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A protein conjugation system essential for autophagy


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Autophagy is a process for the bulk degradation of proteins, in which cytoplasmic components of the cell are enclosed by double-membrane structures known as autophagosomes for delivery to lysosomes or vacuoles for degradation. This process is crucial for survival during starvation and cell differentiation. No molecules have been identified that are involved in autophagy in higher eukaryotes. We have isolated 14 autophagy-defective (apg) mutants of the yeast Saccharomyces cerevisiae and examined the autophagic process at the molecular level. We show here that a unique covalent-modification system is essential for autophagy to occur. The carboxy-terminal glycine residue of Apg12, a 186-amino-acid protein, is conjugated to a lysine at residue 149 of Apg5, a 294-amino-acid protein. Of the apg mutants, we found that apg7 and apg10 were unable to form an Apg5/Apg12 conjugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-E1-like enzyme. This conjugation can be reconstituted in vitro and depends on ATP. To our knowledge, this is the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system. Furthermore, Apg5 and Apg12 have mammalian homologues, suggesting that this new modification system is conserved from yeast to mammalian cells.
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Nature © Macmillan Publishers Ltd 1998
Received 21 May; accepted 21 July 1998.
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Acknowledgements. We thank D. Smith and J. Olesker for assistancein writing the manuscript; T. So
J. E. Rothman, P. Szabo, G. van Meer, D. Nikolov, A. Koff, G. Bacher, and members of the Wiedmann,
Duvoisin and Rothman laboratories for discussions and comments; N. Min and L. Cohen-Gould for
performing the initial immunohistochemistry and electron microscopy experiments. We thank P. Marks
and R. Rifkind for suggesting the eye lens experiments. This work was supported by the Memorial Sloan-
Kettering Cancer Center, a Fellowship by the Deutsche Forschungsgemeinschaft (to K.v.L.), and by the
Samuel and May Rudin Foundation and a Tolly Vinik Pilot Grant Award (to R.M.D.).
Correspondence and requests for materials should be addressed to M.W.
letters to nature
VOL 395
24 SEPTEMBER 1998 395
A protein conjugation system
essential for autophagy
Noboru Mizushima*, Takeshi Noda*, Tamotsu Yoshimori*,
Yae Tanaka
, Tomoko Ishii
, Michael D. George
Daniel J. Klionsky
, Mariko Ohsumi
& Yoshinori Ohsumi*
* Department of Cell Biology, National Institute for Basic Biology,
Okazaki 444-8585, Japan
Department of Biosciences, Teikyo University of Science & Technology,
Yamanashi 409-0193, Japan
Section of Microbiology, University of California, Davis, California 95616, USA
Autophagy is a process for the bulk degradation of proteins, in
which cytoplasmic components of the cell are enclosed by double-
membrane structures known as autophagosomes for delivery to
lysosomes or vacuoles for degradation
. This process is crucial
for survival during starvation and cell differentiation. No mol-
ecules have been identified that are involved in autophagy in
higher eukaryotes. We have isolated 14 autophagy-defective (apg)
mutants of the yeast Saccharomyces cerevisiae
and examined the
autophagic process at the molecular level
. We show here that a
unique covalent-modification system is essential for autophagy to
occur. The carboxy-terminal glycine residue of Apg12, a 186-
amino-acid protein, is conjugated to a lysine at residue 149 of
Apg5, a 294-amino-acid protein. Of the apg mutants, we found
that apg7 and apg10 were unable to form an Apg5/Apg12 con-
jugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-
E1-like enzyme. This conjugation can be reconstituted in vitro
and depends on ATP. To our knowledge, this is the first report of a
protein unrelated to ubiquitin that uses a ubiquitination-like
conjugation system. Furthermore, Apg5 and Apg12 have mam-
malian homologues, suggesting that this new modification system
is conserved from yeast to mammalian cells.
In yeast, autophagy is induced by various starvation conditions,
and its progression is easily monitored under a light microscope
when wild-type cells were cultured under nitrogen-starvation con-
ditions in the presence of phenylmethylsulphonyl fluoride (PMSF),
autophagic bodies accumulated in the vacuoles (arrows in Fig. 1a).
The apg12-1 mutant did not accumulate autophagic bodies during
starvation. We cloned the APG12 gene by the method described
. APG12 encodes a hydrophilic protein of 186 amino
acids with a predicted relative molecular mass (M
) of 21K (Fig. 1b).
Figure 1 Cloning of APG12 and phenotype of apg12 disruptant. a, Wild-type,
apg12-1 mutant and Dapg12 cells were cultured in nitrogen-starvation medium
containing 1 mM PMSF. After incubation for 6 h, cells were observed under a
phase-contrast microscope. Arrows indicate autophagic bodies. b, Amino-acid
sequence of Apg12. c, Wild-type (squares) and Dapg12 (circles) were cultured in
nitrogen-starvation medium and their viability was determined by phloxine B
. d, Quantification of autophagic activity of wild-type and Dapg12 cells by
alkaline phosphatase (ALP) assay before (black bars) and after (white bars)
nitrogen starvation for 4 h. Error bars indicate s.d. of three independent
experiments. e, Homology between Apg12 and potential human and C. elegans\-
counterparts. C. elegans U32305 is 46% similar and 22% identical to amino acids
67–186 of yeast Apg12. A human cDNA (THC173313) encodes a protein that is
59% similar and 32% identical to amino acids 102186 of Apg12.
Nature © Macmillan Publishers Ltd 1998
letters to nature
VOL 395
Gene disruption experiments revealed that APG12 is not essential
for growth (data not shown) but is essential for autophagy (Fig. 1a)
and for maintaining viability during starvation (Fig. 1c). We
confirmed this in an assay system for measuring autophagic activity
(Fig. 1d), in which a truncated form of pro-alkaline phosphatase
expressed in the cytoplasm was delivered to vacuoles in an autop-
hagy-dependent manner and processed to the active enzyme
. A
vacuolar enzyme, aminopeptidase I, is delivered from the cytoplasm
to vacuoles constitutively to yield the mature, active enzyme
. This
‘Cvt pathway’ is closely linked to the autophagic process
, and all
apg mutants
, including Dapg12 cells, show defects in this pathway
(see Fig. 3d). The amino-acid sequence of Apg12 did not provide
any insight into its function, but a BLAST search identified a
potential Caenorhabditis elegans homologue whose function is
unknown (Fig. 1e). In addition, a search of the EST (expressed-
sequence tag) database identified several cDNA fragments encoding
parts of a potential human homologue (Fig. 1e).
To detect Apg12, we constructed a 3 × haemagglutinin(HA)-
tagged APG12. On immunoblotting, Apg12 presented as a ladder of
bands between 31K32.5K (Fig. 2a). As phosphatase treatment of
the lysate yielded a single band at 31K representing tagged Apg12
(data not shown), we concluded that Apg12 is phosphorylated in
vivo. Furthermore, we found that about half of the Apg12 was
present as a much larger band of ,70K (asterisked in Fig. 2a, b).
Although the 31K Apg12 was detected in all apg mutant strains, the
Dapg5, apg7-1 and apg10-1 strains did not show the 70K band
(Fig. 2b; Dapg1 is representative of the other mutants). These results
indicate that these three APG products are essential for the genera-
tion of the 70K band.
We have previously shown that the APG5 gene encodes a 294-
amino-acid protein
. Immunoblot analysis of 1 × HA-tagged Apg5
indicated that it also generated two bands in nearly equal amounts,
one of the size of tagged Apg5 (32.5K) and the other at about 70K
(Fig. 2c). In the Dapg12 strain, the higher band was not seen,
whereas the 32.5K band of Apg5 was slightly increased (Fig. 2c).
Immunoprecipitation analysis revealed that the 70K band included
both Apg5 and Apg12 (Fig. 2d). We concluded that it was a one-to-
one conjugate of Apg5 and Apg12.
To characterize the 70K band further, we did mutagenic analysis
of Apg12 (Fig. 3a). We found that the carboxy-terminal portion of
Apg12 was important for the conjugation (Fig. 3b: D57 and D121).
HA-APG5 + + -
Figure 2 Apg12 is conjugated to Apg5. a, b, Lysates from Dapg12 cells carrying
only vector or 3 × HA-APG12 (a), and Dapg5, apg7-1, apg10-1 and Dapg1 cells
carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody.
The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated. c,
Immunoblot analysis of wild-type and Dapg12 cells harbouring HA-APG5 plasmid.
d, Dapg5 Dapg12 cells were co-transformed with Myc-APG12 and HA-APG5.
Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and
detected by immunoblotting using anti-Myc antibody. The position of the
crossreacting IgG heavy chain is indicated.
Apg12 WT
Apg12 121
Apg12 57
Apg12 G
Apg12 G186A
ALP activity (U)
Figure 3 The C-terminal Gly residue of Apg12 is essential for interaction with
Apg5 and for autophagy. a, Diagram of Apg12 C-terminal mutants. b, Dapg12 cells
were transformed with the mutant plasmids and their lysates were immunoblotted
with anti-HA antibody. c, Autophagic activity was measured as described for Fig.1d.
d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-
API antiserum. The positions of pro-API and mature API are indicated.
vector WT K149R
ALP activity (U)
Figure 4 Apg5
is unable to generate Apg5/Apg12 conjugate and is defective
in autophagy. a, Position of the putative Apg12-interacting Lys residue. Dapg5
cells were transformed with vector alone, wild-type APG5 or APG5
, and then
immunoblotted with anti-HA (b) and anti-API (d). Autophagic activity was
determined by alkaline phosphatase assay (c).
Nature © Macmillan Publishers Ltd 1998
letters to nature
VOL 395
24 SEPTEMBER 1998 397
Even a single Gly 186 deletion at the C terminus (Apg12DG) caused
complete loss of the Apg12/Apg5 conjugate, although free Apg12DG
was detected in an amount comparable to that in the wild type
(Fig. 3b: DG). Apg12
, in which the Gly 186 is replaced by
alanine, was incorporated into the higher band inefficiently, but still
significantly (Fig. 3b: G186A). This indicates that Gly 186 is impor-
tant for Apg5/Apg12 conjugation. We next assessed the functional
activities of these mutants. Apg12DG showed an Apg-negative
phenotype (Fig. 3c), and was also unable to produce mature
aminopeptidase I (Fig. 3d), indicating that the Apg5/Apg12 con-
jugate is required for both autophagy and cytosol-to-vacuole
targeting of this enzyme. The Apg12
mutant showed an
almost normal phenotype for autophagy and for maturation of
aminopeptidase I (Fig. 3c, d), suggesting that a small amount of
Agp5/Apg12 conjugate is enough for it to function normally.
By analogy with ubiquitin
, conjugation of Apg5 and Apg12
probably occurs through formation of an isopeptide bond between
the C-terminal Gly 186 of Apg12 and an e-amino group of one of the
19 lysine residues in Apg5. To test this, we systematically replaced
each lysine residue of Apg5 with arginine. Both free Apg5 and the
Apg5/Apg12 conjugate were detected in 18 mutants (data not
shown). The Apg5
variant had no conjugate at all, but a
higher amount of free Apg5
(Fig. 4a, b), indicating that the
Lys 149 residue of Apg5 is the acceptor site for Apg12 conjugation.
As expected, Apg5
was defective in both autophagy and in
generating mature aminopeptidase I (Fig. 4c, d), whereas the other
18 mutants were normal (data not shown). Starvation did not alter
the relative amounts of free Apg5, free Apg12 or of the Apg5/Apg12
conjugate. We conclude that the conjugate functions as a common
machinery in both pathways: for the autophagic pathway during
starvation and for the Cvt pathway in the growing phase.
As shown in Fig. 2, the apg7 and apg10 mutants failed to
conjugate Apg5 and Apg12, suggesting that these two APG products
may function as an enzyme system for conjugation. Cloning of the
APG7 gene revealed that it encodes a 630-amino-acid protein with
predicted M
of 71.4K (Fig. 5a). The region containing amino acids
322392 of Apg7 shows significant homology with the correspond-
ing region in E1, the ubiquitin-activating enzyme in S. cerevisiae
(Fig. 5b) and in other species (data not shown). This region
encompasses a putative ATP-binding site (GxGxxG)
, suggesting
that Apg7 may be an Apg12-activating enzyme. Although the
sequence around the active-site cysteine is less conserved, align-
ments between Apg7 and other E1-like enzymes indicate that
Cys 507 is a putative active-site cysteine (Fig. 5b). Apg10 might be
an E2 ubiquitin-conjugating enzyme type of protein because its size
is similar to various E2 enzymes and one of its cysteine residues is
essential for its function (T. Shintani et al., unpublished results). We
reconstituted the conjugation reaction in vitro. Lysates of Dapg5
cells and Dapg12 cells were mixed in vitro and incubated with or
without ATP. Figure 5c shows that the 70K band appeared in a time-
dependent and ATP-dependent manner. The conjugation was
sensitive to 1 mM N-ethylmaleimide (data not shown). These
results show that the Apg12 conjugation pathway contains an
ATP-dependent step, which is probably the activation of Apg12 by
Autophagy involves a dynamic membrane rearrangement
Morphological studies have indicated that all APG products func-
tion at or before the autophagosome formation step (M. Baba and
Y.O., unpublished results). Some Apg proteins are present on
membrane structures
. Most of the Apg5 and Apg5/Apg12 con-
jugate, and more than half of the free Apg12, were present in
100,000g pellet fractions (data not shown), suggesting that they
associate with some membrane compartments. We therefore exam-
ined their intracellular localization by sucrose density-gradient
centrifugation analysis and found that free Apg5 and the Apg5/
Apg12 conjugate co-fractionated (Fig. 6); in contrast, most of the
0 2 10 30 30 (min)
1 2 3 4 5 6 7
| |:|| |:|:| :|| : : ||: | | || :: || || |: :| || |:| || :: : |
506 MCTV Apg7
599 LCTL Uba1
176 VCTI Uba2
167 MCTI Uba3
Figure 5 Apg7 is an E1-like protein and Apg12 is conjugated to Apg5 in an ATP-
dependent manner. a, Amino-acid sequence of Apg7. b, Homology between
Apg7 and Uba1, S. cerevisiae E1 enzyme. Black circles indicate a putative ATP-
binding site (GxGxxG). The putative active-site Cys residue of Apg7 is indicated. c,
In vitro conjugation of Apg5 and Apg12. A cell lysate of Dapg12 carrying HA-APG5
(lane 1) was incubated with an equal amount of lysate from Dapg5 carrying HA
APG12 (2 m plasmid) (lane 2) at 30 8C with (lane 46) or without (lane 7) 5 mM ATP.
Samples were mixed with SDS-sample buffer at the times indicated.
Fraction number
ADH ALP Kex2 Sec12
Figure 6 Apg5/Apg12 conjugate co-fractionates with free Apg5 but not free
Apg12. Spheroplasts were generated from cells expressing either HA-Apg12 or
HA-Apg5. Their lysates were mixed and layered on top of a 10-step (1854 % w/w)
sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions
were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12
conjugate were examined by western blotting. The peak fractions of alcohol
dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endo-
plasmic reticulum) are indicated by arrows.
Figure 7 Model of the Apg12-conjugation system.
Nature © Macmillan Publishers Ltd 1998
letters to nature
VOL 395
Apg12 was in the denser fractions. These results indicate that the
conjugation of Apg5 and Apg12 is associated with a change in the
subcellular localization of Apg12.
We have described a new covalent modification system that is
required for autophagy in yeast. Four of 14 APG products function
in this pathway. Our model is shown in Fig. 7: Apg12 is activated by
binding to Apg7 via a high-energy thioester bond; through transfer
to an E2-like molecule (possibly Apg10), Apg12 is finally conjugated
to Lys 149 of Apg5 via an isopeptide bond. Although the steps in this
conjugation pathway are similar to those that occur in ubiquitina-
and in the modification by other ubiquitin-like proteins
such as SUMO-1 (refs 1821), Smt3 (ref. 22), Rub1 (refs 23, 24) and
Nedd8 (ref. 25), Apg12 has several unique features. It has no
significant homology to ubiquitin and is much larger than ubiquitin
and ubiquitin-related modifiers
. Only a single specific substrate,
Apg5, has been found. Apg12 homologues in human and C. elegans
have a glycine residue at the C terminus (Fig. 1c). We have cloned
human Apg12 and found that it is conjugated to human Apg5
(N.M., H. Sugita, T.Y. and Y.O., manuscript in preparation). Human
Apg5 was recently cloned as ‘apoptosis specific protein by another
, although its physiological significance is not clear yet. This
conjugation system is conserved from yeast to mammalian cells, and
may be critical for autophagy in every eukaryote.
. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yeast strains. The Saccharomyces cerevisiae strains used for cloning and
immunochemical analysis were MT3-4-4(MATa apg12-1 ura3), MT87-4-
5(MATa apg7-1 ura3), MT91-4-2(MATa apg10-1 ura3), SKD5-1D(MATa ura3
leu2 trp1 Dapg5::LEU2) and YYK36(MATa ura3 leu2 trp1 his3 Dapg1::LEU2).
Gene disruptions of APG5 and APG12 were performed with YW5-1B(MATa
ura3 leu2 trp1) or KA31(MATa ura3 leu2 trp1 his3).
Alkaline phosphatase assay. The APG12 or APG5 gene was disrupted in
TN125(MATa ura3 leu2 trp1 his3 ade2 lys2 PHO8::pho8D60), and the assay was
done as described
Immunochemical procedures. Whole-cell extracts were prepared by
suspending cells in 0.2M NaOH, 0.5% b-mercaptoethanol, and precipitated
with acetone. Extracts were separated by SDSPAGE, followed by immuno-
blotting using anti-HA antibody (16B12, BAbCO) or anti-API (aminopepti-
dase I) polyclonal antibody. Immunoprecipitation was done as described
using 16B12 or anti-Myc antibody (9E10).
Site-directed mutagenesis. Mutation and deletion constructs were generated
by PCR-based site-directed mutagenesis and confirmed by automated DNA
In vitro Apg12 conjugation assay. Total cell lysates were prepared from
Dapg12 strain expressing HA-Apg5 and Dapg5 strain expressing HA-Apg12
after spheroplasting. Both lysates (30 mg ml
) were mixed in 50 mM Tris (pH
7.5), 100 mM NaCl, 10 mM MgCl
, 1 mM DTT, 0.3 mM PMSF and 2mg ml
pepstatin, and incubated at 30 8C for the indicated times with or without 5 mM
ATP. The reaction was stopped by mixing with SDSPAGE buffer and boiling.
Received 20 May; accepted 29 June 1998.
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function of the SCF
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25. Osaka, F. et al. A new Nedd8-ligating system for cullin-4A. Genes Dev. (in the press).
26. Hammond, E. M. et al. Homology between a human apoptosis specific protein and the product of
APG5, a gene involved in autophagy in yeast. FEBS Lett. 425, 391395 (1998).
27. Noda, T. & Ohsumi, Y. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J.
Biol. Chem. 273, 39633966 (1998).
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function and localizes in a novel Golgi-like distribution. Mol. Biol. Cell 3, 633654 (1992).
Acknowledgements. We thank Y. Wada for the genomic library. N.M. is a research fellow of the Japan
Society for the Promotion of Science.
Correspondence and requests for materials should be addressed to Y.O. (e-mail:
The sequences of APG12 and APG7 are available from GenBank under accession numbers Z36086 (ORF
YBR217w) and U00027 (ORF YHR171w), respectively.
Retinoid-X receptor signalling
in the developing spinal cord
Ludmila Solomin*, Clas B. Johansson
, Rolf H. Zetterstro
Reid P. Bissonnette§, Richard A. Heyman§, Lars Olson
Urban Lendahl
, Jonas Frise
& Thomas Perlmann*
* The Ludwig Institute for Cancer Research, Stockholm Branch, PO Box 240,
S-171 77 Stockholm, Sweden
Departments of
Cell and Molecular Biology and
Karolinska Institute, S-171 77 Stockholm, Sweden
§ Ligand Pharmaceuticals, 10275 Science Center Drive, San Diego,
California 92121, USA
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Retinoids regulate gene expression through the action of retinoic
acid receptors (RARs) and retinoid-X receptors (RXRs), which
both belong to the family of nuclear hormone receptors
. Reti-
noids are of fundamental importance during development
, but it
has been difficult to assess the distribution of ligand-activated
receptors in vivo. This is particularly the case for RXR, which is a
critical unliganded auxiliary protein for several nuclear receptors,
including RAR
, but its ligand-activated role in vivo remains
uncertain. Here we describe an assay in transgenic mice, based
on the expression of an effector fusion protein linking the ligand-
binding domain of either RXR or RAR to the yeast Gal4 DNA-
binding domain, and the in situ detection of ligand-activated
effector proteins by using an inducible transgenic lacZ reporter
gene. We detect receptor activation in the spinal cord in a pattern
that indicates that the receptor functions in the maturation of
limb-innervating motor neurons. Our results reveal a specific
activation pattern of Gal4RXR which indicates that RXR is a
critical bona fide receptor in the developing spinal cord.
Ligands for retinoid receptors are all-trans retinoic acid (RA),
which binds to RAR, and 9-cis RA, which binds both RAR and

Supplementary resources (2)

... Additionally, the key to autophagosome development in humans is class III phosphoinositide 3-kinases Vps34 and Vps35, which, along with Beclin-1 (human homolog of Atg6), mediate the "nucleation" step. Elongation of the isolation membrane is mediated by two ubiquitin-like conjugation systems that are key to the autophagosome expansion: one system where Atg7 (an E1-like protein) and Atg10 (E2-like) act to conjugate Atg5-Atg12, and another where the Atg5-Atg12 conjugate (an E3-like protein) acts in concert with Atg7 and Atg3 (E2-like) to conjugate Atg8 to phosphatidylethanolamine, PE (so-called lipidation" of (LC3) for insertion into the membranes of the growing autophagosome [17][18][19]. LC3-I is uniformly distributed in the cell when autophagy levels are low, whereas upon induction of autophagy, lipidation of LC3 causes its relocalization to the autophagosome, which can be visualized and quantified by counting LC3 spots using immunofluorescence microscopy or by its differential migration in SDS-PAGE gels by Western blotting [20]. P62, also called sequestosome 1 (p62/SQSTM1), is a ubiquitin-binding scaffold protein, and is a classical selective autophagy receptor, but it also has roles in the ubiquitin-proteasome system, cellular metabolism, signaling, and apoptosis. ...
... During autophagy, Atg4 removes the Cterminal arginine of Atg8, which is a crucial step required for Atg8 to covalently link to phosphatidylethanolamine (PE) on isolation membranes [143]. ATG5 Atg5 is a protein involved in the early stages of autophagosome formation [19]. Atg5 binds with Atg12 and is catalyzed by Atg7 and Atg10. ...
Full-text available
Autophagy is a highly conserved lysosomal degradation pathway active at basal levels in all cells. However, under stress conditions, such as a lack of nutrients or trophic factors, it works as a survival mechanism that allows the generation of metabolic precursors for the proper functioning of the cells until the nutrients are available. Neurons, as post-mitotic cells, depend largely on autophagy to maintain cell homeostasis to get rid of damaged and/or old organelles and misfolded or aggregated proteins. Therefore, the dysfunction of this process contributes to the pathologies of many human diseases. Furthermore, autophagy is highly active during differentiation and development. In this review, we describe the current knowledge of the different pathways, molecular mechanisms, factors that induce it, and the regulation of mammalian autophagy. We also discuss its relevant role in development and disease. Finally, here we summarize several investigations demonstrating that autophagic abnormalities have been considered the underlying reasons for many human diseases, including liver disease, cardiovascular, cerebrovascular diseases, neurodegenerative diseases, neoplastic diseases, cancers, and, more recently, infectious diseases, such as SARS-CoV-2 caused COVID-19 disease.
... Autophagy requires two ubiquitin-like conjugation systems. The first is the Atg12~Atg5 conjugation system primarily involved in expansion of the isolation membrane, in which ubiquitin-like Atg12 is activated by E1-like Atg7, transferred to E2-like Atg10, and then covalently conjugated to Atg5 [39]. Atg12~Atg5 then associates with dimeric Atg16 to form a multimeric membrane-associated complex [133]. ...
... Atg5 was suggested to be membrane-associated using subcellular fractionation [39,40]. Liposome sedimentation assays further confirmed the binding of Atg5 to Folch liposomes in vitro, where the addition of Atg12 reduced the binding, which was then restored upon further addition of Atg16 [54]. ...
Cells rely on autophagy to degrade cytosolic material and maintain homeostasis. During autophagy, content to be degraded is encapsulated in double membrane vesicles, termed autophagosomes, which fuse with the yeast vacuole for degradation. This conserved cellular process requires the dynamic rearrangement of membranes. As such, the process of autophagy requires many soluble proteins that bind to membranes to restructure, tether, or facilitate lipid transfer between membranes. Here, we review the methods that have been used to investigate membrane binding by the core autophagy machinery and additional accessory proteins involved in autophagy in yeast. We also review the key experiments demonstrating how each autophagy protein was shown to interact with membranes.
... Lacking BRAP homologous protein led to enhanced autophagy activity ATG5 is an essential factor for autophagy. It is constantly conjugated to ATG12 (Mizushima et al, 1998) and is involved in autophagic vesicle formation. We put forward a hypothesis that the mechanisms underlying the regulatory effect of BRAP on BLM induced lung injury might also be via the autophagy pathway by interacting with ATG5. ...
Full-text available
Bombesin receptor–activated protein (BRAP) was found to express in the interstitial cells of human fibrotic lungs with unknown function. Its homologous protein, encoded by BC004004 gene, was also present in mouse lung tissues. We used BC004004 −/− mice which lack BRAP homologous protein expression to establish a bleomycin-induced lung fibrotic model. After bleomycin treatment, BC004004 −/− mice exhibited attenuation of pulmonary injury and less pulmonary fibrosis. Fibroblasts from BC004004 −/− mice proliferated at a lower rate and produced less collagen. Autophagy-related gene 5 (ATG5) was identified as a partner interacting with human BRAP. Lacking BRAP homologous protein led to enhanced autophagy activity in mouse lung tissues as well as in isolated lung fibroblasts, indicating a negative regulatory role of this protein in autophagy via interaction with ATG5. Enhanced autophagy process in fibroblasts due to lack of BRAP homologous protein might contribute to the resistance of BC004004 −/− mice to pulmonary fibrosis.
... The Atg16L1 complex is recruited to the pre-autophagosomal structure (PAS) by PtdIns3P binding protein such as WIPI2 (Polson et al., 2010;Dooley et al., 2014). LC3 is processed into LC3-II with the assistance of Atg7, Atg4, and Atg3 (Glick et al., 2010), and then LC3-II binds with phosphatidylethanolamine (PE), which is essential for the expansion and completion of the autophagic membrane (Mizushima et al., 1998;Glick et al., 2010). Finally, the autophagosome fuses with the lysosome to form an autolysosome with the involvement of some proteins, including the small GTPase Rab7, syntaxin 17, vesicle-associated membrane protein (VAMP7, VAMP8) (Jager et al., 2004;Itakura et al., 2012;Wang et al., 2016). ...
Full-text available
Autophagy is an immune homeostasis process induced by multiple intracellular and extracellular signals. Inflammation is a protective response to harmful stimuli such as pathogen microbial infection and body tissue damage. Porphyromonas gingivalis infection elicits both autophagy and inflammation, and dysregulation of autophagy and inflammation promotes pathology. This review focuses on the interaction between autophagy and inflammation caused by Porphyromonas gingivalis infection, aiming to elaborate on the possible mechanism involved in the interaction.
... The process of expansion and maturation of the autophagosome membrane involves two ubiquitin-like conjugation systems: the conjugation of ATG12 to ATG5, and the conversion of LC3 I to LC3 II (Mizushima, 2020). The conjugation of Atg12 to Atg5 occurs at Lys130 through the activation of E1 enzyme Atg7 and the E2-like Atg10 (Mizushima et al., 1998;Otomo et al., 2013). The Atg12-Atg5 conjugate then forms a large protein complex with Atg16, acts as the E3 ligase for the conjugation of LC3 I to PE (phosphatidylethanolamine). ...
Full-text available
Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder, which is caused by mutations in the PKD1 and PKD2 genes, characterizing by progressive growth of multiple cysts in the kidneys, eventually leading to end-stage kidney disease (ESKD) and requiring renal replacement therapy. In addition, studies indicate that disease progression is as a result of a combination of factors. Understanding the molecular mechanisms, therefore, should facilitate the development of precise therapeutic strategies for ADPKD treatment. The roles of epigenetic modulation, interstitial inflammation, and regulated cell death have recently become the focuses in ADPKD. Different epigenetic regulators, and the presence of inflammatory markers detectable even before cyst growth, have been linked to cyst progression. Moreover, the infiltration of inflammatory cells, such as macrophages and T cells, have been associated with cyst growth and deteriorating renal function in humans and PKD animal models. There is evidence supporting a direct role of the PKD gene mutations to the regulation of epigenetic mechanisms and inflammatory response in ADPKD. In addition, the role of regulated cell death, including apoptosis, autophagy and ferroptosis, have been investigated in ADPKD. However, there is no consensus whether cell death promotes or delays cyst growth in ADPKD. It is therefore necessary to develop an interactive picture between PKD gene mutations, the epigenome, inflammation, and cell death to understand why inherited PKD gene mutations in patients may result in the dysregulation of these processes that increase the progression of renal cyst formation.
... In the mammalian cells, the mATG8 family consist of six orthologs, including the LC3 (LC3A, LC3B, and LC3C) and GABARAP (GABARAP, GABARAPL1, and GABARAPL2) subfamilies. The lipidation of mATG8s relies on the following two Ub-like conjugation systems [25][26][27] . In the ATG12 system, ATG12 is activated by the E1-like ATG7 before it is transferred to the E2-like ATG10, followed by its covalent conjugation to ATG5. ...
Full-text available
PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses. The mechanisms underlying mitochondrial quality control are not fully understood. Here the authors identify a switch from degradative to secretory autophagy in the absence of the mATG8-conjugation system, termed Autophagic Secretion of Mitochondria.
... Next, to confirm specifically that autophagy and LAP were not responsible for the clearance of intracellular leptospires, we used siRNA targeting the autophagy-related protein Atg5, essential for canonical autophagosome nucleation (Mizushima et al., 1998). After infection with L. interrogans strain L495 and gentamicin-protection assay, we observed no difference between control and atg5 siRNA conditions and witnessed the same reduction in leptospiral loads between 3 and 6 h ( Figure 4E, upper panel). ...
Full-text available
Leptospira interrogans are pathogenic bacteria responsible for leptospirosis, a zoonosis impacting 1 million people per year worldwide. Leptospires can infect all vertebrates, but not all hosts develop similar symptoms. Human and cattle may suffer from mild to acute illnesses and are therefore considered as sensitive to leptospirosis. In contrast, mice and rats remain asymptomatic upon infection, although they get chronically colonized in their kidneys. Upon infection, leptospires are stealth pathogens that partially escape the recognition by the host innate immune system. Although leptospires are mainly extracellular bacteria, it was suggested that they could also replicate within macrophages. However, contradictory data in the current literature led us to reevaluate these findings. Using a gentamicin–protection assay coupled to high-content (HC) microscopy, we observed that leptospires were internalized in vivo upon peritoneal infection of C57BL/6J mice. Additionally, three different serotypes of pathogenic L. interrogans and the saprophytic L. biflexa actively infected both human (PMA differentiated) THP1 and mouse RAW264.7 macrophage cell lines. Next, we assessed the intracellular fate of leptospires using bioluminescent strains, and we observed a drastic reduction in the leptospiral intracellular load between 3 h and 6 h post-infection, suggesting that leptospires do not replicate within these cells. Surprisingly, the classical macrophage microbicidal mechanisms (phagocytosis, autophagy, TLR–mediated ROS, and RNS production) were not responsible for the observed decrease. Finally, we demonstrated that the reduction in the intracellular load was associated with an increase of the bacteria in the supernatant, suggesting that leptospires exit both human and murine macrophages. Overall, our study reevaluated the intracellular fate of leptospires and favors an active entrance followed by a rapid exit, suggesting that leptospires do not have an intracellular lifestyle in macrophages.
LC3-associated phagocytosis (LAP) is a noncanonical autophagy process reported in recent years and is one of the effective mechanisms of host defense against bacterial infection. During LAP, bacteria are recognized by pattern recognition receptors (PRRs), enter the body, and then recruit LC3 onto a single-membrane phagosome to form a LAPosome. LC3 conjugation can promote the fusion of the LAPosomes with lysosomes, resulting in their maturation into phagolysosomes, which can effectively kill the identified pathogens. However, to survive in host cells, bacteria have also evolved strategies to evade killing by LAP. In this review, we summarized the mechanism of LAP in resistance to bacterial infection and the ways in which bacteria escape LAP. We aim to provide new clues for developing novel therapeutic strategies for bacterial infectious diseases.
Autophagy is an evolutionarily conserved multistep degradation mechanism in eukaryotes, that maintains cellular homoeostasis by replenishing cells with nutrients through catabolic lysis of the cytoplasmic components. This critically coordinated pathway involves sequential processing events that begin with initiation, nucleation, and elongation of phagophores, followed by the formation of double‐membrane vesicles known as autophagosomes. Finally, autophagosomes migrate towards and fuse with lysosomes in mammals and vacuoles in yeast and plants, for the eventual degradation of the intravesicular cargo. Here, we review the recent advances in our understanding of the molecular events that define the process of autophagy.
Autophagy is a highly conserved self-degradation process of eukaryotic cells which is required for the effective elimination of damaged and unnecessary cytosolic constituents. Defects in the process can cause the intracellular accumulation of such damages, thereby leading to the senescence and subsequent loss of the affected cell. Defective autophagy hence is implicated in the development of various degenerative processes, including cancer, neurodegenerative diseases, diabetes, tissue atrophy and fibrosis, and immune deficiency, as well as in accelerated aging. The autophagic process is mediated by numerous autophagy-related (ATG) proteins, among which the ATG8/LC3/GABARAP (Microtubule-associated protein 1A/1B-light chain 3/Gammaaminobutyric acid receptor-associated protein) superfamily has a pivotal role in the formation and maturation of autophagosome, a key (macro) autophagic structure (the autophagosome sequesters parts of the cytoplasm which are destined for breakdown). While in the unicellular yeast there is only a single ATG8 protein, metazoan systems usually contain more ATG8 paralogs. ATG8 paralogs generally display tissue-specific expression patterns and their functions are not strictly restricted to autophagy. For example, GABARAP proteins also play a role in intracellular vesicle transport, and, in addition to autophagosome formation, ATG8 also functions in selective autophagy. In this review, we summarize the functional diversity of ATG8/LC3/GABARAP proteins, using tractable genetic models applied in autophagy research.
Full-text available
We have investigated the role of the essential Rho1 GTPase in cell integrity signaling in budding yeast. Conditional rho1 mutants display a cell lysis defect that is similar to that of mutants in the cell integrity signaling pathway mediated by protein kinase C (Pkc1), which is suppressed by overexpression of Pkc1. rho1 mutants are also impaired in pathway activation in response to growth at elevated temperature. Pkc1 co-immunoprecipitates with Rho1 in yeast extracts, and recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon Pkc1 the ability to be stimulated by phosphatidylserine, indicating that Rho1 controls signal transmission through Pkc1.
Full-text available
Although LOX mRNA accumulates early during differentiation, a differentiation control element in its 3′ untranslated region confers translational silencing until late stage erythropoiesis. We have purified two proteins from rabbit reticulocytes that specifically mediate LOX silencing and identified them as hnRNPs K and E1. Transfection of hnRNP K and hnRNP E1 into HeLa cells specifically silenced the translation of reporter mRNAs bearing a differentiation control element in their 3′ untranslated region. Silenced LOX mRNA in rabbit reticulocytes specifically coimmunoprecipitated with hnRNP K. In a reconstituted cell-free translation system, addition of recombinant hnRNP K and hnRNP E1 recapitulates this regulation via a specific inhibition of 80S ribosome assembly on LOX mRNA. Both proteins can control cap-dependent and internal ribosome entry site–mediated translation by binding to differentiation control elements. Our data suggest a specific cytoplasmic function for hnRNPs as translational regulatory proteins.
Full-text available
Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.
Full-text available
The RUB1/NEDD-8 family of ubiquitin-related genes is widely represented among eukaryotes. Here we report that Cdc53p in Saccharomyces cerevisiae, a member of the Cullin family of proteins, is stably modified by the covalent attachment of a single Rub1p molecule. Two genes have been identified that are required for Rub1p conjugation to Cdc53p. The first gene, designated ENR2, encodes a protein with sequence similarity to the amino-terminal half of the ubiquitin-activating enzyme. By analogy with Aos1p, we infer that Enr2p functions in a bipartite Rub1p-activating enzyme. The second gene is SKP1, shown previously to be required for some ubiquitin-conjugation events. A deletion allele of ENR2 is lethal with temperature-sensitive alleles of cdc34 and enhances the phenotypes of cdc4, cdc53, and skp1, strongly implying that Rub1p conjugation to Cdc53p is required for optimal assembly or function of the E3 complex SCFCdc4. Consistent with this model, both enr2delta and an allele of Cdc53p that is not Rub1p modified, render cells sensitive to alterations in the levels of Cdc4p, Cdc34p, and Cdc53p.
The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping , mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquiti-nated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE) , where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.
The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.
Stress conditions lead to a variety of physiological responses at the cellular level. Autophagy is an essential process used by animal, plant, and fungal cells that allows for both recycling of macromolecular constituents under conditions of nutrient limitation and remodeling the intracellular structure for cell differentiation. To elucidate the molecular basis of autophagic protein transport to the vacuole/lysosome, we have undertaken a morphological and biochemical analysis of this pathway in yeast. Using the vacuolar hydrolase aminopeptidase I (API) as a marker, we provide evidence that the autophagic pathway overlaps with the biosynthetic pathway, cytoplasm to vacuole targeting (Cvt), used for API import. Before targeting, the precursor form of API is localized mostly in restricted regions of the cytosol as a complex with spherical particles (termed Cvt complex). During vegetative growth, the Cvt complex is selectively wrapped by a membrane sac forming a double membrane-bound structure of ∼150 nm diam, which then fuses with the vacuolar membrane. This process is topologically the same as macroautophagy induced under starvation conditions in yeast (Baba, M., K. Takeshige, N. Baba, and Y. Ohsumi. 1994. J. Cell Biol. 124:903–913). However, in contrast with autophagy, API import proceeds constitutively in growing conditions. This is the first demonstration of the use of an autophagy-like mechanism for biosynthetic delivery of a vacuolar hydrolase. Another important finding is that when cells are subjected to starvation conditions, the Cvt complex is now taken up by an autophagosome that is much larger and contains other cytosolic components; depending on environmental conditions, the cell uses an alternate pathway to sequester the Cvt complex and selectively deliver API to the vacuole. Together these results indicate that two related but distinct autophagy-like processes are involved in both biogenesis of vacuolar resident proteins and sequestration of substrates to be degraded.
Lysosomes play a central role in the degradation of extracellular and intracellular macromolecules. These organelles contain hydrolytic enzymes capable of degrading proteins, proteoglycans, nucleic acids, and lipids. The mechanisms involved in the delivery of such intracellular compounds to the lysosome have been characterized in several recent studies. The sequestration of intracellular macromolecules for intralysosomal degradation can occur by crinophagy, hsc73-mediated carrier transport, or autophagy. The major route of delivery of cellular proteins and RNA into lysosomes is by autophagy. Furthermore, autophagy is regulated by nutrients and hormones, thus allowing the cell to adjust its degradative state to environmental changes.