 of Atg8L is essential for
cleavage
The C-termini of yeast Atg8, mammalian LC3,
GABA-RAP and GATE-16 are post-translationally cleaved to
expose a Gly residue, which is essential for ubiquitin-like
modification [3,10,12] This consensus Gly116 is
con-served in Atg8 and its mammalian homologs [21], and is
present in Atg8L To determine whether the C-terminus
of Atg8L is post-translationally cleaved, we constructed
an Atg8L expression vector tagged with a Myc epitope
at its N-terminus and a 3xFLAG epitope at its
C-termi-nus (Fig 1A, Myc–Atg8Lwt)3xFLAG) Following
transfection of HEK293 cells with this construct, the
lysates were analyzed by SDS⁄ PAGE A protein of
18 kDa corresponding to Myc–Atg8L, a C-terminal
cleaved form of Myc–Atg8Lwt)3xFLAG, was
recog-nized by immunoblotting with anti-Myc, but not with
anti-FLAG (Fig 1B)
We next investigated whether the Gly116 residue of
Atg8L is essential for cleavage of its C-terminus by
changing Gly116 of Myc–Atg8Lwt)3xFLAG to Ala
by site-directed mutagenesis (Fig 1A,
Myc–Atg8L-GA)3xFLAG), and expressing the mutant protein in
HEK293 cells (Fig 1B, GA) The mobility of mutant
Myc–Atg8LGA)3xFLAG on SDS ⁄ PAGE was slower than that of wild-type Myc–Atg8Lwt)3xFLAG More-over, the mutant protein was recognized by immuno-blotting with both anti-Myc and anti-FLAG (Fig 1B) Similar results were obtained when the Gly116 residue was deleted by site-directed mutagenesis (Fig 1A, Myc–Atg8LDG)3xFLAG; Fig 1B, DG) These results suggest that the C-terminus of Atg8L is post-transla-tionally cleaved in HEK293 cells, and that Gly116 of Atg8L is essential for the cleavage of its C-terminus The cleaved form of Atg8L was designated Atg8L-I
Atg4B cleaves the C-terminus of Atg8L in vitro The three previously identified mammalian Atg8 homo-logs, LC3, GABARAP, and GATE-16, were shown to
be cleaved by human Atg4B (hAtg4B) in vitro [13,15] Although we showed that the C-terminus of Atg8L was cleaved soon after its translation in HEK293 cells, the enzyme responsible for this activity could not be identi-fied Therefore, we examined whether Atg4B has pro-teolytic activity on the C-terminus of Atg8L FLAG– hAtg4B was expressed in HEK293 cells, the cells were lysed, and the lysate was fractionated by ultracentrifuga-tion, with the resulting supernatant used as the enzyme mixture FLAG–hAtg4B in the supernatant was recog-nized by immunoblotting with anti-FLAG (Fig 1C, FLAG–hAtg4B) The substrate, wild-type thioredoxin (TRX)–Atg8L)3xFLAG, consisting of Atg8L with TRX at its N-terminus and the 3xFLAG epitope at its C-terminus, was expressed in Escherichia coli, and the supernatant of this cell lysate was used as the substrate solution When we incubated the two supernatants con-taining FLAG–hAtg4B with TRX–Atg8L)3xFLAG,
we found that the C-terminus of Atg8L was cleaved, as shown by immunoblotting with anti-FLAG (Fig 1C) Using anti-TRX, we confirmed that the N-terminal TRX tag within each substrate remains unchanged, indicating that the C-terminal FLAG tag was cleaved
by hAtg4B (Fig 1C) When an active site mutant of hAtg4B, hAtg4BC74A[15], was used instead of wild-type hAtg4B, little cleavage occurred (Fig 1D, lane 2 versus lane 1) When a mutant in which the Gly116 residue of Atg8L had been deleted, TRX–Atg8LDG)3xFLAG, was employed instead of the wild type, little C-terminal cleavage occurred (Fig 1D, lane 5 versus lane 1)
In addition to hAtg4B, hAtg4A⁄ hApg4A ⁄ autophag-in-2, hAtg4C⁄ hAutl1 ⁄ autophagin-3 and hAtg4D⁄ autophagin-4 have also been reported [16,22] Of these three Atg4 homologs, hAtg4A has been shown to cleave the C-terminus of GATE-16 [16], and autophag-in-3⁄ hAtg4C ⁄ hAutl1, but not hAtg4D, has been shown
to exert N-ethylmaleimide-sensitive proteolytic activity
Trang 3on the synthetic substrate
Mca-Thr-Phe-Gly-Met-Dpa-NH2 [22] Therefore, hAtg4A and hAtg4C may also
cleave the C-terminus of Atg8L When hAtg4A or
hAtg4C was used instead of hAtg4B for in vitro
diges-tion, little cleavage of the C-terminal 3xFLAG tag of
TRX–Atg8L)3xFLAG occurred (Fig 1, lanes 3 and 4
versus lane 1) These results indicate that Atg4B
cleaves the C-terminus of Atg8L in vitro
Atg8L-I forms an E1-substrate intermediate with
human Atg7C572S
In the ubiquitlyation-like modification steps, an active
site Cys within an E1 enzyme temporally conjugates to
a substrate to form an E1-substrate intermediate via a
thiol–ester bond Owing to the rapid turnover of an
E1 reaction, it is difficult to recognize such an interme-diate in sufficient quantity When an active site Cys residue of human Atg7 is changed to Ser, a stable O-ester bond instead of a thiol–ester bond will be formed between the enzyme and substrate(s) Previ-ously, we showed that the mammalian Atg8 homologs LC3, GATE-16 and GABARAP form E1-substrate intermediates with an active site mutant human Atg7C572S, in which the active site Cys572 of human Atg7 was changed to Ser12 If Atg8L-I is a substrate
of human Atg7, Atg8L-I will form an E1-substrate intermediate with human Atg7C572S Therefore, we investigated whether Atg8L-I also forms an intermedi-ate with human Atg7C572S When Myc–Atg8Lwt )3x-FLAG and Atg7C572S were expressed together, an Atg7C572S–Myc–Atg8L (E1-substrate) intermediate was observed (Fig 2) This intermediate was also detected
by immunoblotting with Myc, but not with
anti-Fig 1 The C-terminus of Atg8L is cleaved in vivo and in vitro (A)
Schematic representation of mutant proteins of Myc–Atg8L
)3x-FLAG The arrowhead indicates a Gly residue predicted to be
essential for ubiquitylation-like reactions Myc–Atg8Lwt )3xFLAG
represents wild-type Atg8L protein tagged with the Myc epitope at
its N-terminus and with the 3xFLAG epitope at the C-terminus.
Myc–Atg8LGA )3xFLAG represents a mutant protein in which the
Gly116 of Atg8L was changed to Ala, and Myc–Atg8LDG )3xFLAG
represents a mutant protein in which the Gly116 of Atg8L was
deleted (B) Cleavage of the C-terminus of Atg8L Wild-type or
mutant Myc–Atg8L )3xFLAG proteins were transiently expressed in
HEK293 cells The cells were lysed, total proteins were separated
by SDS⁄ PAGE, and wild-type and mutant Myc–Atg8L )3xFLAG
were recognized by immunoblotting with Myc (a-Myc) and
anti-FLAG (a-anti-FLAG) wt, GA, and DG were the same as in (A) (C) In
vit-ro assay for Atg4B cleavage of the C-terminus of Atg8L Wild-type
TRX–Atg8L )3xFLAG was expressed in Escherichia coli; the cells
were lysed and centrifuged, and the supernatants were used as
the substrate The supernatants of HEK293 cells transfected with
pTag2B–hATG4B (hAtg4B wild) were used as enzymes; the
negat-ive control consisted of the supernatants of HEK293 cells
transfect-ed with pCMV–Tag2B (control) Following incubation of 1 lg of
enzyme solution with 10 lg of substrate solution for the indicated
times (incubation time), the reactions were stopped, and the total
proteins in each mixture were separated by SDS ⁄ PAGE TRX–
Atg8L )3xFLAG was recognized by immunoblotting with
anti-thiore-doxin (TRX), and FLAG–hAtg4B and the C-terminal 3-FLAG tag of
TRX–Atg8L )3xFLAG were recognized with anti-FLAG TRX–
Atg8L )3xFLAG, uncleaved form of TRX–Atg8L)3xFLAG; TRX–
Atg8L-I, cleaved form of TRX–Atg8L )3xFLAG; FLAG–hAtg4B,
FLAG-tagged Atg4B cysteine protease (D) In vitro assay for the
cleavage of the C-terminus of Atg8L by an active site mutant
hAtg4 C74A or other Atg4 homologs As an enzyme solution,
supern-atants of HEK293 cells expressing an active site mutant
hAtg4B C74A (hAtg4B C74A), wild-type hAtg4A (hAtg4A wild) or
wild-type hAtg4C (hAtg4C) were employed instead of wild-type
hAtg4B (hAtg4B wild) Supernatant containing mutant TRX–
Atg8LDG )3xFLAG (DG), in which the Gly116 of Atg8L was deleted,
was used as the substrate The enzyme solution and the substrate
solution were mixed and incubated for 30 min.
A
B
C
D
Trang 4FLAG These results indicate that, like the other Atg8
homologs, Atg8L-I forms an E1-substrate intermediate
with human Atg7C572S
Atg8L-I forms an E2-substrate intermediate with
green fluorescent protein (GFP)–Atg3C264S; this is
dependent on Atg7
Owing to the rapid turnover of the E2 reaction, it is
difficult to recognize such an E2-substrate intermediate
with an E2-like enzyme When the active site Cys
resi-due of the E2-like enzyme is replaced by Ser, a stable
O-ester bond instead of a thiol–ester bond will be
formed between the enzyme and substrate(s); this is
dependent on an E1-like enzyme Previously, we
showed that LC3, GABARAP and GATE-16 form
E2-substrate intermediates with an active site mutant human Atg3C264S and that this activity was dependent
on human Atg7 [19] If Atg8-L is a substrate of Atg3, Atg8L-I will form an E2-substrate intermediate with human Atg3C264S that is dependent on Atg7 There-fore, we investigated whether Atg8L-I also forms an Atg7-dependent E2-substrate intermediate with human Atg3 and whether formation of this intermediate occurs via the Gly116 in Atg8L When wild-type Myc–Atg8Lwt)3xFLAG, GFP–Atg3C264S and wild-type Atg7 were expressed in HEK293 cells, a GFP– Atg3C264S–Myc–Atg8L (E2-substrate) intermediate was formed (Fig 3, lane 2) When mutant Myc– Atg8LDG)3xFLAG was substituted for wild-type Atg8L (Fig 3, lane 3), no intermediate was observed,
as demonstrated by immunoblotting with anti-GFP When Atg7 was not overexpressed (endogenous Atg7 alone), no intermediate was observed, while endo-genous Atg7 was detected by anti-Atg7 (Fig 3, lane
Fig 2 Atg8L-I forms an E1-substrate intermediate with human
Atg7C572S Myc-tagged Atg8L )3xFLAG (Myc–Atg8L)3xFLAG) was
transiently expressed with wild-type (Atg7 wt) or mutant human
Atg7 (Atg7 C572S) The cells were lysed, the proteins were
separ-ated by SDS ⁄ PAGE, and human Atg7 was recognized by
immuno-blotting with anti-human Atg7 (WB:a-Atg7), while Myc–Atg8L-I was
recognized by immunoblotting with anti-Myc (WB:a-Myc) Atg7–
Atg8L-I intermediate, E1-substrate intermediate between human
Atg7C572Sand Atg8L; Atg7, human Atg7; Myc–Atg8L-I, Myc-tagged
Atg8L-I.
Fig 3 Atg8L-I forms an E2-substrate intermediate with human Atg3 C264S , which is dependent on human Atg7 Myc–Atg8Lwt )3x-FLAG (wt) was transiently expressed together with green fluores-cent protein (GFP)–Atg3C264S in the presence (Atg7+) or absence (Atg7–) of human Atg7 DG represents mutant Myc–Atg8LDG )3x-FLAG lacking the Gly116 of Atg8L (see Fig 1A) After lysing the cells, total proteins were separated by SDS ⁄ PAGE Atg7 was recognized by immunoblotting with anti-human Atg7 (WB:a-Atg7), GFP–Atg3 C264S and its E2-substrate intermediate were recognized with anti-GFP (WB:a-GFP), and Myc-tagged Atg8L proteins were recognized by immunoblotting with anti-Myc (WB:a-Myc) The faint band in lane 3 (DG) of the middle panel (WB:a-GFP) is GFP– Atg3 C264S -endogenous Atg8 homolog intermediate(s).
Trang 51) These results indicate that Atg8L-I forms an
Atg7-dependent E2-substrate intermediate with human Atg3
via its Gly116
Atg8L-II is increased in the presence of E64d and
pepstatin A, inhibitors of lysosomal proteases,
in HeLa cells
LC3-I and GABARAP-I have been shown to be
modi-fied by Atg7 and Atg3 to form the respective protein–
phospholipid conjugates, LC3-II and GABARAP-PL
[15] We have reported the accumulation of LC3-II
and GABARAP-PL in HeLa cells incubated with
E64d [23], a membrane-permeable inhibitor of
cathep-sins B, H, and L, and pepstatin A [24], an inhibitor of
cathepsins D and E, for 24 h under nutrient-rich
con-ditions [15,25] These results suggest that a modified
form of Atg8L-I may accumulate under the same
con-ditions When HeLa cells expressing Myc–Atg8L were
incubated with E64d and pepstatin A for 24 h under
nutrient-rich conditions, LC3-II and GABARAP-PL
accumulated (Fig 4A, WB:a-LC3 and a-GABARAP)
Under these conditions, we observed two bands by
immunoblotting with anti-Myc (Fig 4A, WB:a-Myc)
One band corresponded to Myc–Atg8L-I, while the
other showed faster mobility This second band,
repre-senting a modified form of Atg8L, was designated
Atg8L-II
We have also shown that the modified forms of
LC3 and GABARAP, LC3-II and GABARAP-PL,
are present in the pellet after subcellular
fraction-ation [15] Therefore, we hypothesized that, if
Atg8L-II is a modified form and not a degradation
product of Atg8L-I, it would be present in the pellet
of inhibitor-treated HeLa cells Therefore, we
centri-fuged cell lysates at 100 000 g for 1 h and examined
the subcellular localization of Myc–Atg8L-I and
Myc–Atg8L-II by immunoblotting with anti-Myc
Our results indicated that Myc–Atg8L-II was present
in the pellet, whereas Myc–Atg8L-I was present in
the supernatant (Fig 4B), suggesting that Myc–
Atg8L-II is a modified form of Myc–Atg8L-I, and
not a degradation product
We next investigated the intracellular localization of
Atg8L-II under these conditions using cyan fluorescent
protein (CFP)–Atg8L HeLa cells expressing both
CFP–Atg8L and yellow fluorescent protein (YFP)–
LC3 were cultured under nutrient-rich conditions in
the presence of E64d and pepstatin A for 24 h, and
punctate signals of both CFP–Atg8L (Fig 4C,e,h,k)
and YFP–LC3 (Fig 4C,d,g,j) were observed by
fluor-escent microscopy Like Myc–Atg8L, CFP–Atg8L can
form E1-substrate and E2-substrate intermediates with
human Atg7C572S and human Atg3C264S, respectively (Fig 4D,E), and little degradation of CFP–Atg8L was observed even in the presence of these inhibitors for
24 h under nutrient-rich conditions (Fig 4F), indica-ting that the fluorescence signal reflects intact tagged protein Merging of the images indicated that most of the puncta of CFP–Atg8L were colocalized with those
of YFP–LC3 (Fig 4,f,I,l) In the absence of inhibitors, only a few puncta of either type were observed (Fig 4C,a–c)
Little Atg8L-II accumulates under conditions of starvation even in the presence of E64d and pepstatin A in HeLa cells
LC3-I is significantly lipidated to form LC3-II under conditions of starvation [10], and LC3-II is degraded
in the lysosome [25] Therefore, in the presence of E64d and pepstatin A, LC3-II shows significant accu-mulation under conditions of starvation [25] It is poss-ible that, like LC3, Atg8L is modified to Atg8L-II under conditions of starvation Therefore, we investi-gated whether Atg8L-II accumulates in HeLa cells expressing Myc–Atg8L)3xFLAG incubated in Krebs– Ringer buffered medium for 4 h under conditions of starvation in the presence of E64d and pepstatin A, conditions under which LC3-II has been reported to accumulate [25] (Fig 5, LC3-II) However, little Atg8L-II accumulation was observed (Fig 5, lane 4 versus lane 3) Even when cells were incubated with the inhibitors under nutrient-rich conditions for a short time—4 h compared with 24 h (Fig 4A)—little accumulation of Atg8L-II occurred (Fig 5, lane 2)
Discussion
Here, we have shown that Atg8L is a substrate of reac-tions mediated by human Atg4B, Atg7, and Atg3 We found that the C-terminus of Atg8L is post-transla-tionally cleaved and that the Gly116 in Atg8L is essen-tial for this reaction, which is mediated by human Atg4B, a cysteine protease, in vitro We also found that Atg8L forms an E1-substrate intermediate with an E1-like enzyme, the active site mutant Atg7C572S, and
an E2-substrate intermediate with an E2-like enzyme, the active site mutant Atg3C264S, with the latter reac-tion dependent on Atg7 All these reacreac-tions are similar
to a series of reactions of three other mammalian Atg8 homologs: LC3, GABARAP, and GATE-16 In addi-tion, we showed that a modified form of Atg8L, Atg8L-II, accumulates in HeLa cells in the presence of lysosomal protease inhibitors under nutrient-rich con-ditions, comparable to the accumulation of LC3-II
Trang 6B
C
Fig 4 Atg8L-II, a modified form of Atg8L, accumulates in HeLa cells in the presence of inhibitors of lysosomal proteases, E64d and pepstatin A (A) Accumulation of Atg8L-II in the presence of E64d and pepstatin A Myc–Atg8L )3xFLAG was transiently expressed in HeLa cells, and the transfectants were incubated for 24 h in the presence (+) or absence (–) of E64d and pepstatin A The cells were lysed, and total proteins were separated by SDS ⁄ PAGE Endogenous LC3 and GABARAP were recognized by immunoblotting with anti-LC3 and 4-aminobutyrate A -receptor associated protein (GABARAP), respectively (WB:a-LC3 and a-GABARAP) Myc–Atg8L was recognized with anti-Myc (WB:a-Myc) (B) Subcellular localization of Myc–Atg8L-II Cell lysates of inhibitor-treated HeLa cells (A, E64d and pepstatin A+) were fractionated into pellet (Ppt) and soluble (Sup) fractions by ultracentrifugation at 100 000 g for 1 h [12,15] LC3-I, soluble unlip-idated form of LC3; LC3-II, lipunlip-idated membrane-bound form of LC3; GABARAP-I, unlipunlip-idated form of GABARAP; GABARAP-PL, lipunlip-idated form of GABARAP; Myc–Atg8L-I, soluble form of Myc–Atg8L; Myc–Atg8L-II, membrane-bound form of Myc–Atg8L (C) Intracellular local-ization of cyan fluorescent protein (CFP)–Atg8L HeLa cells expressing both CFP–Atg8L and yellow fluorescent protein (YFP)–LC3 were cultured in the absence (dimethylsulfoxide (DMSO)) or presence of E64d and pepstatin A (E64d and pepstatin A) for 24 h After fixing, cyan and yellow fluorescence in HeLa cells were observed with a Zeiss Axioplan2 fluorescence microscope with filters XF114-2 and XF104-2 The deconvoluted images are shown in (C) In a, d, g, and j, yellow fluorescent images correspond to YFP–LC3 In b, e, h, and
k, cyan fluorescent images correspond to CFP–Atg8L Merged images (Merge) are shown in e, f, i, and l Arrowheads indicate colocaliz-ation of YFP–LC3 and CFP–Atg8L (D) Formcolocaliz-ation of an E1-substrate intermediate with Atg7C572Sand CFP–Atg8L CFP–Atg8L was trans-iently expressed with wild-type (Atg7 wt) or mutant human Atg7 (Atg7 C572S) The cells were lysed, the proteins were separated by SDS ⁄ PAGE, and human Atg7 was recognized by immunoblotting with anti-human Atg7 (WB:a-Atg7), and CFP–Atg8L was recognized by immunoblotting with anti-GFP (WB:a-GFP) Atg7–Atg8L intermediate, E1-substrate intermediate between human Atg7C572S and Atg8L; Atg7, human Atg7; CFP–Atg8L, CFP-tagged Atg8L (E) Formation of an E2-substrate intermediate with Atg3 C264S S and CFP–Atg8L CFP– Atg8L was transiently expressed together with GFP–Atg3 C264S in the presence of human Atg7 (Atg7+) After lysing the cells, total pro-teins were separated by SDS ⁄ PAGE Atg7 was recognized by immunoblotting with anti-human Atg7 (WB:a-Atg7), GFP–Atg3 C264S and its E2-substrate intermediate were recognized with anti-Atg3 (WB:a-Atg3), and CFP–Atg8L was recognized by immunoblotting with anti-Atg8L (WB:a-Atg8L) (F) There was little degradation of CFP–Atg8L in the presence of E64d and pepstatin A for 24 h under nutrient-rich condit-ions CFP–Atg8L and YFP–LC3 were transiently expressed in HeLa cells, and the transfectant was incubated for 24 h in the presence (+)
or absence (–) of E64d and pepstatin A The cells were lysed, and total proteins were separated by SDS ⁄ PAGE YFP–LC3 and CFP–Atg8L were recognized by immunoblotting with anti-GFP (WB:a-GFP) CFP–Atg8L was recognized with anti-Atg8L (WB:a-Atg8L) Positions of molecular weight markers for SDS ⁄ PAGE are indicated on the right of the panel.
Trang 7and GABARAP-PL under identical conditions
More-over, we found that Atg8L-II was fractionated in the
pellet These results indicate that Atg8L is a substrate
of Atg4B, Atg7, and Atg3, and that Atg8L is the
authentic fourth modifier in the mammalian Atg8
con-jugation system Previously, we showed that LC3-II
and GABARAP-PL are protein–phospholipid
conju-gates in vivo [15], and that these two conjuconju-gates, as
well as GATE-16-II, are localized to a membrane
compartment [10,12] Like LC3-II and
GABARAP-PL, Atg8L-II was fractionated in the pelletable
fraction, suggesting that Atg8L-II may be a
protein–phospholipid conjugate that is localized to a membrane compartment
The modified form, Myc–Atg8L-II, was observed only in the presence of E64d and pepstatin A for 24 h under nutrient-rich conditions (Fig 4A), whereas little Myc–Atg8L was observed for a shorter period (4 h) even in the presence of these inhibitors In contrast, LC3-II accumulates in the presence of E64 and pepstatin
A within only 4 h under both nutrient-rich and starva-tion condistarva-tions There are two possible reasons for the low level of Myc–Atg8L-II accumulation for 4 h in the presence of these inhibitors The first is that
F
Fig 4 (Continued).
Trang 8tion of Atg8L-I to Atg8L-II may occur much more
slowly than that of LC3-I to LC3-II Therefore, Myc–
Atg8L-II was observed only after a longer incubation
time (24 h) in the presence of E64d and pepstatin A
The other possibility is that Myc–Atg8L-II may be very
unstable compared with LC3-II We have reported that
Atg4B is a delipidating enzyme for LC3-II and
GABA-RAP-PL in addition to being a cysteine protease that
cleaves the C-termini of mammalian Atg8 homologs
Therefore, if Atg4B delipidates Myc–Atg8L-II more
effectively than LC3-II, there will be less accumulation
of Myc–Atg8L-II compared with LC3-II These points
will be clarified in future studies by in vitro assays of
lipidation and delipidation of Atg8L
Experimental procedures
Materials, and biochemical and molecular
biological techniques
Molecular biological and biochemical techniques were
per-formed as described [25] To clone mouse Atg8L cDNA by
PCR, we used high-fidelity KOD plus DNA polymerase (Toyobo, Osaka, Japan) A plasmid containing mouse Atg8L cDNA was transfected into HEK293 cells with Lipofectamine 2000 reagent according to the manufac-turer’s protocol (Invitrogen, Carlsbad, CA) E coli strain DH5a cells, the hosts for plasmids and protein expression, were grown in Luria broth medium in the presence of anti-biotics as required Restriction enzymes were purchased from Toyobo and New England Biolabs (Beverly, MA) Oligonucleotides were synthesized by Invitrogen pGEM-T was purchased from Promega (Madison, WI), and p3xFLAG–CMV14 was obtained from Sigma-Aldrich (St Louis, MO)
Cloning of mouse Atg8L cDNA and site-directed mutagenesis
A DNA fragment encoding the entire open reading frame
of ATG8L, according to a cDNA sequence in GenBank (accession number: BG244294) [21], was amplified by high-fidelity PCR from a Marathon ready mouse brain cDNA library (BD Biosciences Clontech, Palo Alto, CA) and inserted into pGEM-T The resulting plasmid was designa-ted pGEM–mATG8L
Using the Gene-Editor in vitro site-directed mutagenesis system (Promega) and the plasmid pGEM–mATG8L, Gly116 in mouse Atg8L was replaced by Ala in accordance with the manufacturer’s protocol, and the resulting plasmid was designated pGEM–mATG8LG116A Gly116 in mouse Atg8L of pGEM–mATG8L was deleted by site-directed mutagenesis, and the resulting plasmid was designated pGEM–mATG8LDG Mammalian expression vectors for N-terminal Myc-tagged and C-terminal 3xFLAG-tagged versions of Atg8L, Myc–Atg8Lwt)3xFLAG,
Myc–Atg8L-GA)3xFLAG and Myc–Atg8LDG)3xFLAG were con-structed from pGEM–mATG8L and p3xFLAG–CMV14 by high-fidelity PCR, and designated pMyc–mATG8L) 3xFLAG, pMyc–mATG8LGA)3xFLAG, and pMyc– mATG8LDG)3xFLAG, respectively An expression vector for N-terminal TRX-tagged and C-terminal 3xFLAG-tagged Atg8L in E coli, TRX–Atg8L)3xFLAG, was con-structed based on pThioHisA (Invitrogen) and designated pTRX–ATG8L)3xFLAG A mammalian expression vector for N-terminal CFP-tagged Atg8L designated pCFP– ATG8L was generated by inserting the open reading frame
of mAtg8L excised from pGEM–mATG8L into pCFP-C1 (BD Biosciences Clontech) The DNA sequences of new constructs were confirmed using a ABI PRISMTM310 Gen-etic Analyzer (Applied Biosystems, Foster City, CA) The expression vectors pCMV–hAPG7, pCMV–hAPG7CS, pGFP–hAPG3, pGFP–hAPG3CS, pTag2B–HsATG4B, pTag2B–HsATG4BC74A, pTag2B–HsATG4A and pTag2B– HsAUTL1 for human Atg7, mutant Atg7C572S, GFP–Atg3, mutant GFP–Atg3C264S, wild-type FLAG–hAtg4B, mutant FLAG–hAtg4BC74A, type FLAG–hAtg4A and
wild-Fig 5 There was little modification of Atg8L under conditions of
starvation, even in the presence of inhibitors of lysosomal proteases.
Myc–Atg8L )3xFLAG was transiently expressed in HeLa cells The
transfectant was transferred to Krebs–Ringer buffered medium
(KRB, 4 h, starvation) or Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (DMEM, 10% fetal
bovine serum, nutrient-rich), and incubated for 4 h in the presence
(+) or absence (–) of inhibitors Little Myc–Atg8L-II was detected by
immunoblotting with anti-Myc under either condition Endogenous
LC3 was recognized by immunoblotting with antibodies to LC3 As a
control, the membrane was stained with Coomassie Brilliant Blue.
LC3-II, LC3–phospholipid conjugate.
Trang 9type FLAG–hAtg4C⁄ AutL1, respectively, were as described
[15,17,19]
Cell culture
The HEK293 and HeLa cell lines were purchased from the
ATCC (Manassas, VA) and cultured in 60-mm dishes in
Dulbeccos’s modified Eagles’s medium (DMEM,
Invitro-gen) containing 10% fetal bovine serum (InvitroInvitro-gen) in a
humidified 5% CO2atmosphere at 37C
Antibodies
For preparation of antiserum against mouse Atg8L, rabbits
were immunized with a TRX–Atg8L fusion protein
Anti-human GABARAP, LC3, Atg7 and Atg3 were prepared
and purified as described [12,17,19,26] Anti-Myc was
pur-chased from Cell Signaling (Beverly, MA), anti-FLAG
M2 was purchased from Sigma-Aldrich, anti-TRX was
pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA),
and anti-GFP was purchased from BD Biosciences Clontech
Immunoblotting analyses
Protein concentrations were determined using the
bicincho-ninic acid protein assay reagent (Pierce, Rockford, IL)
Immunoblotting analyses were carried out according to
standard protocols using a chemiluminescent method with
SuperSignal West Dura Extended Duration Substrate or
SuperSignal West Pico Chemiluminescent Substrate (Pierce)
In vitro assay for cleavage of the C-terminus of
TRX–Atg8L)3xFLAG by Atg4B
The in vitro assay for cleavage of the C-terminus of mouse
Atg8L was performed as previously described [15]
Fluorescence microscopy
HeLa cells expressing YFP–hLC3 and CFP–mAtg8L were
fixed according to the manufacturer’s protocol (BD
Bio-sciences Clontech) Briefly, after transfection of pYFP–LC3
and pCFP–ATG8L into HeLa cells using
Lipofecta-mine2000 (Invitrogen), cells were incubated for 24 h under
nutrient-rich conditions in the presence or absence of E64d
and pepstatin A Thereafter, cells were washed twice with
NaCl⁄ Pi, and fixed in NaCl⁄ Pi containing 4%
paraformal-dehyde for 10 min at room temperature Fixed cells were
washed three times with NaCl⁄ Pi, and then mounted on
slide-glasses with SlowFade Light antifade reagent (50%
glycerol solution) (Invitrogen) Fluorescence of YFP and
CFP in the cells was monitored with a Zeiss Axioplan2
fluorescence microscope (Carl Zeiss, Thornwood, NY)
equipped with an ORCA-ER CCD camera (Hamamatsu
Photonics, Tokyo, Japan) For deconvolution of the ima-ges, a Zeiss Axioplan2 fluorescence microscope (Carl Zeiss) and an Aqua C-imaging system (Hamamatsu Photonics) were employed
Acknowledgements
This work was supported in part by grants-in-aid
15590254 (to IT), 09680629 (to TU) and 12470040 (to EK) for Scientific Research, and grant-in-aid 12146205 (to EK) for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Cul-ture of Japan, and The Science Research Promotion Fund from the Japan Private School Promotion Foun-dation (to EK)
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