Control of autophagy initiation by phosphoinositide 3-phosphatase Jumpy

Article (PDF Available)inThe EMBO Journal 28(15):2244-58 · August 2009with53 Reads
DOI: 10.1038/emboj.2009.159 · Source: PubMed
Abstract
The majority of studies on autophagy, a cytoplasmic homeostasis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3-kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3-phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI-1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.
Control of autophagy initiation by
phosphoinositide 3-phosphatase jumpy
Isabelle Vergne
1,
*, Esteban Roberts
1
,
Rasha A Elmaoued
1
, Vale
´
rie Tosch
2
,
Mo
´
nica A Delgado
1
, Tassula Proikas-
Cezanne
3
, Jocelyn Laporte
2
and
Vojo Deretic
1,4,
*
1
Department of Molecular Genetics and Microbiology, University of New
Mexico School of Medicine, Albuquerque, NM, USA,
2
Departments of
Neurobiology and Genetics, IGBMC, INSERM U596, CNRS UMR,
Universite
´
Louis Pasteur de Strasbourg, Colle
`
ge de France, Illkirch,
France,
3
Department of Molecular Biology, University of Tuebingen,
Tuebingen, Germany and
4
Department of Cell Biology and Physiology,
University of New Mexico School of Medicine, Albuquerque, NM, USA
The majority of studies on autophagy, a cytoplasmic
homeostatis pathway of broad biological and medical
significance, have been hitherto focused on the phospha-
tidylinositol 3-kinases as the regulators of autophagy.
Here, we addressed the reverse process driven by phos-
phoinositide phosphatases and uncovered a key negative
regulatory role in autophagy of a phosphatidylinositol
3-phosphate (PI3P) phosphatase Jumpy (MTMR14).
Jumpy associated with autophagic isolation membranes
and early autophagosomes, defined by the key factor
Atg16 necessary for proper localization and development
of autophagic organelles. Jumpy orchestrated orderly suc-
cession of Atg factors by controlling recruitment to autop-
hagic membranes of the sole mammalian Atg factor that
interacts with PI3P, WIPI-1 (Atg18), and by affecting the
distribution of Atg9 and LC3, the two Atg factors control-
ling organization and growth of autophagic membranes. A
catalytically inactive Jumpy mutant, R336Q, found in
congenital disease centronuclear myopathy, lost the ability
to negatively regulate autophagy. This work reports for
the first time that initiation of autophagy is controlled not
only by the forward reaction of generating PI3P through a
lipid kinase but that its levels are controlled by a specific
PI3P phosphatase, which when defective can lead to
human disease.
The EMBO Journal (2009) 28, 2244–2258. doi:10.1038/
emboj.2009.159; Published online 9 July 2009
Subject Categories: differentiation & death; molecular biology
of disease
Keywords: Atg18; autophagy; LC3; myopathy;
PI3P phosphatase
Introduction
Autophagy is an ancient, highly conserved eukaryotic intra-
cellular homeostatic process carrying out degradation of
cytoplasmic components including damaged or superfluous
organelles, toxic protein aggregates and intracellular patho-
gens (Levine and Deretic, 2007; Levine and Kroemer, 2008;
Mizushima et al, 2008). Autophagy takes place at basal levels
in all eukaryotic cells, turning over long-lived macromole-
cules and large supra-molecular structures including whole
organelles. In addition to its housekeeping role, autophagy
can be upregulated during metabolic, genotoxic or hypoxic
stress conditions and acts as an adaptive mechanism essen-
tial for cell survival (Levine and Kroemer, 2008). Dysfunc-
tional autophagy, when defective or excessive, has been
linked to human pathologies ranging from neurodegeneration
and myopathies to cancer and inflammatory diseases (Levine
and Deretic, 2007; Mizushima et al, 2008). The autophagy-
associated clinical conditions can be due to increased or
decreased autophagy levels, underscoring the need to under-
stand both the induction of the autophagy pathway and its
downregulation. Thus far, identifying the brakes in the sys-
tem once it is set in motion by the upstream Tor-dependent
signalling systems has eluded a proper definition. And yet,
the autophagic process must be tightly regulated to support
cell survival when needed but also to avoid cell death and
injury through excessive autophagy.
A key signalling regulator of autophagy is the Akt/mTOR
pathway. Inhibition of mTOR kinase by specific inhibitor,
rapamycin or nutrient deprivation results in activation of
autophagy (Mizushima et al, 2008). Once triggered, the
morphologically detectable phase of autophagic execution
can be divided in several stages, beginning with formation
of isolation membrane and its elongation, followed by com-
pletion of an autophagosome and finally maturation through
fusion with lysosomes and degradation of the lumenal con-
tent (Mizushima et al, 2008). More than 31 autophagy-related
genes (ATG) have been identified in yeast with the core ATG
subset being conserved in mammalian cells (Xie and
Klionsky, 2007). Formation and elongation of an autophagic
isolation membrane (phagophore) requires two unique pro-
tein conjugation systems, resulting in the formation of an
Atg5–Atg12 conjugate, in a noncovalent complex with Atg16,
and in the C-terminally lipid-conjugated LC3 (Atg8) (Xie and
Klionsky, 2007; Yoshimori and Noda, 2008). These two
systems cooperate in phagophore expansion allowing it to
engulf cytosolic targets, to finally close and form a double-
membrane organelle termed the autophagosome. Once an
autophagosome is formed, the Atg5–Atg12/Atg16 complex is
released, whereas a portion of LC3 remains trapped within
the autophagosome and is degraded on autophagosome–
lysosome fusion to form the lytic organelle called autolyso-
some in a process referred to as maturation or flux. The
degradation of LC3 during flux could provide one level of
feedback inhibition in the system.
Received: 11 January 2009; accepted: 18 May 2009; published online:
9 July 2009
*Corresponding authors. I Vergne or V Deretic, Department of Molecular
Genetics and Microbiology, University of New Mexico School of
Medicine, Health Sciences Center, 915 Camino de Salud, Albuquerque,
NM 87131-001, USA. Tel.: þ 1 505 272 9579;
Fax: þ 1 505 272 6029; E-mail: ivergne@salud.unm.edu or
Tel.: þ 1 505 272 0291; Fax: þ 1 505 272 5309;
E-mail: vderetic@salud.unm.edu
The EMBO Journal (2009) 28, 2244–2258
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2009 European Molecular Biology Organization
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THE
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THE
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In addition to the above conjugation systems, phosphati-
dylinositol 3-phosphate (PI3P) has been implicated in the
autophagy pathway based on the pivotal role of the type III
PI3Kinase hVps34, an enzyme that generates PI3P, and its
autophagy-regulatory partner Beclin-1 (Atg6) (Liang et al,
1999; Levine and Kroemer, 2008; Yoshimori and Noda, 2008).
Inhibition of hVps34 blocks formation of autophagosomes,
whereas exogenous delivery of PI3P to cells stimulates au-
tophagy (Kabeya et al, 2000; Petiot et al, 2000). The Beclin-
1–hVps34 complex is additionally modified by Atg14 or
Vps38 (UVRAG) to promote autophagy initiation and matura-
tion stages, respectively (Itakura et al, 2008; Liang et al,
2008). Thus, much has been learned about the activation of
the autophagic pathway. In sharp contrast, little is known
about its intensity modulation and downregulation once
hVps34 and Beclin-1 are activated (Wei et al, 2008). The
location of PI3P formation and its enzymatic turnover in the
context of autophagy in mammalian cells remain undefined.
Furthermore, a role for a PI3P phosphatase has not been
implicated in any of the autophagy systems, possibly given
that Ymr1, the sole yeast myotubularin-like protein (Laporte
et al, 1998) (myotubularins in mammalian cells act as PI3P
phosphatases), has not been identified in screens for Atg
proteins. Considering the apparent absence of a role for a
PI3P turnover in yeast, in which nearly all known key
autophagy factors have been pioneered and identified, it is
possible that PI3P turnover might not be of significance for
autophagy in mammalian systems and in general.
Myotubularins (MTM) are a family of lipid phosphatases
(Figure 1A) that contain the consensus signature of the
tyrosine and dual-specificity phosphatase, His-Cys-X
2
-Gly-
X
2
-Arg. They specifically dephosphorylate PI3P and
PI(3,5)P
2
at the D3 position (Laporte et al, 2003; Clague
and Lorenzo, 2005; Robinson and Dixon, 2006). Sixteen
MTM family members (MTM1, MTMR1-15) have been iden-
tified in humans, but only nine of them seem to have catalytic
activity (Alonso et al, 2004). PI3P synthesis takes place on
membranes of diverse intracellular organelles; therefore, it is
likely that different myotubularins control specific cognate
intracellular pools of PI3P (Laporte et al, 2003; Clague and
Lorenzo, 2005; Robinson and Dixon, 2006). However, the
intracellular site of action and specific function of myotubu-
larins remain largely unknown. Here, we tested the hypo-
thesis that one or more myotubularins may control autop-
hagy in mammalian cells. We screened all active myotubu-
larin family members by siRNA knock-downs for their effects
on autophagy. We found that Jumpy (also known as
MTMR14) and, to a lesser extent MTMR6, affect autophagy.
We show that Jumpy inhibits autophagy by acting at the
autophagic isolation membrane stage, and that this function
is associated with a specific form of human genetic disease
called centronuclear myopathy (Tosch et al, 2006).
Results
PI3P phosphatase screen identifies Jumpy and MTMR6
as autophagy regulators
One of the key assays to monitor autophagy is LC3 immuno-
blotting (Kabeya et al, 2000). On autophagy induction, LC3-I
is C-terminally lipidated with phosphatidylethanolamine to
form LC3-II. The LC3-I to LC3-II conversion can be conve-
niently monitored by electrophoretic mobility shift from a
slower migrating form, LC3-I, to the faster migrating form
LC3-II. LC3 conversion depends on PI3kinase activity
(Kabeya et al, 2000). To test whether myotubularins are
involved in autophagic processes, the enzymatically active
members of the myotubularin family were knocked-down
with siRNA in RAW 264.7 macrophages and baseline or
induced LC3-II levels analysed by immunoblotting. RAW
264.7 macrophages were transfected for 48 h with control
(scramble) siRNA or siRNA specific for each of the myotu-
bularins, incubated for 2 h in full or starvation media and
probed for LC3 conversion by quantifying LC3-II relative to
the loading control. Among the eight catalytically active
murine myotubularins (MTM1, MTMR1, 2, 3, 4, 6, 7 and
Jumpy) (Figure 1A), only MTMR6, and Jumpy knock-downs
changed LC3-II/loading control ratios (Supplementary Figure
S1A–C). Knock-down of individual myotubularins were con-
firmed by RT–PCR or by immunoblotting (Supplementary
Figure S2) except for MTMR7. Treatment with siRNA against
Jumpy (Supplementary Figure S1B and D) and MTMR6
(Supplementary Figure S1C and F) increased LC3-II/GAPDH
after 2 h starvation in comparison to control siRNA.
To discern whether LC3-II increase following Jumpy or
MTMR6 knock-downs was due to autophagy stimulation or
inhibition of LC3-II degradation, Bafilomycin A1 (BafA1) and
lysosomal protease inhibitors were added during the 30-min
treatment (Mizushima and Yoshimori, 2007). Jumpy and
MTMR6 knock-downs increased LC3-II in starvation media
in the presence of inhibitors in excess to cells transfected with
control siRNA (Figure 1B). The effect of Jumpy knock-down
on LC3-II was more pronounced than MTMR6. Of note is that
increase in basal autophagy (full medium) as a result of
Jumpy knock-down equaled or exceeded in magnitude LC3-
II conversion caused by starvation (Figure 1B, graph). Thus,
Jumpy is needed to prevent spurious activation of autophagy
when it is not physiologically induced (e.g. by starvation).
The effects of Jumpy siRNA on enhanced LC3-II levels were
also pronounced in starvation media (Figure 1B, starvation
lanes). These results indicate that Jumpy knock-down
increases both basal and starvation-mediated autophagy.
Importantly, the effect of Jumpy knock-down was stronger
than or as strong as the effect of starvation (Figure 1B,
graph), which is used as a key physiological inducer of
autophagy applied as the gold standard in nearly all autop-
hagic studies (Levine and Deretic, 2007; Levine and Kroemer,
2008; Mizushima et al, 2008). Next, we determined whether
the raise in LC3-II level was accompanied by changes in
autophagic flux. LC3-II levels were measured in the presence
or absence of flux inhibitors. Jumpy siRNA increased autop-
hagic flux both in full and starvation media (Supplementary
Figure S3), indicating that Jumpy acts as a suppressor of the
autophagy pathway.
Jumpy controls autophagosome formation
and maturation in macrophages
As Jumpy siRNA showed the strongest effect on LC3-II levels,
we focused on this PI3P phosphatase. To determine whether
the increase in autophagosome formation caused by Jumpy-
knock-down also led to maturation into autolysosomes, we
used mRFP-GFP-LC3 probe developed to differentiate early
from late autophagic organelles (Kimura et al, 2007). On
autophagy induction, the lipidated LC3-II associates with
autophagosomal membranes, resulting in the formation of
Control of autophagy initiation by phosphoinositide 3-phosphatase jumpy
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punctate organelles that can be quantified by fluorescence
microscopy (Kabeya et al, 2000). mRFP-GFP-LC3 allows
distinction between early autophagic organelles (GFP
þ
RFP
þ
puncta) and mature, acidified autolysosomes (GFP
RFP
þ
puncta) as the GFP signal is quenched in acidic
compartments (Filimonenko et al, 2007; Kimura et al,
2007). RAW 264.7 macrophages knocked-down for Jumpy
and transfected with mRFP-GFP-LC3 displayed increased
total (GFP
þ
RFP
þ
plus GFP
RFP
þ
) LC3 puncta per cell in
both full and starvation media (Figure 1C and D;
Supplementary Figure S4). The number of GFP
þ
RFP
þ
punc-
ta per cell remained the same in full media and decreased in
starvation media with Jumpy siRNA-treated cells in compar-
ison to control (Figure 1C and D; Supplementary Figure S4).
In contrast, Jumpy knock-down resulted in increased
GFP
RFP
þ
puncta representing maturing autophagosomes
(Figure 1C and D; Supplementary Figure S4). In conclusion,
Jumpy knock-down increased the total number of autophagic
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organelles, indicating that reduced Jumpy expression stimu-
lated autophagosome formation. Furthermore, Jumpy knock-
down facilitated maturation of the newly formed autophago-
somes resulting in increased autolysosome numbers instead
of accumulation of autophagosomes. These data show that
Jumpy affects formation of autophagosomes and their ma-
turation into autolysosomes.
Jumpy inhibits autophagy in C2C12 cells
We next confirmed that the effects of Jumpy knock-downs on
autophagy detected in macrophages applied to other cell
types, by testing HeLa cells, mouse neuroblast Neuro-2A,
MEF cells (Supplementary Figures S5 and S6A) and myoblast
cell line C2C12 (Figure 1E–F; Supplementary Figure S6C).
Muscle cells were of particular interest, as missense muta-
tions in human populations within the Jumpy gene are
associated with sporadic cases of centronuclear myopathy
(Tosch et al, 2006). The mutations seen in patients result in a
loss or decrease in Jumpy PI3P phosphatase activity (Tosch
et al, 2006). Furthermore, overexpression of Jumpy results in
cellular PI3P decrease (Tosch et al, 2006). Although Jumpy
enzymatic activity is well defined (Tosch et al, 2006), its
cellular function and the cell biological consequences of its
loss are completely unknown. Therefore, we focused on
testing further whether and how Jumpy regulated autophagy,
by using mouse myoblast cell line C2C12. C2C12 myoblasts
were transfected for 48 h with Jumpy siRNA and incubated in
full or starvation media for 4 h in the presence or absence of
BafA1. In both full and starvation media, Jumpy siRNA
increased LC3-II levels compared with control samples. This
was also observed in the presence of autophagic flux inhi-
bitor, BafA1 (Supplementary Figure S6B and C), indicating an
increase in autophagosome formation.
We next assessed the maturation state of the autophago-
somes in C2C12 cells using the mRFP-GFP-LC3 construct
(Kimura et al, 2007). C2C12 were transfected for 48 h with
control or Jumpy siRNA, followed by a second transfection as
above plus mRFP-GFP-LC3 and incubated for 4 h in full or
starvation media. Although a knock-down of Jumpy did not
change the number of GFP
þ
RFP
þ
puncta (early autophagic
organelles) per cell in comparison to control, Jumpy siRNA
resulted in an increase in total and GFP
RFP
þ
(mature) LC3
puncta (Figure 1E and F; Supplementary Figure S6D and E).
The results indicate that, as in RAW 264.7 macrophages,
Jumpy inhibits basal and starvation-induced autophagy
levels in C2C12 cells, and that its loss results in increased
autophagy.
To show that Jumpy siRNA-induced LC3-II increase
required autophagy machinery, Atg5, protein essential for
LC3-II formation (Mizushima et al, 2001), was knocked-down
and LC3-II level measured after 48 h transfection with control
(sc) and/or Atg5 and Jumpy siRNA (Figure 1). Atg5 knock-
down inhibited LC3-II increase observed with Jumpy siRNA
treatment (Figure 1) showing that Jumpy knock-down en-
hances autophagy in Atg5-dependent manner. As expected
with MEF Atg7
/
, no LC3-II was observed in cells transfected
with control or Jumpy siRNA (Supplementary Figure S5).
Jumpy controls autophagic degradation
To test whether the increase of autophagy associated with
Jumpy knock-down resulted in a functional autophagic pro-
cess, we examined effects on long-lived proteins, a canonical
substrate for autophagic turnover. Transfection with Jumpy
siRNA resulted in increased rate of proteolysis in C2C12 cells
observed both in full and starvation media compared with
control siRNA (Figure 2A).
A common adapter for autophagic substrates, p62 (Bjorkoy
et al, 2005), is degraded along with the substrates it delivers
to autophagosomes (Bjorkoy et al, 2005; Filimonenko et al,
2007; Komatsu et al, 2007). We tested whether Jumpy
affected p62 levels. After 48 h transfection with Jumpy
siRNA, p62 protein levels were decreased by 20
±
2% com-
pared with control samples (Figure 2B). This decrease was
rescued after 4 h treatment with BafA1, indicating that the
degradation was lysosomal in nature, consistent with p62
proteolysis in autolysosomes (Figure 2C). When we knocked-
down Beclin-1, a key autophagy regulator (Liang et al, 1999)
acting in complexes with hVps34, this completely erased the
incremental increase in p62 degradation observed in cells
treated with Jumpy siRNA (Figure 2D), indicating that Jumpy
knock-down resulted in elevated p62 degradation through
autophagy. These data along with the increased LC3-I to
LC3-II conversion detected by immunoblotting, elevated
LC3 puncta formation, increased autophagic flux and in-
creased stable protein degradation show that Jumpy nega-
tively controls autophagy.
Figure 1 Screening of active members of the myotubularin family identifies Jumpy as a negative regulator of autophagy. (A) Domains and
members of catalytically active myotubularins. Asterisks, Jumpy mutations found in patients with centronuclear myopathy. C330S, R336Q and
Y462C, Jumpy mutants used in this study. (B) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated
for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 mg/ml E64d and 10 mg/ml pepstatin, then incubated for 30 min in full or starvation media in
the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti-LC3 or anti-actin. Densitometric LC3-II/
actin ratios are shown underneath the blot. Graph: LC3-II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3-II/actin ratio
for control siRNA (sc) in starvation medium. (C, D) RAW 264.7 cells were transfected for 36 h with control (scramble) or Jumpy siRNA,
transfected once more with corresponding siRNA and mRFP-GFP-LC3 DNA construct overnight and incubated for 2 h in full or starvation
media. Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy. (C) Representative confocal images of RAW 264.7 cells
in full or starvation media after transfection with control (scramble) or Jumpy siRNA (siJumpy). Red and yellow arrows indicate GFP
RFP
þ
and GFP
þ
RFP
þ
puncta, respectively. (D) Quantitation of number of LC3 puncta per cell, total puncta per cell (GFP
þ
RFP
þ
and GFP
RFP
þ
puncta) GFP
þ
RFP
þ
puncta per cell and GFP
RFP
þ
puncta per cell. Data, mean
±
s.e.m. n ¼ 5 independent experiments, 30 cells per
experiments. *Po 0.05, **Po0.01, ***Po0.001 (t-test). Scale bars, 5 mm. (E, F) C2C12 myoblasts were transfected for 48 h with control
(scramble) or Jumpy siRNA, followed by a second transfection overnight with corresponding siRNA and mRFP-GFP-LC3 DNA construct. Cells
were fixed and LC3 puncta were counted by confocal fluorescence microscopy. (E) Representative confocal images of C2C12 cells in full media
after transfection with control (scramble) or Jumpy siRNA (siJumpy). (F) Quantification of the number of LC3 puncta per cell. Data,
mean
±
SEM for n ¼ 3 (independent experiments), 30 cells per experiments. *P o0.05 (t-test). Scale bars, 10 mm. (G, H) C2C12 cells were
transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same
siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 mg/ml
E64d and 10 mg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti-LC3 or anti-actin (G). (H) Densitometric LC3-II/
actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5–8). Inset shows Atg5 knock-down by immunoblotting.
Control of autophagy initiation by phosphoinositide 3-phosphatase jumpy
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Figure 2 Jumpy blocks autophagic degradation. (A) Proteolysis of long-lived proteins in C2C12 myoblasts. C2C12 cells were transfected with
control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [
3
H] leucine, washed, incubated for 2 h in complete
media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the
tricarboxylic acid-soluble form relative to total cell radioactivity. Results shown represent mean
±
s.e.m. for combined data from three
independent experiments. (B) Quantitation of p62 protein levels. C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA and
p62 levels analysed by immunoblotting, quantitated by densitometry and represented as a percentage of control. Data are mean
±
s.e.m.
(n ¼ 4 independent experiments). (C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without
100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated
(mean
±
s.e.m., n ¼ 3). (D) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2) or Beclin siRNA (lanes 3, 4) followed by a second
transfection for 48 h with same siRNA and control (lanes 1, 3) or Jumpy siRNA (lanes 2, 4). Cells were lysed, probed for p62, Beclin and actin
by immunoblotting and percentage of p62 were quantitated (mean
±
s.e.m., n ¼ 5). (EM) C2C12 cells were transfected for 48 h with GFP, GFP-
Jumpy (Jumpy), GFP-MTM1 (MTM1) or GFP-MTMR2 (MTMR2), fixed and immunostained with anti-p62 antibody (red). p62 puncta were
quantitated by confocal fluorescence microscopy. Representative confocal images of GFP (E), GFP-Jumpy (F), GFP-MTM1 (G), GFP-MTMR2
(H) transfected cells, immunostained for p62 (I), (J), (K) and (L), respectively. Scale bars, 5 mm. White lines represent outline of the cells.
Quantitation of p62 puncta per cell (M). Bars, SEM (n ¼ 3 independent experiments, with an average of 30 cells per experiments). (N, O) C2C12
cells were transfected for 48 h with GFP, GFP-Jumpy (Jumpy), GFP-MTM1 (MTM1) or GFP-MTMR2 (MTMR2), lysed, analysed for p62, GFP
and actin by immunoblotting (N) and percentage of p62 were quantitated (mean
±
s.e.m., n ¼ 3) (O). (P, Q) C2C12 cells were transfected for
48 h with GFP or GFP-Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and
actin by immunoblotting (P) and percentage of p62 were quantitated (mean
±
s.e.m., n ¼ 4) (Q). *Po0.05, ***Po0.001, wns (t-test).
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Jumpy but not other myotubularins inhibits
p62 degradation
We next tested, in an experimental set up converse to siRNA
knock-down studies, whether Jumpy overexpression inhib-
ited autophagy. As p62-positive protein aggregates increase
with decreased autophagy (Filimonenko et al, 2007; Komatsu
et al, 2007), we monitored the number of p62 puncta per cell.
C2C12 myoblasts were transfected with GFP or GFP-Jumpy
expression constructs and immunostained for endogenous
p62. Cells transfected with Jumpy showed an eight-fold
increase in p62 aggregates compared with controls (Figure
2E, F, I, J and M). To establish a specificity for Jumpy, C2C12
cells were also transfected with GFP-MTM1 or GFP-MTMR2.
Overexpression of MTM1 and MTMR2 led to fewer p62
aggregates than Jumpy overexpression (Figure 2G, H, K, L
and M), indicating that Jumpy specifically affects p62 degra-
dation relative to other myotubularins tested. In contrast to
MTM1- and MTMR2-transfected cells used as comparison
controls, p62 did not appear in Lamp1-positive compartments
in Jumpy-transfected even after addition of BafA1
(Supplementary Figure S7), showing that the p62 aggregates
induced by Jumpy overexpression were not delivered to
lysosomes. Accumulation of p62 was also observed by
immunoblotting in lysates from cells overexpressing Jumpy
(Figure 2N and O). Again, MTM1 and MTMR2 showed no
effect like Jumpy in increasing levels of p62, supporting the
immunofluorescence findings. To verify that accumulation of
p62 was due to inhibition of autophagy, cells transfected
with GFP or GFP-Jumpy were treated with BafA1 for 4 h. In
contrast to control samples, BafA1 did not increase p62 levels
in GFP-Jumpy-transfected cells (Figure 2P and Q), indicating
that in cells overexpressing Jumpy autophagic degradation of
p62 was already inhibited. The p62 accumulation observed in
Jumpy-transfected cells correlated with an increase in large
tdTomato-LC3 puncta (Supplementary Figure S8) similar to
the large LC3 puncta accumulating in Atg14-depleted cells
(Zhong et al, 2009). Collectively, our data unequivocally
show that the PI3P phosphatase Jumpy inhibits autophagy.
Jumpy localizes to autophagic isolation membranes
and autophagoso mes
Next, we asked whether Jumpy acted directly on autophagic
membranes, by examining the localization of Jumpy relative
to autophagic organelles in C2C12 cells. As GFP-Jumpy WT
was found mainly diffuse in the cytosol of C2C12 myoblasts
(Figure 2F), we considered the possibility that Jumpy recruit-
ment to autophagic membranes might be transient. C2C12
myoblasts were transfected with GFP-Jumpy WT and Cherry-
LC3, autophagy was induced by starvation, and GFP-Jumpy
WT dynamics relative to Cherry-LC3 autophagosomes was
monitored by live confocal fluorescence microscopy. We
detected association of GFP-Jumpy WT with Cherry-LC3
puncta (Figure 3A; Supplementary Movie S1), suggesting
that Jumpy is physically present, albeit transiently, on autop-
hagic organelles. The mean duration of Jumpy association
with autophagosomes was 7.5 min (
±
2.5 min; range)
(Figure 3A, graph), during which a cycle of adsorption and
desorbtion of GFP-Jumpy WT was completed.
It has been reported that the catalytically inactive Jumpy
variant C330S (Jumpy CS, expected to act as a substrate-
locked mutant), in contrast to the diffuse cytosolic localiza-
tion of Jumpy WT localizes to distinct intracellular compart-
ments, that is, Golgi and undefined peripheral punctate
structures (Tosch et al,2006).Tocharacterizethenatureof
the peripheral Jumpy CS profiles (Figure 3B) and given our
functional findings, we tested whether they might be autopha-
gosomal in nature. We first ascertained that GFP-Jumpy CS is
not a substrate for autophagy (Supplementary Figure S6F).
Next, we examined Jumpy CS localization relative to LC3.
C2C12 cells were transfected with GFP-Jumpy CS and
tdTomato-LC3 and immunostained for Golgi marker G58K. As
anticipated (Tosch et al, 2006), GFP-Jumpy CS partially colo-
calized with the Golgi marker G58K but was also present on
peripheral structures as noted earlier (Tosch et al, 2006) (Figure
3B–I; Supplementary Table S1). Importantly, we found that the
peripheral Jumpy CS-positive G58K-negative structures were
positive for LC3 (Figure 3B–I; Supplementary Table S1).
A similar colocalization between tdTomato-LC3 and Jumpy
CS was observed in RAW 264.7 macrophages (Supplementary
Figure S9A–C). The localization of Jumpy CS to LC3-positive
organelles was not due to Jumpy CS being an autophagic
substrate (e.g. as a protein aggregate) as GFP-Jumpy CS was
not a substrate for autophagy (Supplementary Figure S6F).
To determine how early Jumpy acts in autophagic orga-
nelle development, we analysed Jumpy CS localization rela-
tive to autophagic isolation membrane (phagophore)
markers, Atg16 and Atg12. Atg16 forms a multimeric com-
plex with Atg5–Atg12 and is found exclusively on phago-
phores in mammalian cells or the preautophagosomal
structures in (PAS) yeast (Mizushima et al, 2003; Fujita
et al, 2008). C2C12 cells were transfected with GFP-Jumpy
CS and tdTomato-LC3 and immunostained for endogenous
Atg16. A portion of Jumpy CS-positive structures were posi-
tive for both LC3 and Atg16, consistent with the phagophore
stage (Figure 4A–H; Supplementary Table S1). Another sub-
set of profiles was only positive for Jumpy CS and LC3 (i.e.
were Atg16 negative), indicating that Jumpy CS was also
present on complete autophagosomes. Of further note is that
in cells transfected with Jumpy CS, Atg16, predominantly
present in the cytosol, redistributed to the perinuclear region
profiles that were LC3 negative (Figure 4A, B, I and J).
To firm up the notion that Jumpy CS associates with
autophagic isolation membranes, we next analysed Jumpy
CS localization relative to endogenous Atg12. A portion of
Jumpy CS-positive structures were positive for both
tdTomato-LC3 and Atg12 (Figure 4K–R; Supplementary
Table S1), whereas another subset of profiles were only
positive for Jumpy CS and LC3 (i.e. were Atg12 negative)
(Figure 4K–R), confirming that Jumpy CS is present on both
autophagic isolation membranes (phagophores) and autop-
hagosomes. A similar colocalization was observed among
B10-tagged Jumpy CS, GFP-LC3 and Atg12 (Supplementary
Figure S10A), validating that Jumpy CS localizes to isolation
membranes irrespective of its tag. Furthermore, those Jumpy
CS-positive structures that were positive for tdTomato-LC3
did not colocalize with Lamp1 (Figure 4S–ZA; Supplementary
Table S1), showing that Jumpy CS is not present on auto-
lysosomes. Furthermore, GFP-Jumpy CS did not colocalize
with the endosomal PI3P-binding protein, EEA1
(Supplementary Figure S10B and Table S1), indicating that
Jumpy CS associates specifically with autophagic mem-
branes. Collectively, these data show that Jumpy has a
function in autophagy by associating with early autophagic
organelles, acting as early as at the phagophore stage.
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Figure 3 Jumpy localizes to autophagosomes. (A) Transient association of Jumpy WT with LC3
þ
organelles. Time lapse sequence of C2C12
myoblasts transfected for 24 h with GFP-Jumpy WT and Cherry-LC3 and analysed by live confocal microscopy in a 5LIVE Zeiss microscope.
EBSS was added to the cells and z-stacks collected at 3-min intervals for a total of 45 min. The collected images were processed to generate a
maximum projection (collapsing a 3D image into an x–y projection) for each time point. Arrows indicate colocalization of GFP-Jumpy WT with
LC3. Graph: Plots of relative fluorescence intensity (GFP fluorescence pixel intensity) over time of GFP-Jumpy WT colocalizing with Cherry-
LC3
þ
-positive puncta indicated by arrows. (BI) Association of Jumpy CS mutant with autophagic organelles. C2C12 cells were transfected for
24 h with GFP-Jumpy C330S (Jumpy CS) and tdTomato-LC3 (LC3), fixed and immunostained with anti-G58K (Alexa 648-labelled secondary
antibody). Jumpy CS (green), LC3 (red) and G58K (white in D, H or blue in E, I). Boxed areas (B–E) are shown at higher magnification in the
corresponding panel below (F–I). Scale bars, 2 mm. White arrows indicate colocalization between Jumpy CS, LC3 but not G58K, blue arrow
indicates colocalization between Jumpy CS and G58K but not LC3.
Figure 4 Jumpy localizes to autophagic isolation membranes. (AH) C2C12 cells were transfected for 24 h with GFP-Jumpy C330S (Jumpy CS)
and tdTomato-LC3 (LC3), fixed and immunostained with anti-Atg16 (Alexa 648-labelled secondary antibody). Jumpy CS (green), LC3 (red) and
Atg16 (white in B and F or blue in D and H). Boxed areas (A–D) are shown at higher magnification in the corresponding panel below (E–H).
White arrows indicate colocalization among Jumpy CS, LC3 and Atg16, yellow arrow indicates colocalization between Jumpy CS and LC3 but
not Atg16, blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3. (I, J) C2C12 cells were transfected for 24 h with GFP
(I), fixed and immunostained with anti-Atg16 (J). (KR) C2C12 cells were transfected for 24 h with GFP-Jumpy C330S (Jumpy CS) and
tdTomato-LC3 (LC3), fixed and immunostained with anti-Atg12(M) antibody. Jumpy CS (green), LC3 (red) and Atg12 (white in L, P or blue in
N, R). Boxed areas (K–N) are shown at higher magnification in the corresponding panel below (O–R). White arrows indicate colocalization
among Jumpy CS, LC3 and Atg12, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12, blue arrow indicates
colocalization between Jumpy CS and Atg12 but not LC3. (SZA) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry-LC3 (LC3),
fixed and immunostained with anti-lamp1. Jumpy CS (green), LC3 (red) and Lamp1 (white in T, X or blue in V, ZA). Boxed areas (S–V) are
shown at higher magnification in the corresponding panel below (W–ZA). Yellow arrows indicate colocalization between Jumpy CS and LC3
but not Lamp1. Scale bars, 2 mm.
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Jumpy regulates recruitment of the PI3P-binding
autophagy factor WIPI-1 (Atg18) to autophagic membranes
WIPI-1 is the mammalian orthologue of the yeast Atg18, the
only thus far characterized autophagic PI3P-binding protein
that participates in the formation of autophagosomes
(Proikas-Cezanne et al, 2007; Xie and Klionsky, 2007).
WIPI-1 partially colocalizes with LC3 positive membranes
but not with mature autophagosomes (Proikas-Cezanne et al,
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2004, 2007). As Jumpy is present on Atg16-positive mem-
branes and the relationship between Atg16 and Atg18 is not
known, we first examined intracellular distribution of WIPI-1
relative to Atg16. We found that WIPI-1 structures colocalized
with Atg16 (Figure 5A–C). Thus, WIPI-1 puncta represents
autophagic isolation membranes. As WIPI-1 is recruited to
autophagic membranes in a PI3P-dependent manner
(Proikas-Cezanne et al, 2007) and both WIPI-1 and Jumpy
were present on autophagic isolation membranes, we hy-
pothesized that Jumpy levels might regulate WIPI-1 puncta
formation. We monitored GFP-WIPI-1 distribution in C2C12
myoblast cells by measuring the number of WIPI-1 puncta
per cell. Starvation induced a 10-fold increase in the number
of WIPI-1 puncta (Figure 5D, E and I). The WIPI-1 puncta
formation was inhibited by wortmannin (Figure 5F and I)
confirming that the formation of WIPI-1 puncta required
PI3P synthesis and that they were not the result of nonspecific
GFP-WIPI-1 protein aggregation. Next, the effect of Jumpy
knock-down was examined. C2C12 cells were transfected with
control or Jumpy siRNA, followed by second transfection with
GFP-WIPI-1, and autophagy was induced by starvation. Jumpy
siRNA resulted in a five-fold increase in the number of GFP-
WIPI-1 puncta per cell in full media (Figure 5D, G and I) and a
50% increase in starvation media (Figure 5E, H and I). These
data show that Jumpy prevents PI3P-dependent WIPI-1 re-
cruitment to autophagic membranes.
Jumpy increases Atg9 association with autophagic
organelles
In yeast, Atg18 has a function in the retrieval of Atg9 from
PAS (Xie and Klionsky, 2007). Atg9, the only known mam-
malian transmembrane Atg protein, is required for autopha-
gosome formation and is present on PAS in yeast and on LC3-
positive compartments in mammalian cells (Noda et al, 2000;
Young et al, 2006). The Atg9 cycling is believed to be essential
for autophagosome formation (Xie and Klionsky, 2007). As
Jumpy impaired WIPI-1 (Atg18) recruitment to autophagic
membranes, we next asked whether Jumpy affected Atg9
Figure 5 Jumpy siRNA increases recruitment of PI3P-binding Atg factor WIPI-1 (Atg18) to autophagic membranes. (AC) C2C12 were
transfected for 48 h with Jumpy siRNA, followed by a second transfection overnight with Jumpy siRNA and GFP-WIPI-1, incubated for 4 h in
starvation media, fixed and immunostained with anti-Atg16 antibody (red). GFP-WIPI1-transfected cell (A), endogenous Atg16 immunostain-
ing (B), colocalization of GFP-WIPI-1 and Atg16 is indicated in yellow in merge image (C). Boxed areas are shown at higher magnification as
inset. Scale bars, 10 and 5 mm in insets. (DI) C2C12 cells were transfected for 48 h with control (scramble) (D, E) or Jumpy siRNA (F–H),
followed by a second transfection overnight with corresponding siRNA and GFP-WIPI-1. Cells were incubated for 4 h in full (D, G) or starvation
media (E, F, H) in presence (F) or absence (E–H) of 100 nM wortmannin, fixed and analysed by confocal fluorescence microscopy. Quantitation of
WIPI-1 puncta per cell (I). Bars, s.e.m. (n ¼ 3 independent experiments, with an average of 30 cells per experiment). *Po0.05, **Po0.01 (t-test).
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distribution vis-a-vis autophagic organelles. First, we exam-
ined the localization of Jumpy CS relative to exogenously
expressed Atg9. C2C12 cells were transfected with GFP-
Jumpy CS, tdTomato-LC3 and HA-Atg9 and immunostained
for HA. We found more than 50% colocalization between
Jumpy CS and Atg9 with a portion of Jumpy CS- and Atg9-
positive structures colocalizing with LC3 (Figure 6A–H).
Next, we measured the percentage of LC3 puncta colocalizing
with Atg9 in cells transfected with GFP, GFP-Jumpy CS or
GFP-Jumpy WT. Both Jumpy WT and Jumpy CS increased by
three-fold the percentage of LC3 puncta that were Atg9
positive compared with control GFP-transfected cells
(Figure 6I–U). This shows that Jumpy causes Atg9 accumula-
tion on autophagic organelles most likely by blocking its PI3P
and Atg18-dependent cycling.
Jumpy variant associated with centronuclear myopathy
is defective in downregulating autophagy
Two Jumpy missense variants have been described in pa-
tients with centronuclear myopathy (Tosch et al, 2006) (i) a
missense mutation, R336Q (RQ), within the catalytic site that
abrogates Jumpy phosphatase activity and (ii) the missense
Figure 6 Jumpy colocalizes with and regulates Atg9 distribution to autophagic organelles. (AT) C2C12 cells were transfected for 24 h with
GFP, GFP-Jumpy WT or GFP-Jumpy C330S (Jumpy CS) and Tomato-LC3 (LC3) and HA-Atg9, fixed and immunostained with anti-HA. GFP (I),
Jumpy WT (M), Jumpy CS (green) (A, E, Q), LC3 (red) (C, G, J, N, R) and Atg9 (white in B, F, K, O, S or blue in D, H, I, P, T). Boxed areas (A–D)
are shown at higher magnification in the corresponding panel below (E–H). White arrows indicate colocalization between Jumpy CS, LC3 and
Atg9, blue arrow indicates colocalization between Jumpy CS and Atg9 but not LC3 and yellow arrow indicates colocalization between Jumpy
CS and LC3 but not Atg9. White boxes (I–T) show LC3 puncta location. Scale bars, 5 mm. (U) Percentage of LC3 puncta colocalizing with Atg9.
n, number of puncta counted.
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Y462C (YC) mutation outside of the phosphatase domain,
which has a mild effect (80% of wild-type phosphatase
activity) (Tosch et al, 2006). We tested these naturally occur-
ring Jumpy variants for their effects on autophagy. We first
measured p62 puncta accumulation in C2C12. Both Jumpy
WT and Jumpy YC increased the number of p62 puncta
compared with control (YFP) (Figure 7A, B, D, F, G, I and
K). In contrast, Jumpy RQ did not promote accumulation of
p62 puncta and showed a similar number of puncta as seen in
controls (YFP) (Figure 7A, E, F, J and K). These data suggest
that a loss of Jumpy PI3P phosphatase activity associated
with the R336Q Jumpy allele leads to increased autophagy.
Strikingly, overexpression of Jumpy CS promoted accumu-
lation of p62 puncta and large tdTomato-LC3 puncta as in the
case of Jumpy WT overexpression (Figure 7B, C, G, H and K;
Supplementary Figure S8). One explanation for this finding is
that Jumpy CS acts as a substrate trap and thus blocks PI3P
accessibility to its effectors which results in inhibition of
autophagy ostensibly similar to the effects of Jumpy WT.
Consistent with this possibility, similar mutations in the
active site of tyrosine phosphatases result in catalytically
inactive phosphatases with substrate-trapping properties
(Garton et al, 1996; Flint et al, 1997). This also agrees with
the findings that Jumpy CS, in contrast to Jumpy WT, YC and
RQ, is mostly found on membranes and not in the cytosol
(Figure 7A–E) and that Jumpy CS as Jumpy WT increase
number of large LC3 puncta and promote Atg9 accumulation
on autophagic organelles (Supplementary Figure S8; Figure
6I–U), supporting the idea that this mutant remains asso-
ciated with membrane sites in which unimpeded access to
specific PI3P patches is needed to promote the PI3P-depen-
dent stages of autophagy.
The effects of Jumpy WT and RQ on LC3 lipidation were
compared next. Cells were transfected with YFP-Jumpy WTor
YFP-Jumpy RQ construct, autophagy induced in the presence
of BafA1 to prevent autophagic degradation of LC3-II, and
Figure 7 Jumpy R336Q mutant associated with centronuclear myopathy is defective in inhibiting autophagy. (AK) C2C12 cells were
transfected for 48 h with YFP, YFP-Jumpy WT (Jumpy WT), YFP-Jumpy C330S (Jumpy CS), YFP-Jumpy Y462C (Jumpy YC) or YFP-Jumpy
R336Q (Jumpy RQ), fixed and immunostained with anti-p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy.
Representative confocal images of YFP (A), YFP-Jumpy WT (B), YFP-Jumpy C330S (C), YFP-Jumpy Y462C (D) and YFP-Jumpy R336Q (E)
transfected cells, immunostained for p62 (F), (G), (H), (I) and (J), respectively. Scale bars, 5 mm. Quantitation of p62 puncta per cell (K). Bars,
s.e.m. (n ¼ 3 independent experiments, 30 cells per experiments). *Po0.05 (t-test), ns: nonsignificant. (L) HeLa cells were transfected for 24 h
with YFP, YFP-Jumpy WT (Jumpy WT) or YFP-Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/ml rapamycin (BafA1 (100 nM) was
present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3-
II/actin ratios are shown underneath the blot.
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levels of LC3-II assessed by immunoblotting. Overexpression
of Jumpy WT inhibited LC3-II conversion associated with
autophagy induction in HeLa (Figure 7L) or C2C12 cells
(Supplementary Figure S11), in keeping with the role of
Jumpy phosphatase activity in inhibition of early stages of
autophagy. In contrast, overexpression of the inactive Jumpy
RQ mutant did not decrease LC3-II conversion but instead
increased LC3-II levels, suggesting that Jumpy RQ acts as a
dominant negative mutant (Figure 7L; Supplementary Figure
S11). These experiments show that the Jumpy RQ variant
associated with centronuclear myopathy is unable to down-
regulate autophagy.
Discussion
Our study establishes that the PI3P phosphatase Jumpy
negatively regulates autophagy. This represents a paradigm
shift from the notion that autophagy initiation in mammalian
cells is controlled exclusively by regulating PI 3-kinase activ-
ity, a concept that now has to account for the role of a specific
PI3P phosphatase. Jumpy acts on discrete domains of nascent
autophagic membranes, and orchestrates succession of Atg
factors controlling autophagy initiation. At a macro-level,
Jumpy acts as a ‘brake’ in the autophagy pathway. This
brake is applied at the PI3P-dependent initiation stage of
the execution phase of autophagy. A model emerges in which
it is the balance between the PI3P production (regulated by
hVp34) and the PI3P hydrolysis (regulated by Jumpy) that
determines induction and baseline levels of autophagy, rather
than the forward reaction alone, which has been thus far the
nearly exclusive view of the process. In addition, our findings
link a human myopathy to altered regulation of autophagy by
Jumpy, highlighting the significance of autophagosomal PI3P
regulation by a phosphatase in a physiologically and medi-
cally relevant context.
Despite its critical regulatory role, the mechanistic details
of PI3P function during initiation of autophagy are surpris-
ingly lacking (Yoshimori and Noda, 2008). Both in yeast and
mammalian cells, PI3P production is necessary for autophagy
initiation, the Atg16-directed recruitment of the Atg5–Atg12
complex to PAS or autophagic isolation membranes (Fujita
et al, 2008) and for the linked conversion of LC3-I into LC3-II
(Kabeya et al, 2000; Xie and Klionsky, 2007). More recently,
PI3P-enriched compartments, derived from endoplasmic re-
ticulum, have been proposed to possibly have a function in
autophagosome biogenesis (Axe et al, 2008). However, how
PI3P is connected to these molecular events is far from being
understood. In yeast, PI3P is enriched on inner surfaces of
isolation membranes and autophagosomes (Obara et al,
2008) and its production is required for the proper recruit-
ment to autophagic membranes of Atg18, the only thus far
characterized PI3P effector among the Atg factors (Reggiori
et al, 2004). Atg18 is implicated in the so-called process of
retrieval of Atg9 from PAS to peripheral compartments
(Reggiori et al, 2004). The purpose of this Atg9 cycling during
autophagy remains unclear (Young et al, 2006; Suzuki and
Ohsumi, 2007), although it clearly has a function in auto-
phagosome formation (Xie and Klionsky, 2007). In resting
mammalian cells, Atg9 is in an equilibrium between the
trans-Golgi network and late endosomes, the locations from
where it promptly redistributes to LC3
þ
autophagic orga-
nelles, ranging from nascent phagophores to early degrada-
tive but still LC3-positive autophagic vesicles (Young et al,
2006). Our data now indicate that overexpression of Jumpy
wild type or its CS mutant, freezes autophagic organelles in
an Atg9
þ
and LC3
þ
dual-positive state. Thus, one can infer
that Jumpy regulates specific PI3P domains on early autop-
hagic membranes, at the site and time when key molecular
decisions are made to initiate the process of autophagosome
formation.
The function of WIPI-1, the mammalian orthologue of
yeast Atg18, while presently unknown holds promise in
deciphering the precise role of PI3P in autophagy initiation.
WIPI-1 is recruited to autophagic membranes after autophagy
induction showing partial colocalization with LC3 and Atg16
(observed in this study) and accumulation on cup-shaped
membranes (Proikas-Cezanne et al, 2004). As with yeast
Atg18, the association of WIPI-1 to autophagic membranes
requires PI3P (Proikas-Cezanne et al, 2007). As Jumpy as-
sociates with autophagic organelles, in particular phago-
phores, and regulates WIPI-1 and Atg9 trafficking, we
conclude that Jumpy inhibits autophagy by dephosphorylat-
ing a pool of PI3P directly involved in WIPI-1 (Atg18)
recruitment to autophagic membranes. One of the candidate
processes affected by Jumpy through WIPI-1 is the cycling of
Atg9, but additional roles in positioning and organization of
phagophores cannot be excluded.
Although autophagic machinery is conserved from yeast to
humans, Jumpy is not found in yeast (Tosch et al, 2006).
Noteworthy, yeast encodes a myotubularin-like protein, Ymr1
(Laporte et al, 1998), although its role in autophagy has not
been examined. It remains possible that Ymr1 has a function
in autophagy, perhaps observable only under certain physio-
logical conditions similarly to one of the myotubularins
identified here as affecting autophagy: the mammalian myo-
tubularins affecting autophagy include Jumpy characterized
here, the yet to be examined in full MTMR6, and potentially
MTMR7. MTMR7 did not show any effects in the siRNA
screen shown in Supplementary Figure S1; it turned out,
however, that the mRNA entry for MTMR7 used in the initial
screen was based on unstable mRNA species, and when a
more stable mRNA species was targeted with siRNAs,
MTMR7 mRNA knock-down could be documented and
showed some effects on LC3-II (Supplementary Figure S13).
The extra layers of regulation in mammalian cells may be
necessary to accommodate the additional functions of autop-
hagy in mammals including development of metazoan-spe-
cific tissues (Cecconi and Levine, 2008) such as muscles. Of
note, autophagosomes are noticeably smaller in skeletal
muscles than in other tissues (Mizushima et al, 2004), thus,
another role of myotubularins could be the regulation of
autophagosomal size, which may be tissue specific and
thus perhaps idiosyncratic to multicellular eukaryotes.
Autophagy has been implicated in several types of myo-
pathies, such as Danon disease, Pompe disease and X-linked
myopathy with excessive autophagy (Kundu and Thompson,
2008; Levine and Kroemer, 2008; Mizushima et al, 2008).
Recently, autophagy has been reported to have an important
function during denervation- or fasting-induced skeletal mus-
cle wasting and atrophy (Mammucari et al, 2007; Zhao et al,
2007). Given the general importance of autophagy in muscle
physiology and pathology, and the unanticipated link uncov-
ered here between autophagy and Jumpy as a myopathy risk
locus, we propose that excessive or improperly regulated
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autophagy contributes to the pathogenesis of centronuclear
myopathy. Centronuclear myopathies are congenital disor-
ders characterized by muscle atrophy and by a peculiar
positioning of the nuclei in the center of the skeletal muscle
fibres (Pierson et al, 2005). Although, several genes involved
in the disease, including dynamin 2, amphiphysin 2, Jumpy
and MTM1, have been identified, their cellular functions in
relationship to muscle physiology are not understood
(Blondeau et al, 2000; Taylor et al, 2000; Bitoun et al, 2005;
Tosch et al, 2006; Nicot et al, 2007). Here, we have shown
that Jumpy R336Q, a naturally occurring mutation in centro-
nuclear myopathy (Tosch et al, 2006), disables Jumpy in
suppressing autophagy. This suggests that autophagy dysre-
gulation may be an important pathogenesis determinant in
centronuclear myopathy. Missense mutations in the MTM1
gene, which encodes another prominent member of the
myotubularin family, is the most common cause leading to
centronuclear myopathy (Laporte et al, 2003). MTM1 protein
is found on ruffles at the plasma membrane (Blondeau et al,
2000) and on endocytic compartments (Cao et al, 2007).
Although, our screen in macrophages did not identify
MTM1 as an autophagy regulator, we cannot exclude the
possibility that in muscle cells MTM1 contributes to the
regulation of the autophagy pathway.
In summary, our study shows a role for the PI3P phospha-
tase Jumpy in regulating the early stage of autophagy,
Furthermore, we have uncovered a link between a disease-
associated Jumpy mutation and autophagy. These findings
represent advance in our understanding of the regulation
autophagy at the level of PI3P on autophagic membranes
during initiation, which now no longer involves only the
forward reaction mediated by the hVPS34–Beclin-1 complex
but also depends on the Jumpy-dependent turnover of PI3P
pools that are relevant for autophagy.
Materials and methods
Cell cultures
C2C12 muscle myoblast, and murine RAW264.7 macrophage cell
lines were from ATCC and were maintained in Dulbecco’s modified
Eagle’s medium (DMEM; Invitrogen, CA, USA) supplemented with
10% fetal bovine serum (FBS) and
L-glutamine (complete media).
HeLa cells were maintained in DMEM, 10% FBS.
Chemicals, antibodies and plasmid constructs
All chemicals were purchased from Sigma-Aldrich except for BafA1
and rapamycin (LC laboratories). Generation of EYFP-Jumpy-Wild-
Type, -C330S, -R336Q and -Y462C, EGFP-Jumpy-Wild-Type, -C330S,
B10-Jumpy CS, EGFP-MTM1, EGFP-MTMR2 (Tosch et al, 2006) and
GFP-WIPI-1 (Proikas-Cezanne et al, 2007) plasmid constructs are
described elsewhere. tdTomato-LC3 and Cherry-LC3 were from T.
Johansen (Bjorkoy et al, 2005; Pankiv et al, 2007). mRFP-GFP-LC3
and HA-mAtg9 were from T. Yoshimori (Kimura et al, 2007) and S.
Tooze (Young et al, 2006), respectively. The following antibodies
were used in this study: LC3 (Sigma or from T Ueno), Atg12 (M)
and Atg16 from N. Mizushima (Mizushima et al, 2001, 2003), Atg12
(C) (Cell signaling), Beclin-1 (Santa Cruz), Lamp1 (Clone 1D4B,
RDI /Fitzgerald), G58K (Sigma), Atg5 (Novus), p62 (Progen), GFP
and Actin (Abcam). The specificity of Atg16, Atg12 (M) and Atg12
(C) antibody staining were validated in C2C12 with Atg16 and
Atg12 staining colocalizing with GFP-LC3 under starvation condi-
tions (Supplementary Figure S12).
Transient transfection by nucleoporation
RAW 264.7, C2C12, Neuro2A, MEF cells, and HeLa cells were
harvested at day 2 of culture and re-suspended in the appropriate
electroporation buffer (Amaxa Biosystems, MD). A measure of
5–10 mg plasmid DNA and/or 1.5 mg siRNA were mixed with 0.1 ml
of cell suspension, transferred to an electroporation cuvette, and
nucleofected with Amaxa Nucleofector apparatus (Amaxa Biosys-
tems, MD). Jumpy, Atg5 and Beclin-1 knock-downs were achieved
by using siGENOME SMARTpool reagent (Dharmacon) specific for
Mus musculus for RAW 264.7, C2C12, Neuro2A and MEF cells
transfection or for Homo Sapiens for HeLa transfection (Dharma-
con). All effects of myotubularins, Jumpy, Atg5 or Beclin siRNAs
were compared with siCONTROL Nontargeting siRNA pool (Dhar-
macon), which is labelled as scramble or sc in figures.
Fluorescence confocal microscopy and Immunoblotting
Procedures for fluorescence confocal microscopy, immunofluores-
cence and western immunoblotting were described elsewhere
(Vergne et al, 2005; Delgado et al, 2008). Co-localization of GFP-
Jumpy CS with different cellular markers was defined using the
Zeiss LSM 510 co-localization co-efficient program (0 being no
colocalization and 1 total colocalization). Only pixels above
cytosolic fluorescence were evaluated using LSM 510 co-localization
and crosshair functions.
p62 analysis
p62 protein levels were determined by immunoblotting, measured
using NIH Image J software, and expressed as a percentage of
control (scramble siRNA, GFP or YFP-transfected cells). p62
accumulation was analysed by immunofluorescence microscopy
and quantitated by counting the number of endogenous p62 puncta
(XI mm) per cell using Zeiss LSM Image Browser software
(Filimonenko et al, 2007).
Proteolysis of long-lived proteins
C2C12 myoblast cells were transfected with siRNA and seeded in
12-well plates (8 10
4
cells per well). Four hours later, cells were
labelled overnight in media containing 1 mCi/ml 3Hleucine, washed
to remove unincorporated label, and pulsed for 2 h in full media
containing cold leucine to allow degradation of short-lived proteins.
Finally, cells were incubated in full or starvation media for 4 h.
Trichloroacetic acid (TCA)-precipitable radioactivity of the cells
monolayers and the TCA-soluble radioactivity in the media were
determined. Leucine release (a measure of proteolysis of stable
proteins) was calculated as a ratio between TCA-soluble super-
natant and total cell-associated radioactivity.
Live confocal fluorescence microscopy
Recruitment of GFP-Jumpy WT was analysed by live confocal
microscopy using a 5LIVE Zeiss microscope. EBSS was added to the
cells and z-stacks collected at 3-min intervals for a total of 45 min.
The collected images were processed to generate a maximum
projection (collapsing a 3D image into an x–y projection) for each
time point, as described earlier for 4D live confocal imaging (Chua
and Deretic, 2004; Kyei et al, 2006; Roberts et al, 2006). Jumpy
association was quantified as relative fluorescence units (RFUs) of
GFP-Jumpy on LC3
þ
-puncta substracted for RFU of GFP-Jumpy in
cytosol and normalized to RFU
c
–RFU
p
at time corresponding to
925 s.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
This work was supported by grants AI45148 from the National
Institutes of Health to VD, INSERM, CNRS, Colle
`
ge de France,
and grants from AssociationFranc¸aise contre les myopathies,
Agence Nationale de la Recherche and Foundation pour la
recherche Me
´
dicale to JL, and grant SFB773 from the Deutsche
Forschungsgemeinschaft to TP-C. This project was supported in
part by the Dedicated Health Research Funds from the University of
New Mexico School of Medicine to IV.
Conflict of interest
The authors declare that they have no conflict of interest.
Control of autophagy initiation by phosphoinositide 3-phosphatase jumpy
I Vergne et al
The EMBO Journal VOL 28
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NO 15
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2009 & 2009 European Molecular Biology Organization2256
References
Alonso A, Sasin J, Bottini N, Friedberg I, Friedberg I, Osterman A,
Godzik A, Hunter T, Dixon J, Mustelin T (2004) Protein tyrosine
phosphatases in the human genome. Cell 11 7 : 699–711
Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL,
Habermann A, Griffiths G, Ktistakis NT (2008) Autophagosome
formation from membrane compartments enriched in phosphati-
dylinositol 3-phosphate and dynamically connected to the endo-
plasmic reticulum. J Cell Biol 182: 685–701
Bitoun M, Maugenre S, Jeannet PY, Lacene E, Ferrer X, Laforet P,
Martin JJ, Laporte J, Lochmuller H, Beggs AH, Fardeau M,
Eymard B, Romero NB, Guicheney P (2005) Mutations in dyna-
min 2 cause dominant centronuclear myopathy. Nat Genet 37:
1207–1209
Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M,
Overvatn A, Stenmark H, Johansen T (2005) p62/SQSTM1 forms
protein aggregates degraded by autophagy and has a protective
effect on huntingtin-induced cell death. J Cell Biol 171:
603–614
Blondeau F, Laporte J, Bodin S, Superti-Furga G, Payrastre B,
Mandel JL (2000) Myotubularin, a phosphatase deficient in
myotubular myopathy, acts on phosphatidylinositol 3-kinase
and phosphatidylinositol 3-phosphate pathway. Hum Mol Genet
9: 2223–2229
Cao C, Laporte J, Backer JM, Wandinger-Ness A, Stein MP (2007)
Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid
kinase complex on endosomes. Traffic 8: 1052–1067
Cecconi F, Levine B (2008) The role of autophagy in mammalian
development: cell makeover rather than cell death. Dev Cell 15:
344–357
Chua J, Deretic V (2004) Mycobacterium tuberculosis reprograms
waves of phosphatidylinositol 3-phosphate on phagosomal orga-
nelles. J Biol Chem 279: 36982–36992
Clague MJ, Lorenzo O (2005) The myotubularin family of lipid
phosphatases. Traffic 6: 1063–1069
Delgado MA, Elmaoued RA, Davis AS, Kyei G, Deretic V (2008) Toll-
like receptors control autophagy. EMBO J 27: 1110–1121
Filimonenko M, Stuffers S, Raiborg C, Yamamoto A, Malerod L,
Fisher EM, Isaacs A, Brech A, Stenmark H, Simonsen A (2007)
Functional multivesicular bodies are required for autophagic
clearance of protein aggregates associated with neurodegenera-
tive disease. J Cell Biol 179: 485–500
Flint AJ, Tiganis T, Barford D, Tonks NK (1997) Development of
‘substrate-trapping’ mutants to identify physiological substrates
of protein tyrosine phosphatases. Proc Natl Acad Sci USA 94:
1680–1685
Fujita N, Itoh T, Omori H, Fukuda M, Noda T, Yoshimori T (2008)
The Atg16 L complex specifies the site of LC3 lipidation for
membrane biogenesis in autophagy. Mol Biol Cell 19: 2092–2100
Garton AJ, Flint AJ, Tonks NK (1996) Identification of p130(cas) as
a substrate for the cytosolic protein tyrosine phosphatase PTP-
PEST. Mol Cell Biol 16: 6408–6418
Itakura E, Kishi C, Inoue K, Mizushima N (2008) Beclin 1 forms two
distinct phosphatidylinositol 3-kinase complexes with mamma-
lian Atg14 and UVRAG. Mol Biol Cell 19: 5360–5372
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T,
Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian
homologue of yeast Apg8p, is localized in autophagosome mem-
branes after processing. EMBO J 19: 5720–5728
Kimura S, Noda T, Yoshimori T (2007) Dissection of the autophago-
some maturation process by a novel reporter protein, tandem
fluorescent-tagged LC3. Autophagy 3: 452–460
Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, Mizushima
N, Iwata J, Ezaki J, Murata S, Hamazaki J, Nishito Y, Iemura S,
Natsume T, Yanagawa T, Uwayama J, Warabi E, Yoshida H, Ishii
T, Kobayashi A et al. (2007) Homeostatic levels of p62 control
cytoplasmic inclusion body formation in autophagy-deficient
mice. Cell 131: 1149–1163
Kundu M, Thompson CB (2008) Autophagy: basic principles and
relevance to disease. Annu Rev Pathol 3: 427–455
Kyei GB, Vergne I, Chua J, Roberts E, Harris J, Junutula JR,
Deretic V (2006) Rab14 is critical for maintenance of
Mycobacterium tuberculosis phagosome maturation arrest.
EMBO J 25: 5250–5259
Laporte J, Bedez F, Bolino A, Mandel JL (2003) Myotubularins, a
large disease-associated family of cooperating catalytically active
and inactive phosphoinositides phosphatases. Hum Mol Genet 12
(Spec No 2): R285–R292
Laporte J, Blondeau F, Buj-Bello A, Tentler D, Kretz C, Dahl N,
Mandel JL (1998) Characterization of the myotubularin dual
specificity phosphatase gene family from yeast to human. Hum
Mol Genet 7: 1703–1712
Levine B, Deretic V (2007) Unveiling the roles of autophagy in
innate and adaptive immunity. Nat Rev Immunol 7: 767–777
Levine B, Kroemer G (2008) Autophagy in the pathogenesis of
disease. Cell 132: 27–42
Liang C, Lee JS, Inn KS, Gack MU, Li Q, Roberts EA, Vergne I,
Deretic V, Feng P, Akazawa C, Jung JU (2008) Beclin1-binding
UVRAG targets the class C Vps complex to coordinate auto-
phagosome maturation and endocytic trafficking. Nat Cell Biol
10: 776–787
Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh
H, Levine B (1999) Induction of autophagy and inhibition of
tumorigenesis by beclin 1. Nature 402: 672–676
Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del
Piccolo P, Burden SJ, Di Lisi R, Sandri C, Zhao J, Goldberg AL,
Schiaffino S, Sandri M (2007) FoxO3 controls autophagy in
skeletal muscle in vivo. Cell Metab 6: 458–471
Mizushima N, Kuma A, Kobayashi Y, Yamamoto A, Matsubae M,
Takao T, Natsume T, Ohsumi Y, Yoshimori T (2003) Mouse
Apg16L, a novel WD-repeat protein, targets to the autophagic
isolation membrane with the Apg12-Apg5 conjugate. J Cell Sci 11 6
(Pt 9): 1679–1688
Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Auto-
phagy fights disease through cellular self-digestion. Nature 451:
1069–1075
Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y,
Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T (2001) Dissection of
autophagosome formation using Apg5-deficient mouse embryo-
nic stem cells. J Cell Biol 152
: 657–668
Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y
(2004) In vivo analysis of autophagy in response to nutrient
starvation using transgenic mice expressing a fluorescent autop-
hagosome marker. Mol Biol Cell 15: 1101–1111
Mizushima N, Yoshimori T (2007) How to interpret LC3 immuno-
blotting. Autophagy 3: 542–545
Nicot AS, Toussaint A, Tosch V, Kretz C, Wallgren-Pettersson C,
Iwarsson E, Kingston H, Garnier JM, Biancalana V, Oldfors A,
Mandel JL, Laporte J (2007) Mutations in amphiphysin 2 (BIN1)
disrupt interaction with dynamin 2 and cause autosomal reces-
sive centronuclear myopathy. Nat Genet 39: 1134–1139
Noda T, Kim J, Huang WP, Baba M, Tokunaga C, Ohsumi Y,
Klionsky DJ (2000) Apg9p/Cvt7p is an integral membrane protein
required for transport vesicle formation in the Cvt and autophagy
pathways. J Cell Biol 148: 465–480
Obara K, Noda T, Niimi K, Ohsumi Y (2008) Transport of phospha-
tidylinositol 3-phosphate into the vacuole via autophagic
membranes in Saccharomyces cerevisiae. Genes Cells 13:
537–547
Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H,
Overvatn A, Bjorkoy G, Johansen T (2007) p62/SQSTM1 binds
directly to Atg8/LC3 to facilitate degradation of ubiquitinated
protein aggregates by autophagy. J Biol Chem 282: 24131–24145
Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P (2000)
Distinct classes of phosphatidylinositol 3
0
-kinases are involved in
signaling pathways that control macroautophagy in HT-29 cells. J
Biol Chem 275: 992–998
Pierson CR, Tomczak K, Agrawal P, Moghadaszadeh B, Beggs AH
(2005) X-linked myotubular and centronuclear myopathies. J
Neuropathol Exp Neurol 64: 555–564
Proikas-Cezanne T, Ruckerbauer S, Stierhof YD, Berg C, Nordheim
A (2007) Human WIPI-1 puncta-formation: a novel assay to
assess mammalian autophagy. FEBS Lett 581: 3396–3404
Proikas-Cezanne T, Waddell S, Gaugel A, Frickey T, Lupas A,
Nordheim A (2004) WIPI-1alpha (WIPI49), a member of the
novel 7-bladed WIPI protein family, is aberrantly expressed in
human cancer and is linked to starvation-induced autophagy.
Oncogene 23: 9314–9325
Reggiori F, Tucker KA, Stromhaug PE, Klionsky DJ (2004) The Atg1-
Atg13 complex regulates Atg9 and Atg23 retrieval transport from
the pre-autophagosomal structure. Dev Cell 6: 79–90
Control of autophagy initiation by phosphoinositide 3-phosphatase jumpy
I Vergne et al
& 2009 European Molecular Biology Organization The EMBO Journal VOL 28
|
NO 15
|
2009 2257
Roberts EA, Chua J, Kyei GB, Deretic V (2006) Higher order Rab
programming in phagolysosome biogenesis. JCellBiol174: 923–929
Robinson FL, Dixon JE (2006) Myotubularin phosphatases: policing
3-phosphoinositides. Trends Cell Biol 16: 403–412
Suzuki K, Ohsumi Y (2007) Molecular machinery of autophago-
some formation in yeast, Saccharomyces cerevisiae. FEBS Lett
581: 2156–2161
Taylor GS, Maehama T, Dixon JE (2000) Inaugural article: myotubular-
in, a protein tyrosine phosphatase mutated in myotubular myopathy,
dephosphorylates the lipid second messenger, phosphatidylinositol
3-phosphate. Proc Natl Ac ad Sci USA 97: 8910–8915
Tosch V, Rohde HM, Tronchere H, Zanoteli E, Monroy N, Kretz C,
Dondaine N, Payrastre B, Mandel JL, Laporte J (2006) A novel
PtdIns3P and PtdIns(3,5)P2 phosphatase with an inacti-
vating variant in centronuclear myopathy. Hum Mol Genet 15:
3098–3106
Vergne I, Chua J, Lee HH, Lucas M, Belisle J, Deretic V (2005)
Mechanism of phagolysosome biogenesis block by viable
Mycobacterium tuberculosis. Proc Natl Acad Sci USA 102: 4033–4038
Wei Y, Sinha S, Levine B (2008) Dual role of JNK1-mediated
phosphorylation of Bcl-2 in autophagy and apoptosis regulation.
Autophagy 4: 949–951
Xie Z, Klionsky DJ (2007) Autophagosome formation: core machin-
ery and adaptations. Nat Cell Biol 9: 1102–1109
Yoshimori T, Noda T (2008) Toward unraveling membrane biogen-
esis in mammalian autophagy. Curr Opin Cell Biol 20: 401–407
Young AR, Chan EY, Hu XW, Kochl R, Crawshaw SG, High S, Hailey
DW, Lippincott-Schwartz J, Tooze SA (2006) Starvation and
ULK1-dependent cycling of mammalian Atg9 between the TGN
and endosomes. J Cell Sci 11 9 (Pt 18): 3888–3900
Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, Lecker SH,
Goldberg AL (2007) FoxO3 coordinately activates protein degra-
dation by the autophagic/lysosomal and proteasomal pathways
in atrophying muscle cells. Cell Metab 6: 472–483
Zhong Y, Wang QJ, Li X, Yan Y, Backer JM, Chait BT, Heintz N, Yue Z
(2009) Distinct regulation of autophagic activity by Atg14 L and
Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase
complex. Nat Cell Biol 11: 468–476
Control of autophagy initiation by phosphoinositide 3-phosphatase jumpy
I Vergne et al
The EMBO Journal VOL 28
|
NO 15
|
2009 & 2009 European Molecular Biology Organization2258
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    • "These puncta were colocalized with LAMP1 (Fig 3D,upper panel), suggesting that a fraction of INPP5E is localized to lysosomes . This localization pattern was not altered even under autophagy induced condition in both MEFs and N1E-115 cells (Appendix Fig S7A and B ). Catalytically inactive variants of phosphatases such as MTMR14/Jumpy and MTMR3, which are thought to behave as substrate-locked mutants, exhibit a punctate localization , in contrast to the diffuse cytoplasmic localization of the respective wild-type proteins (Vergne et al, 2009; Taguchi-Atarashi et al, 2010). Hence, we also examined a catalytically inactive mutant of INPP5E, D477N. "
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