Download Inhibition of Target of Rapamycin Signaling and

Inhibition of Target of Rapamycin Signaling and Stress
Activate Autophagy in Chlamydomonas reinhardtii1[W]
Marı́a Esther Pérez-Pérez, Francisco J. Florencio, and José L. Crespo*
Instituto de Bioquı́mica Vegetal y Fotosı́ntesis, Consejo Superior de Investigaciones Cientı́ficas-Universidad
de Sevilla, 41092 Seville, Spain
Autophagy is a catabolic membrane-trafficking process whereby cells recycle cytosolic proteins and organelles under stress
conditions or during development. This degradative process is mediated by autophagy-related (ATG) proteins that have been
described in yeast, animals, and more recently in plants. In this study, we report the molecular characterization of autophagy in
the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) is
functionally conserved and may be used as a molecular autophagy marker. Like yeast ATG8, CrATG8 is cleaved at the
carboxyl-terminal conserved glycine and is associated with membranes in Chlamydomonas. Cell aging or different stresses such
as nutrient limitation, oxidative stress, or the accumulation of misfolded proteins in the endoplasmic reticulum caused an
increase in CrATG8 abundance as well as the detection of modified forms of this protein, both landmarks of autophagy
activation. Furthermore, rapamycin-mediated inhibition of the Target of Rapamycin signaling pathway, a major regulator of
autophagy in eukaryotes, results in identical effects on CrATG8 and a relocalization of this protein in Chlamydomonas cells
similar to the one observed upon nutrient limitation. Thus, our findings indicate that Chlamydomonas cells may respond to
stress conditions by inducing autophagy via Target of Rapamycin signaling modulation.
Protein turnover is essential for the adaptation
of cells to variable environmental conditions. Similar to other eukaryotes, plants have developed two
distinct mechanisms to regulate protein degradation, a selective ubiquitin/26S proteasome pathway
(Vierstra, 2009) and macroautophagy (hereafter referred
to as autophagy), a nonselective membrane-trafficking
process (Bassham, 2009). During autophagy, a large
number of cytosolic components, including entire organelles, organelle fragments, and protein complexes,
are enclosed in bulk within a double-membrane structure known as the autophagosome and delivered
to the vacuole/lysosome for degradation to recycle
needed nutrients or degrade toxic components (Xie
and Klionsky, 2007; Nakatogawa et al., 2009). The
autophagosomes appear to arise from isolation membranes usually observed in close proximity to the
vacuole called the preautophagosomal structure
(PAS). These membranes expand and fuse to encircle
portions of the cytoplasm, generating an autophagosome that is targeted to the vacuole. The outer membrane of the autophagosome then fuses with the
1
This work was supported by the Spanish Ministry of Science
and Innovation (grant no. BFU2009–07368 to J.L.C. and grant no.
BFU2007–60300 to F.J.F.) and the Junta de Andalucı́a (grant no.
BIO284).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
José L. Crespo ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.152520
1874
vacuole membrane, and the remaining vesicle, known
as the autophagic body, is finally released to the
vacuole for its degradation (Xie and Klionsky, 2007).
The evolutionary conservation of autophagy among
eukaryotes indicates that structural and regulatory components of this cellular process must be
also conserved. Accordingly, a significant number of
autophagy-related (ATG) genes that participate in
autophagy regulation and autophagosome formation
have been identified, initially through genetic approaches in yeast and subsequently in higher eukaryotes, including mammals, insects, protozoa, and
plants (Bassham et al., 2006; Bassham, 2007; Meijer
et al., 2007). In yeast, two protein conjugation systems
composed of the ubiquitin-like proteins ATG8 and
ATG12 and the three enzymes ATG3, ATG7, and
ATG10 play an essential role in autophagosome formation and seem to be conserved through evolution
(Geng and Klionsky, 2008). ATG8 becomes modified
with the lipid molecule phosphatidylethanolamine
(PE) by the consecutive action of the ATG7 and
ATG3 enzymes in a process mechanistically similar
to ubiquitination (Ichimura et al., 2000). Prior to this
modification, ATG8 must be cleaved by the Cys protease ATG4 to expose a C-terminal Gly residue that is
conjugated to PE (Kirisako et al., 2000; Kim et al., 2001).
ATG12 becomes covalently attached to the ATG5 protein in a conjugation reaction that is catalyzed by ATG7
and ATG10 (Mizushima et al., 1998). ATG8-PE and
ATG12-ATG5 conjugates localize to autophagy-related
membranes and are required for the initiation and
expansion of autophagosomal membrane and hemifusion of this membrane with the vacuolar membrane
(Hanada et al., 2007; Nakatogawa et al., 2007, 2009;
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Autophagy Regulation in Chlamydomonas
Fujita et al., 2008; Geng and Klionsky, 2008; Xie et al.,
2008).
Our understanding of the autophagy process has
substantially increased with the development of specific markers for autophagy. In plants, two markers for
autophagosomes have been described, the monodansylcadaverine dye and GFP-ATG8 fusion protein
(Yoshimoto et al., 2004; Contento et al., 2005; Thompson
et al., 2005). As in other species, binding of ATG8 to
autophagosomes has been used to monitor autophagy
in plants. In contrast to yeast, where a single ATG8
gene is present, plants appear to contain a small gene
family with several ATG8 isoforms, suggesting that
autophagy is more complex in these photosynthetic
organisms. For example, Arabidopsis (Arabidopsis
thaliana) and maize (Zea mays) encode nine and five
ATG8 genes, respectively (Doelling et al., 2002; Hanaoka
et al., 2002; Ketelaar et al., 2004; Chung et al., 2009).
However, despite the high complexity of the ATG8conjugating system in plants, important findings have
been recently reported on the molecular characterization of autophagy using ATG8 as an autophagy
marker in these organisms. The use of specific markers
for autophagy in plants has revealed that this process
is active at a basal level under normal growth and is
induced upon nitrogen- or carbon-limiting conditions
as well as in response to oxidative stress (Yoshimoto
et al., 2004; Thompson et al., 2005; Xiong et al., 2005,
2007; Chung et al., 2009). Reverse genetic approaches
have also been applied to a number of Arabidopsis
ATG genes using T-DNA insertional mutants or RNA
interference in order to investigate the physiological
roles of autophagy in plants. The initial characterization of autophagy-deficient plants demonstrated that
the ATG system is not essential under nutrient-rich
conditions. However, a detailed analysis of these mutants indicated that autophagy is required for the
proper response of the plant to nutrient limitation or
pathogen infection. Plants lacking the ATG4, ATG5,
ATG7, ATG9, or ATG10 gene display premature leaf
senescence and are hypersensitive to nitrogen or
carbon limitation (Doelling et al., 2002; Hanaoka
et al., 2002; Yoshimoto et al., 2004; Thompson et al.,
2005; Phillips et al., 2008). Arabidopsis plants with
reduced levels of ATG18, which is required for autophagosome formation, are more sensitive to methyl
viologen treatment and accumulate high levels of
oxidized proteins, demonstrating that autophagic processes participate in the response of the plant to
oxidative stress (Xiong et al., 2005, 2007). Plants deficient in the autophagy genes ATG6/Beclin1, ATG3,
ATG7, and ATG9 exhibit unrestricted hypersensitive
response lesions in response to pathogen infection (Liu
et al., 2005; Hofius et al., 2009). These findings implicate autophagy as a prosurvival mechanism to restrict
programmed cell death associated with the pathogeninduced hypersensitive response in plants. Arabidopsis ATG6 has also been shown to mediate pollen
germination in a manner independent of autophagy
(Fujiki et al., 2007).
As mentioned above, autophagy is triggered among
other factors by a reduction in the availability of
nutrients. This starvation signal is transmitted to the
autophagic machinery by important regulatory
factors, including the Ser/Thr kinases Target of Rapamycin (TOR), ATG1, and SNF1 and the phosphatidylinositol 3-kinase ATG6/Beclin1 (Diaz-Troya et al.,
2008b; Bassham, 2009; Cebollero and Reggiori, 2009).
TOR has been identified as a negative regulator of
autophagy in yeast, mammals, and fruit flies (DiazTroya et al., 2008b). The pharmacological inhibition of
TOR by rapamycin leads to autophagy activation
through a mechanism that requires the activation of
the ATG1 kinase (Kamada et al., 2000). It has been
recently demonstrated in mammals and fruit flies that
a rapamycin-sensitive TOR signaling complex termed
TORC1 directly phosphorylates and inhibits the ATG1
kinase and its regulatory protein ATG13 (Chang and
Neufeld, 2009; Hosokawa et al., 2009; Jung et al., 2009).
These regulatory proteins are conserved in plants,
although except for ATG6 (Liu et al., 2005), there is no
direct evidence for regulation of autophagy by these
signaling pathways.
The unicellular green alga Chlamydomonas reinhardtii
has been used as a model for the study of important
cellular and metabolic processes in photosynthetic
organisms (Harris, 2001). More recently, Chlamydomonas has also been proposed as a useful system for the
characterization of the TOR signaling pathway in
photosynthetic eukaryotes based on the finding that,
unlike plants, Chlamydomonas cell growth is sensitive
to rapamycin (Crespo et al., 2005; Diaz-Troya et al.,
2008a). Treatment of Chlamydomonas cells with rapamycin results in a pronounced increase of vacuole size
that resembles autophagy-like processes (Crespo et al.,
2005). However, a role of TOR in autophagy regulation
could not be demonstrated due to the absence of an
autophagy marker in Chlamydomonas. Actually, no studies have been reported on any autophagy-related protein in green algae, despite the high conservation of
ATG genes in Chlamydomonas (Diaz-Troya et al., 2008b).
This study reports the molecular and cellular characterization of autophagy in the green alga Chlamydomonas. We demonstrate that the ATG8 protein from
Chlamydomonas (CrATG8) may be used as a specific
autophagy marker. Nutrient limitation and cell aging
trigger an autophagic response that can be traced as an
increase at the level of CrATG8, the detection of
modified forms of CrATG8, and a change in the
cellular localization of this protein. Furthermore, we demonstrate that autophagy is inhibited by a rapamycinsensitive TOR cascade in Chlamydomonas.
RESULTS
Identification of Chlamydomonas ATG8 cDNA
A recent survey of the Chlamydomonas genome has
revealed the conservation of a single ATG8 gene in this
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Pérez-Pérez et al.
alga (Diaz-Troya et al., 2008b). The putative CrATG8
gene spans about 2 kb of genomic DNA and consists of
five exons and four introns. The full-length ATG8
cDNA was cloned by PCR from a Chlamydomonas
cDNA library (see “Materials and Methods”). The
CrATG8 gene encodes 134 amino acids with a predicted molecular mass of 15 kD. An evolutionary
study of this protein evidenced a high sequence identity to plant ATG8s that ranges from 47% to 74% (data
not shown). The critical Gly-116 residue of yeast ATG8
that is processed by the ATG4 protease is conserved in
CrATG8 and corresponds to Gly-120 (Fig. 1A). However, a C-terminal extension of 14 residues that is not
usually conserved among ATG8 proteins from other
organisms was found after Gly-120 (Fig. 1A). To characterize CrATG8, we generated a polyclonal antibody
against recombinant CrATG8 (see “Materials and
Methods”) that reveals a protein of about 15 kD from
Chlamydomonas soluble extracts (Fig. 1B). The antiCrATG8 antibody cross-reacted with other putative
ATG8 proteins from Arabidopsis and weakly recognized yeast ATG8 in total extracts (Fig. 1B).
Figure 1. A, Amino acid sequence alignment of the C-terminal segments of the ATG8 protein from Chlamydomonas and other organisms.
Identical residues are shaded. The arrowhead indicates the processing
site recognized by the ATG4 protease that exposes the C-terminal Gly
for lipidation. The conserved Gly is shaded and in boldface, and Gly122, present only in Chlamydomonas, is shown in boldface. The
number of amino acids for each protein is indicated at the end of the
sequence. Species abbreviations are as follows: Cr, Chlamydomonas
reinhardtii; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces
pombe; At, Arabidopsis thaliana; Zm, Zea mays; Hs, Homo sapiens.
The sequence from CrATG8 was taken from the Chlamydomonas
database (http://www.chlamy.org/chlamydb.html). The rest of the sequences were obtained from the National Center for Biotechnology
Information (http://ncbi.nlm.nih.gov). B, Cross-reactivity of anti-CrATG8
antibody with ATG8 proteins from Arabidopsis and yeast. Total extracts from Chlamydomonas, Arabidopsis, or S. cerevisiae (wild-type
[WT] and atg8 strains) were prepared and resolved by 15% SDS-PAGE.
ATG8 proteins were detected by immunoblot analysis with anti-CrATG8.
Molecular mass markers (kD) are indicated on the left.
CrATG8 Functionally Replaces Yeast ATG8
To investigate the possible role of CrATG8 in autophagy, we studied the functionality of this protein
expressed in yeast mutant cells lacking endogenous
ATG8. Yeast ATG8 is essential in both cytoplasm-tovacuole targeting and autophagy pathways; therefore,
atg8 mutant cells fail to deliver proaminopeptidase I
(pAPI) from the cytosol to the vacuole, where pAPI is
processed to its mature form (mAPI; Kirisako et al.,
1999; Huang et al., 2000). Therefore, processing of
pAPI to mAPI in atg8 mutant cells expressing CrATG8
was examined in order to study the functionality of
CrATG8. Our results indicate that CrATG8 is able to
functionally replace yeast ATG8, since mAPI was
detected in atg8 cells expressing CrATG8 (Fig. 2). To
further demonstrate that the mAPI band detected in
atg8 cells expressing CrATG8 is due to the activity of
this protein, processing of pAPI was also examined in
cells transformed with a mutant form of CrATG8
where Gly-120 has been replaced by an Ala residue
(CrG120A). A similar mutation in yeast ATG8 results
in the complete loss of protein activity due to its
inability for membrane attachment (Kirisako et al.,
2000). As expected, no mAPI protein was detected in
atg8 cells expressing CrG120A (Fig. 2). To demonstrate
that wild-type and mutant forms of CrATG8 were
correctly expressed, we analyzed the transformant
yeast extracts by western blot using the anti-CrATG8
antibody. Both CrATG8 and CrG120A were efficiently
expressed, and due to the presence of the C-terminal
extension in CrATG8, it was possible to distinguish the
precursor (pCrATG8) and the mature (mCrATG8)
forms of the protein (Fig. 2). Similar to what has
been reported in yeast ATG8 (Kirisako et al., 2000),
CrATG8 is predominantly processed when expressed
in yeast cells, whereas the CrG120A mutant cannot be
maturated. Overall, these results demonstrate that
CrATG8 is cleaved at Gly-120 and presumably binds
to membranes to functionally replace yeast ATG8 in
delivering pAPI from the cytosol to the vacuole.
In Vitro Processing of CrATG8 by Yeast ATG4 at Gly-120
Yeast complementation assays of atg8 mutant cells
suggested that CrATG8 must be processed by yeast
ATG4 at the conserved Gly-120 residue. To demonstrate that CrATG8 is recognized by yeast ATG4
(ScATG4), in vitro cleavage assays were performed
using purified CrATG8 and ScATG4 proteins. The His6
tag was fused to the N terminus of these proteins, and
recombinant CrATG8 and ScATG4 were purified from
Escherichia coli cells by nickel affinity chromatography
(see “Materials and Methods”). Processing of CrATG8
was monitored by the different mobilities in SDSPAGE of the precursor and mature forms of the
protein. Our results indicate that ScATG4 cleaves
CrATG8, presumably at its C terminus (Fig. 3A). To
investigate the participation of Gly-120 in CrATG8
processing, we studied the in vitro cleavage of the
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Autophagy Regulation in Chlamydomonas
Figure 2. Complementation of the yeast atg8 mutant with CrATG8
from Chlamydomonas. Western-blot analysis of transformants using
anti-pAPI (top panel) or anti-CrATG8 (bottom panel) antibodies. Top,
Wild-type (WT) and atg8 strains were transformed with p426TEF empty
plasmid or the same plasmid containing CrATG8 (ATG8 wild-type
protein) or CrG120A (Gly-to-Ala mutant protein). Wild-type and atg8
yeast strains were used as positive and negative controls, respectively.
The pAPI (precursor protein) and mAPI (mature protein) bands are
marked with arrowheads. Bottom, Western-blot analysis of the same
transformants with anti-CrATG8 antibody. The pCrATG8 (precursor
protein) and mCrATG8 (mature protein) bands are marked with arrowheads. Molecular mass markers (kD) are indicated on the left.
G120A mutant. In agreement with the in vivo analysis
of this mutant form of CrATG8 performed in yeast
(Fig. 2), substitution of Gly-120 by Ala (G120A) completely abrogated processing of this protein by
ScATG4 (Fig. 3B). Moreover, we investigated whether
another Gly residue localized at the C terminus of
CrATG8 at position 122, which is not conserved in
other ATG8 proteins (Fig. 1A), may be recognized by
ScATG4. Cleavage assays revealed that the G122A
mutant was processed by ScATG4 whereas the double
mutant G120/122A was not, indicating that substitution of Gly-122 by Ala has no effect on CrATG8
cleavage by ScATG4 and confirming that Gly-120 is
the ScATG4 target (Fig. 3B).
CrATG8 Is Processed at the C Terminus by an ATG4
Protease Activity in Chlamydomonas
Our results demonstrated that CrATG8 is functional
in a yeast system (Fig. 2). To investigate the functionality of CrATG8 in Chlamydomonas, we designed an
ATG4 proteolytic assay in Chlamydomonas total extracts by taking advantage of the different sizes of the
endogenous CrATG8 protein and recombinant His6tagged CrATG8. Total extracts were obtained from
Chlamydomonas cells grown in acetate medium and
then mixed with a fixed amount of different forms
of recombinant CrATG8. Incubation of wild-type
CrATG8 with Chlamydomonas extract resulted in the
fast processing of the recombinant protein. A band
with the same size as a truncated form of CrATG8
lacking the last 14 residues (6H-G120) was readily
visible, indicating that CrATG8 was processed at Gly120 (Fig. 4A). Mutation of Gly-120 to Ala abolished
CrATG8 cleavage, while the substitution of Gly-122 to
Ala had no effect in CrATG8 processing (Fig. 4A). As
expected, the G120/122A double mutant could not be
processed (Fig. 4A). These results indicated that Chlamydomonas total extracts have a proteolytic ATG4
activity that recognizes and processes the C terminus
of CrATG8 at Gly-120. ATG4 proteases contain a
catalytic Cys involved in substrate hydrolysis and
therefore are sensitive to iodoacetamide (Kirisako
et al., 2000; Kim et al., 2001). To investigate if the
CrATG8 processing detected in Chlamydomonas extracts is mediated by a Cys protease, proteolytic assays
were performed in the presence of iodoacetamide.
CrATG8 cleavage was fully abolished by iodoacetamide, while the Ser protease inhibitor phenylmethanesulfonyl fluoride (PMSF) had no effect (Fig. 4A),
indicating that the CrATG8 proteolytic activity in
Chlamydomonas extracts is mediated by a Cys protease.
Having shown that Chlamydomonas cells have ATG4
activity, we next investigated if the CrATG8 band of 15
kD detected in total extracts corresponds to the mature
form of the protein. To this aim, purified unprocessed
CrATG8 and a C-terminal truncated form of the protein (G120), together with Chlamydomonas total extracts
and extracts of yeast cells expressing wild-type or the
G120A mutant of CrATG8, were resolved by SDSPAGE and analyzed by western blot. Our results
indicate that the band detected in Chlamydomonas
extracts with the anti-CrATG8 antibody corresponds
to the C-terminal mature form of CrATG8 (Fig. 4B).
Moreover, unprocessed CrATG8 was not detected
under these and other experimental conditions (Fig.
4B; data not shown), suggesting that newly synthesized CrATG8 is immediately cleaved by an ATG4
protease to expose the Gly residue, as described previously in yeast and plant cells (Kirisako et al., 2000;
Kim et al., 2001; Yoshimoto et al., 2004).
CrATG8 Is Reversibly Modified during Stationary
Growth Phase
Previous studies in yeast, mammals, and plants
demonstrated that the modified form of ATG8 generated upon autophagy activation migrates faster than
unmodified ATG8 (Kabeya et al., 2000; Kirisako et al.,
2000; Yoshimoto et al., 2004). We observed that besides
the main 15-kD CrATG8 band detected by westernblot analysis in Chlamydomonas cell extracts, two less
abundant bands of lower apparent molecular mass
were visible (Figs. 1B and 4). To investigate whether
these two bands may be generated as a result of
CrATG8 modification, we performed western-blot
analysis of Chlamydomonas cells under different
growth conditions. First, we analyzed CrATG8 modification in exponential and stationary phase cells
in acetate-rich or minimal medium. In addition to
mature CrATG8, two bands became visible as cells
approached the stationary phase in acetate-rich medium, whereas these bands were almost undetectable
in exponential cells (Fig. 5A). Similar to what has been
described in other systems, these forms likely repre-
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Pérez-Pérez et al.
Figure 3. CrATG8 of Chlamydomonas is processed in vitro by yeast
ATG4 at Gly-120. A, SDS-PAGE
analysis of CrATG8 processing
by different amounts of ScATG4
(0.25, 0.50, 1, 2, 5, 10, or 20 mg)
for 3 h. B, Recombinant wildtype (CrATG8) and mutant forms
(G120A, G122A, G120122A, and
G120, where G120 is an ATG8
truncated protein at Gly-120 used
as a processed-like form control) of
Chlamydomonas ATG8 were incubated in an assay mixture that was
protease free (left panel) or in the
presence (right panel) of the protease ScATG4 (5 mg) for 3 h. The
typical reaction mixture contained
5 mg of ScATG4, 5 mg of ATG8,
1 mM 1,4-dithiothreitol, and 1 mM
EDTA in 100 mL of TBS buffer, and
reactions were performed at 30°C
for 3 h. The precursor (p) and mature (m) forms of CrATG8 are
marked with arrowheads. Molecular mass markers (kD) are indicated
on the right.
sent lipidated CrATG8, although we cannot exclude
the possibility that one of these bands may be the
result of a different modification. A significant and
progressive increase in CrATG8 abundance was also
found in stationary phase cells compared with exponential cells, suggesting that the function of this protein is required when cells age. Western-blot analysis
of the cytosolic protein FKBP12 and Coomassie Brilliant Blue staining of the gel were used as loading
controls (Fig. 5A). Modified CrATG8 was also observed in minimal medium, although the faster migrating band was faintly detected (Fig. 5A). As in
acetate-grown cells, the level of CrATG8 increased
during the growth phase in minimal medium (Fig.
5A). Since the lower molecular mass variant of
CrATG8 was concomitantly detected with an increase
in the abundance of mature CrATG8, dilution series of
total extracts obtained from log or stationary cells were
analyzed by western blot in order to have similar
amounts of the mature form from both conditions. Our
results indicated that under uniform levels of mature
CrATG8, modified forms are specifically detected in
stationary cells (Fig. 5B).
To further characterize the process of CrATG8 modification associated with cell growth, CrATG8 was
analyzed by western blot in Chlamydomonas cultures
that have reached the stationary growth phase and
then transferred to fresh medium to initiate a new cell
growth cycle or maintained in the same medium.
While modified CrATG8 was clearly detectable in cells
that remained in the stationary growth phase, CrATG8
was delipidated within 6 h when stationary cells were
transferred to new medium (Fig. 5C, middle and lower
panels). Modified CrATG8 was again detected after
48 h of reinoculation (Fig. 5C, middle panel). None of
the three bands detected correspond to unprocessed
CrATG8, since this protein is immediately cleaved
(Fig. 4). Taken together, these results indicate that
CrATG8 modification can be efficiently reversed under
optimal growth conditions.
Modification of CrATG8 under Different
Stress Conditions
The participation of CrATG8 in the cellular response
to different stresses such as nitrogen or carbon limitation and oxidative stress was examined by monitoring
the modification state of this protein. To investigate
whether nitrogen limitation may induce CrATG8
modification and autophagy activation in Chlamydomonas, exponentially growing cells were subjected to
nitrogen depletion and CrATG8 modification was
tested by western-blot analysis. Compared with untreated control cells, nitrogen limitation resulted in a
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Autophagy Regulation in Chlamydomonas
Figure 4. ATG4 protease activity in cell-free extracts of Chlamydomonas. A, Ten nanograms of
the different recombinant ATG8 proteins
(CrATG8, G120A, G122A, G120122A, or G120)
was incubated with 30 mg of cell-free extracts (SE)
of Chlamydomonas during 45 min on ice. When
required, cell-free extracts were previously incubated with the corresponding protease inhibitor
for 45 min on ice. The reaction was stopped by
the addition of loading buffer and boiling at
100°C. Aliquots of the reaction mixtures were
analyzed by western blotting with the antiCrATG8 antibody. Exogenous, recombinant proteins contained the His6 tag and were labeled as
6H to distinguish them from endogenous
CrATG8. The precursor (p) and mature (m) forms
of CrATG8 are marked with arrowheads. The top
panel corresponds to control (without protease
inhibitor) assays, while the middle and bottom
panels correspond to experiments carried out in
the presence of the protease inhibitor 3 mM
iodoacetamide or 5 mM PMSF, respectively. B,
CrATG8 is constitutively processed in Chlamydomonas. Western-blot analysis of the CrATG8 protein. Lane 1, His tag-free ATG8 purified protein;
lane 2, His tag-free G120 purified protein; lane 3,
total extract from atg8 yeast cells expressing
CrATG8; lane 4, total extract from atg8 yeast cells
expressing G120A; lane 5, cell-free extract from
Chlamydomonas. The precursor (p) and mature
(m) forms of CrATG8 are marked with arrowheads. A molecular mass marker (kD) is indicated
on the right.
substantial increase in CrATG8 levels as well as the
detection of modified CrATG8 forms (Fig. 6A).
Carbon starvation induced by darkness has been
shown to induce autophagy in plants (Thompson
et al., 2005; Phillips et al., 2008; Chung et al., 2009).
To test whether carbon-limiting conditions also trigger
autophagy in Chlamydomonas, cells grown under continuous light in minimal medium were shifted to
darkness or maintained under a short-day photoperiod (8 h of light/16 of h dark) and the presence of
modified CrATG8 was examined by western blot.
Fixed-carbon limitation induced by darkness resulted
in an increase in CrATG8 abundance and in the
detection of modified CrATG8 forms (Fig. 6B). However, no effect was observed in cells grown under a
short-day photoperiod compared with cells maintained under continuous light (Fig. 6B).
Autophagy is involved in degrading oxidized proteins during oxidative stress in plants (Xiong et al.,
2007). The possible participation of CrATG8 in oxidative stress was analyzed by hydrogen peroxide (H2O2)
treatment, an established oxidative stress inducer, in
Chlamydomonas cells. Modified CrATG8 was readily
detected upon treatment with H2O2 (Fig. 6C). More-
over, as in nutrient-starved cells, the level of CrATG8
significantly increased under oxidative stress conditions (Fig. 6C). Therefore, autophagy might contribute
to the degradation of oxidized proteins during oxidative stress in Chlamydomonas.
In addition to nutrient or oxidative stress, we investigated the activation of CrATG8 in response to a
specific stress in the endoplasmic reticulum (ER) by
treating Chlamydomonas cells with tunicamycin, which
raises the level of unfolded polypeptides at this compartment by inhibiting protein glycosylation. Compared with control, untreated cells, tunicamycin
strongly increased CrATG8 lipidation and abundance
(Fig. 6D), indicating that autophagy is involved in the
degradation of unfolded proteins in the ER.
TOR Inhibits Autophagy in Chlamydomonas
In a previous study, we showed that rapamycinmediated inhibition of TOR signaling caused increased vacuolization (Crespo et al., 2005), a typical
morphological autophagy effect. To confirm that the
TOR pathway controls autophagy in Chlamydomonas,
we took advantage of the autophagy monitoring assay
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Pérez-Pérez et al.
Figure 5. Analysis of CrATG8 during the growth phase in Chlamydomonas. A, Induction and modification of CrATG8 during the
growth phase in Chlamydomonas. Thirty micrograms of total extracts of Chlamydomonas cells was resolved by 15% SDS-PAGE
followed by western blotting with the anti-CrATG8 antibody. Cells were grown in acetate medium (Tris-acetate phosphate [TAP])
or minimal medium (Min) from 0.2 3 106 cells mL21 to 8 3 106 cells mL21, and the CrATG8 protein profile was analyzed by
western blotting after SDS-PAGE. Western-blot analyses with anti-FKBP12 and Coomassie Brilliant Blue (CBB) staining were
used as loading controls. B, Dilution series of total soluble extracts obtained from Chlamydomonas cells growing at exponential
(1 3 106 cells mL21) or stationary (8 3 106 cells mL21) phase. Different amounts (2.5, 5, 10, and 20 mg) of soluble extracts were
resolved by SDS-PAGE and subjected to western-blot analysis. C, Autophagy induction is a reversible process in Chlamydomonas. Stationary cells (8 3 106 cells mL21) grown in acetate-rich medium were transferred to a fresh medium to a cell density of
2 3 105 cells mL21 (middle panel), 4 3 105 cells mL21 (bottom panel), or maintained under similar growth conditions (top panel).
Soluble extracts were prepared from samples obtained at different times, and CrATG8 modification was analyzed by western
blotting. CrATG8* denotes modified CrATG8.
using the anti-CrATG8 antibody and examined the
presence of modified CrATG8 forms in rapamycintreated cells. As shown in Figure 7, rapamycin treatment resulted in CrATG8 modification and an increase
in the level of this protein. The rapamycin-induced
effects were more pronounced in a Chlamydomonas
mutant strain (Crespo et al., 2005) that exhibits high
sensitivity to rapamycin (Fig. 7). As expected, no effect
on CrATG8 was observed in the rapamycin-resistant
mutant rap2 strain (Fig. 7), which lacks the rapamycin
primary target FKBP12 (Crespo et al., 2005). Thus, our
results demonstrate that CrATG8 is under the control
of a rapamycin-sensitive TOR signaling pathway in
Chlamydomonas.
ATG8 Associates with Membranes in Chlamydomonas
ATG8 proteins from yeast, mammals, and plants
have been shown to be membrane associated (Kirisako
et al., 1999; Kabeya et al., 2000; Yoshimoto et al., 2004;
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Autophagy Regulation in Chlamydomonas
Figure 6. Analysis of CrATG8 under different autophagy-activating conditions. A, CrATG8 modification upon nitrogen
starvation. Chlamydomonas cells growing exponentially (1 3 106 cells mL21) in Tris-acetate phosphate medium were washed
with a nitrogen-free medium and grown under these conditions for 24 h. Cells grown in nitrogen-containing medium were used
as controls. B, CrATG8 modification under light/dark transitions. Exponential cells grown under continuous light in minimal
medium were maintained under similar conditions (L) or subjected to 8-h-light/16-h-dark cycles (L/D) or continuous darkness
(D). Next, cultures were diluted to 4 3 105 cells mL21 and maintained under the same conditions until a cell density of 1 3 106
cells mL21. C, Analysis of CrATG8 modification under oxidative stress conditions. Chlamydomonas cells growing exponentially
(1 3 106 cells mL21) were treated with 1 or 2 mM H2O2 for 8 h. Untreated cells were used as controls. D, CrATG8 modification in
Chlamydomonas cells subjected to ER stress. Chlamydomonas cells growing at exponential phase (1 3 106 cells mL21) were
treated with 5 mg mL21 tunicamycin (tun) for 8 h. Untreated cells at different times were used as controls. In all experiments, 30
mg of total extracts was resolved by 15% SDS-PAGE followed by western blotting with the anti-CrATG8 antibody. Western-blot
analyses with anti-FKBP12 and Coomassie Brilliant Blue (CBB) staining were used as loading controls. CrATG8* corresponds to
CrATG8 modifications.
Chung et al., 2009). To investigate whether CrATG8 is
associated with membranes, we performed subcellular
fractionation by ultracentrifugation. Total soluble extracts were obtained from exponentially growing
Chlamydomonas cells, and the presence of CrATG8 in
the different soluble and membrane-enriched fractions
was analyzed by western blot. The first fractionation
was performed at low speed (15,000g), and CrATG8 was
detected in the supernatant (S1) and pellet (P1) fractions (Fig. 8A), indicating that part of the protein is
membrane bound. Interestingly, a faster migrating
band likely corresponding to modified CrATG8 was
observed in the membrane fraction but not in the
soluble fraction. A second fractionation was achieved
by ultracentrifugation of S1 to separate soluble (S2)
and microsomal (P2) fractions. CrATG8 was again
detected in both fractions and, as in the low-speed
fractionation, putative modified CrATG8 was found in
the membranous pellet (Fig. 8A). Thus, our results
indicated that there might be at least two different
pools of CrATG8 in exponentially growing Chlamydomonas cells, soluble and membrane-bound CrATG8.
The presence of microsomes in the insoluble fraction
after ultracentrifugation was confirmed by immunodetection of a vacuolar membrane ATPase. Parallel
immunoblot assays with antiserum specific to D1 or
Figure 7. TOR signaling inhibition induces autophagy in Chlamydomonas cells. Chlamydomonas cells from different strains (wild type [WT],
rap2, and rap2 + M52V E54Q) growing exponentially in acetate-rich (Tris-acetate phosphate
[TAP]) or minimal (Min) medium were treated
with 500 nM rapamycin for 16 h. The CrATG8
protein profile was analyzed by western blot with
the anti-CrATG8 antibody. Western blot analyses
with anti-FKBP12 and Coomassie Brilliant Blue
(CBB) staining were used as loading controls. C,
Control cells; R, rapamycin-treated cells.
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Figure 8. Association of CrATG8 with membranes. A, Subcellular distribution of CrATG8. Crude extracts prepared from
Chlamydomonas cells were centrifuged at 15,000g (low speed; LS) for 15 min to generate supernatant (S1) and pellet (P1)
fractions. The resulting supernatant was then centrifuged at 100,000g (high speed; HS) for 2 h to generate further supernatants
(S2) and pellets (P2). The samples were subjected to immunoblot analysis with antibodies raised against CrATG8, CrFKBP12, D1,
and vacuolar membrane (vATPase) proteins. CrATG8* denotes modified CrATG8. B, Solubilization of CrATG8. Chlamydomonas
cells were grown to stationary phase, and total extracts (ET) were prepared by centrifugation at 15,000g for 15 min. This extract
was then centrifuged at 100,000g for 2 h to obtain S2 and P2 fractions. The pellet fractions were incubated on ice (control [C]), in
lysis buffer containing 1% deoxycholate on ice for 1 h (DOC), or treated with PlpD (200 units mL21 final concentration) at 37°C
for 1 h. Samples were further centrifuged at 100,000g for 2 h to obtain membrane fractions with insoluble proteins (P3) and
analyzed by western blot. C, Suc density gradient profiles of CrATG8, vATPase, BiP, FOX1, and FKBP12. Total extracts were
fractionated on a 12-mL, 10% to 32% Suc gradient. Fractions (750 mL) were collected from the top of the gradient and processed
for western-blot analysis.
to CrFKBP12 (Crespo et al., 2005) showed that the
microsome-containing fraction is not significantly contaminated with thylakoidal membranes or soluble
cytoplasmic proteins (Fig. 8A).
To improve the detection of modified CrATG8 associated with membranes, a similar fractionation approach was performed in soluble extracts obtained
from stationary cells, where these forms are more
abundant (Fig. 5). High-speed centrifugation revealed
that both mature and modified forms of CrATG8 are
present in the pellet and soluble fractions (Fig. 8B).
However, treatment of pellet membranes with detergent (1% deoxycholate) resulted in the complete solubilization of CrATG8, indicating that these forms are
associated with the membrane (Fig. 8B). Anchoring of
ATG8 to the membrane occurs through the covalent
binding of the protein to a molecule of PE (Ichimura
et al., 2000). Treatment of the ATG8-PE adduct with
phospholipase D (PlpD) releases phosphatidic acid
from the conjugate and consequently solubilizes
membrane-bound ATG8 (Ichimura et al., 2000; Tanida
et al., 2004; Fujioka et al., 2008; Chung et al., 2009). To
investigate whether CrATG8 might be modified by
lipidation, membrane-associated CrATG8 was treated
with PlpD. Our results show that PlpD treatment
caused the complete solubilization of CrATG8 from
the membranous fraction (Fig. 8B), strongly suggesting that modified forms of CrATG8 are the phos-
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Autophagy Regulation in Chlamydomonas
pholipid conjugates of this protein. To further investigate the membrane association of CrATG8, we performed Suc gradient sedimentation assays. Unlike
the CrFKBP12 soluble protein, CrATG8 cosedimented
with protein membrane markers such as vacuolar
ATPase, BiP, and FOX1 (Fig. 8C). Taken together, our
results are consistent with the notion that CrATG8
associates with membranes through covalent binding
to PE.
Distinct CrATG8 Cellular Localization upon
Autophagy Activation
Immunofluorescence (IF) microscopic studies and
GFP-ATG8 fusion proteins have been utilized to monitor
autophagy in several organisms. We used the antiCrATG8 antibody to examine the subcellular localization of this protein by IF microscopy in Chlamydomonas
cells under different conditions. In cells growing exponentially in acetate-rich medium, CrATG8 was
localized to punctate structures and a single spot
was detected in most of the cells (Fig. 9). Similarly,
the signal of CrATG8 in cells grown in minimal
medium was confined to discrete punctae, although
the number of these spots was usually higher than in
acetate-grown cells, likely due to a higher requirement
of autophagy under these conditions (Supplemental
Fig. S1). The distribution of ATG8 in the cell has been
shown to be modified by the activation of autophagy
(Kirisako et al., 1999; Kabeya et al., 2000; Kim et al.,
2001). Thus, we investigated whether CrATG8 cellular
localization may respond to autophagy induction.
Exponentially growing cells were treated with rapamycin or tunicamycin or subjected to nitrogen limitation or oxidative stress to trigger autophagy, and
CrATG8 localization was examined by IF microscopy.
Treatment of acetate-grown cells with rapamycin resulted in a pronounced detection of CrATG8 in more
punctate structures and an increase in cell size (Fig. 9).
Rapamycin caused a similar effect on CrATG8 localization in minimal medium, although a diffuse
CrATG8 signal could also be detected at the periphery
and the anterior end of the cell (Supplemental Fig. S1).
Accordingly, this region of the cell has been reported to
become vacuolated upon rapamycin treatment under
similar experimental conditions (Crespo et al., 2005).
Nitrogen depletion also resulted in a strong modification of CrATG8 cellular localization, being detected as
multiple small dots all over the cell with a predominant localization around the chloroplast (Fig. 9; Supplemental Fig. S1). Similar to nitrogen starvation,
oxidative stress induced by H2O2 increased the overall
intensity of the CrATG8 signal in the cell, and some
large spots could also be detected (Fig. 9; Supplemental Fig. S1). The effect of tunicamycin on the cellular
distribution of CrATG8 differed in some respects from
other stresses. First, the signal was remarkably stronger, and second, CrATG8 accumulated at a specific
region in the middle of the cell that might correspond
to the ER, in agreement with the localized effect of
tunicamycin at this compartment (Fig. 9; Supplemental Fig. S1). Taken together, these results indicate that
the cellular distribution of CrATG8 strongly depends
on the autophagic requirement of the cell.
DISCUSSION
The Chlamydomonas genome contains potential
homologs to yeast and plant ATG genes, including
those involved in the ATG8/ATG12-conjugating systems (Diaz-Troya et al., 2008b), which strongly suggests that Chlamydomonas might recycle intracellular
components through autophagic processes. In agreement with this hypothesis, it has been recently shown
that autophagosome-like structures are generated as a
response to prolonged salt stress in the unicellular
green alga Micrasterias denticulata (Affenzeller et al.,
2009). However, the molecular features of autophagy
have never been characterized in green algae. Here, we
report the participation of the Chlamydomonas CrATG8
protein in autophagy and demonstrate that this protein may be used to monitor autophagic processes in
this green alga.
Compared with the ATG8 proteins from other organisms, the C-terminal extension downstream of the
conserved Gly-120 of CrATG8 is particularly large
(Fig. 1A). A comparative analysis of the C-terminal
extension of ATG8 proteins from different organisms
indicated that the sequence after the conserved Gly
completely diverged (Kabeya et al., 2000). In Arabidopsis, two of the nine ATG8 homologs, AtATG8h and
AtATG8i, do not possess additional amino acids
downstream of the conserved Gly, while the ATG8
protein from other organisms such as rats or the
symbiotic fungus Laccaria bicolor have large C-terminal
extensions (Kabeya et al., 2000). The C-terminal extension does not seem to be required for the functionality
of the protein, since all nine Arabidopsis ATG8s,
including AtATG8h and AtATG8i, are active in plant
autophagy (Yoshimoto et al., 2004). Unprocessed
CrATG8 could not be detected in Chlamydomonas cells
(Fig. 4), indicating that similar to what has been
described in yeast or plants (Kirisako et al., 2000;
Kim et al., 2001), newly synthesized CrATG8 is immediately processed. Whether the C-terminal extension
of CrATG8 may play a role in the stability of the
protein and/or its processing remains to be investigated.
Functional studies performed in yeast revealed that
CrATG8 is able to restore pAPI processing in an atg8
mutant strain under normal growth conditions (Fig. 2),
demonstrating the functionality of this protein in yeast
ATG8-mediated processes. Yeast complementation assays have also been performed with Arabidopsis and
Trypanosoma cruzi ATG8 homologs, and in contrast to
CrATG8, these proteins were able to functionally replace yeast ATG8 under starvation conditions only
(Ketelaar et al., 2004; Alvarez et al., 2008). Mutation of
the conserved Gly residue to Ala in CrATG8 fully
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Figure 9. IF localization of CrATG8 in Chlamydomonas cells. Cells growing exponentially in acetate
medium were subjected to different treatments as
follows: cells were treated with rapamycin for 16 h
(rap), shifted to nitrogen-free medium for 24 h (2N),
treated with 2 mM H2O2 for 8 h to induce oxidative
stress, or treated with 5 mg mL21 tunicamycin (tun)
for 8 h to trigger ER stress. As a control, cells were
maintained in exponential growth. Cells were collected and processed for IF microscopy analysis as
described in “Materials and Methods.” The signal
recognized by the affinity-purified anti-CrATG8 antibody is shown in green (CrATG8). The same acquisition time was used in all samples except for
tunicamycin-treated cells, where it was reduced to
50% to avoid signal saturation. Bars = 2 mm.
abolished pAPI processing, suggesting that despite the
presence of the large C-terminal extension, CrATG8
could be processed and lipidated in yeast cells (Fig.
2A). Indeed, in vitro cleavage assays demonstrated
that CrATG8 is maturated at Gly-120 by yeast ATG4
(Fig. 3).
Our in vivo and in vitro heterologous studies
strongly suggested that CrATG8 performs a similar
function to yeast ATG8 in Chlamydomonas. Several
lines of evidence support this hypothesis. On the one
hand, CrATG8 is processed in Chlamydomonas at the
conserved Gly, as revealed by ATG4 proteolytic assays
performed in cell-free extracts of Chlamydomonas (Fig.
4). In agreement with findings in plants and T. cruzi,
where ATG4 proteases are constitutively expressed
regardless of nutrient conditions (Yoshimoto et al.,
2004; Alvarez et al., 2008), ATG4 activity was detected
in Chlamydomonas cells under optimal and nutrientlimiting growth conditions (Fig. 4; data not shown).
On the other hand, CrATG8 levels increased upon
autophagy activation. The abundance of plant ATG8s
is up-regulated at the protein and mRNA levels in
response to autophagy induction by nutrient starvation (Yoshimoto et al., 2004; Contento et al., 2005;
Thompson et al., 2005; Chung et al., 2009). Accordingly, a significant increase in CrATG8 protein abundance was detected in Chlamydomonas cells limited for
nutrients (Fig. 6). Entry into stationary phase also
resulted in the up-regulation of CrATG8 levels (Fig. 5),
which links autophagy to cell aging in Chlamydomonas.
Autophagy has been shown to play a role in plant
senescence. The phenotypic analysis of Arabidopsis
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Autophagy Regulation in Chlamydomonas
atg mutants has led to the hypothesis that autophagy
occurs before or during senescence, likely to allow the
plant to accomplish efficient nutrient reallocation
(Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto
et al., 2004; Thompson et al., 2005; Xiong et al., 2005;
Phillips et al., 2008). Similarly, high levels of CrATG8,
and by inference autophagy, might be required for the
metabolic adaptation of Chlamydomonas cells before
entering into the stationary phase. Supporting a role
for CrATG8 in the cell cycle, we found that CrATG8
abundance is down-regulated as soon as stationary
cells are allowed to enter the log phase (Fig. 6C).
Moreover, modified CrATG8 is efficiently delipidated
in exponentially growing cells (Fig. 6C), indicating
that CrATG8 modification is a reversible process in
Chlamydomonas. Deconjugation of ATG8-PE has been
demonstrated in yeast and is mediated by the ATG4
protease (Kirisako et al., 2000). It is likely, therefore,
that CrATG8 deconjugation is catalyzed by a homologous ATG4 protease in photosynthetic organisms.
A common feature of ATG8 cellular localization in
lower and higher eukaryotes seems to be a punctate
pattern under optimal growth that is enhanced upon
autophagy activation. In unicellular organisms such as
yeast or protozoa, ATG8 was localized to tiny dots as
well as larger spots distributed throughout the cytoplasm in rich medium (Kirisako et al., 1999; Kim et al.,
2001; Alvarez et al., 2008). Nutrient depletion resulted
in both organisms in an increase in the number of large
spots, reflecting the activation of autophagy and the
formation of mature autophagosomes. The cellular
distribution of CrATG8 in Chlamydomonas displayed
common features with yeast and protozoa ATG8 localization. In acetate-rich medium, CrATG8 signal was
restricted to discrete punctate structures that may
correspond to PAS. However, autophagy induction
by nutrient limitation, oxidative stress, or tunicamycin
treatment drastically modified the localization of
CrATG8 in the cell. Numerous punctate structures
could be detected all over the cell upon nutrient
starvation or H2O2 treatment, while a specific stress
in the ER caused a massive accumulation of CrATG8 at
this cellular compartment (Fig. 9). Using GFP-ATG8
fusion proteins, it has been shown in plant cells that
the cellular distribution of this protein is modified
upon autophagy activation by nutrient limitation
(Yoshimoto et al., 2004; Contento et al., 2005; Thompson
et al., 2005; Xiong et al., 2005). Oxidative stress also
induces autophagy in plants, since H2O2 or methyl
viologen treatment results in increased autophagosome detection (Xiong et al., 2007). It remains to be
determined whether ER stress may trigger autophagy
in plants as it does in yeast (Yorimitsu et al., 2006) or
algae.
Compared with plants, the global picture of CrATG8
cellular localization and hence autophagosome generation in Chlamydomonas seems to be less complex. In
plants, it has been proposed that autophagy occurs
constantly in significant amounts under optimal
growth based on the presence of ATG8s in many rings
and punctate structures representing autophagosomes
or PAS (Yoshimoto et al., 2004; Thompson et al.,
2005). This model is strengthened by the detection of
most ATG8s in the pellet fraction in plant cells, an
indication of ATG8 association with the autophagosomes (Yoshimoto et al., 2004). In exponentially growing Chlamydomonas cells, mature and modified forms
of CrATG8 were also found in association with
membrane fractions (Fig. 8A), which may imply
the participation of CrATG8 in autophagy or other
membrane-trafficking events under optimal growth
conditions. Upon autophagy induction, CrATG8 becomes lipidated and binds to membrane, although
mature, unmodified CrATG8 could also be detected
with membrane fractions (Fig. 8B). The presence of
unmodified CrATG8 in membranes is not exclusive to
Chlamydomonas, since this phenomenon has also been
reported in mammals (Tanida et al., 2004) and plants
(Chung et al., 2009).
The TORC1 pathway perceives the nutritional status
of the cell and transmits these signals to the autophagic machinery, acting as a negative regulator of autophagy (Noda and Ohsumi, 1998). The molecular
mechanisms by which TORC1 inhibits autophagy
were elucidated from studies performed in yeast
(Noda and Ohsumi, 1998; Kamada et al., 2000; Kabeya
et al., 2005), and significant advances in this regulatory
pathway were recently reported in insects and mammals (Chang and Neufeld, 2009; Hosokawa et al., 2009;
Jung et al., 2009). Components of TORC1, including
the TOR kinase, are functionally conserved in plants
(Menand et al., 2002; Anderson et al., 2005; Deprost
et al., 2005; Mahfouz et al., 2006), although the participation of this signaling pathway in the control of
autophagy has not been shown in photosynthetic
eukaryotes. The data presented in this study demonstrate that TOR signaling inhibits autophagy in Chlamydomonas. Rapamycin-mediated inhibition of CrTOR
resulted in increased CrATG8 modification and a
relocalization of CrATG8 in Chlamydomonas cells similar to the one observed under nutrient-limiting conditions (Figs. 7 and 9). Thus, our results assign a
prominent role to the TOR pathway in the control of
autophagy in photosynthetic organisms. In yeast and
animal cells, the ATG1 kinase and its regulatory proteins ATG11, ATG13, and ATG17 transmit the signal
from TORC1 to the autophagosome-generating machinery. Whether these proteins may perform a similar
function in plants or algae is unknown.
Autophagy has been recently involved in chloroplast degradation and Rubisco mobilization to the
vacuole during plant senescence and stress (Ishida
et al., 2008; Wada et al., 2009). Rubisco and other
stroma-targeted proteins are mobilized from the
chloroplast to the vacuole through an ATG-mediated
autophagic process, which does not implicate entire
chloroplast degradation. In Chlamydomonas, it has been
shown that a substantial amount of chloroplast material, including the large subunit of Rubisco and ribosomes, is transferred to a class of vacuoles distinct
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from contractile vacuoles via protrusion of the outer
membrane of the plastid envelope (Park et al., 1999).
However, the participation of autophagy in this degradative process has not been demonstrated. The
availability of a specific autophagy marker in Chlamydomonas will certainly facilitate the investigation of the
role of autophagy in this and other cellular events.
Yeast Complementation
For expression of CrATG8 in yeast cells, the cDNA of the CrATG8 gene and
the mutant version, G120A (whose Gly-120 was substituted by Ala), was
cloned into the SpeI site of the p426TEF vector under control of the TEF
constitutive promoter (Mumberg et al., 1995). Wild-type and atg8 strains were
transformed with empty vector, the pTEF-CrATG8 plasmid, or the pTEFG120A plasmid and grown in rich or synthetic medium as described (Kim
et al., 2001).
Preparation and Analysis of Proteins
MATERIALS AND METHODS
Strains and Growth Conditions
Chlamydomonas reinhardtii wild-type strain 6C+ was obtained from the
laboratory of J.-D. Rochaix (University of Geneva). rap2 and rap2 + M52V E54Q
strains were characterized previously (Crespo et al., 2005). Chlamydomonas
cells were grown under continuous illumination at 25°C in Tris-acetate
phosphate or high-salt minimal medium as described (Harris, 1989). When
required, 1.2% bacto agar (Difco) was added to the medium. Saccharomyces
cerevisiae wild-type (SEY6210) and atg8 mutant (WPHYD7) strains were
obtained from the laboratory of D.J. Klionsky (University of Michigan). Yeast
cells were grown in rich or synthetic medium as described previously (Kim
et al., 2001). Rapamycin was added to Chlamydomonas to 500 nM from a 1 mg
mL21 stock in 90% ethanol and 10% Tween 20.
DNA and Protein Sequence Alignments
The amino acid Chlamydomonas ATG8 sequence was obtained from the
Chlamydomonas database (www.chlamy.org/chlamydb.html). The ATG8
amino acid sequences from other organisms were obtained from the National
Center for Biotechnology Information (www.ncbi.nlm.nih.gov). The multiple
sequence alignment was performed with the ClustalW2 program (www.ebi.
ac.uk/Tools/clustalw2).
Cloning and Protein Purification
The ATG8 coding region from Chlamydomonas (351 bp) was amplified by
PCR from a Chlamydomonas cDNA library using the following primers:
ATG8For (5#-CCGGCATATGGTTGGCTCCCGACCCCCGACATTC-3#) and
ATG8Rev (5#-CCGGCTCGAGTCACAACGCCAGCTCCTCCACAGG-3#), designed to contain a NdeI or XhoI site (underlined), respectively. The resulting
product was digested with NdeI and XhoI and inserted into pET28a (Novagen)
for expression in Escherichia coli. Expression of the recombinant protein was
induced by adding 1 mM isopropyl-b-D-thiogalactopyranoside for 3 h at 37°C.
The ATG8 His6-tagged protein was purified from the lysates by two consecutive affinity chromatographies on a nickel matrix from Novagen.
The His6-tagged Gly-to-Ala mutants of ATG8, denoted as G120A, G122A,
and G120122A, whose Gly-120, Gly-122, or both were replaced by Ala,
respectively, were obtained by PCR using the wild-type version of the ATG8
gene cloned into pET28a and reverse primers including the proper mutation
as follows: G120ARev (5#-TCACAACGCCAGCTCCTCCACAGGCAGCTGCAGCTGCTCCCCGGCAGCGAACGTGTT-3#) for the G120A mutant,
G122ARev (5#-CCGGCTCGAGTCACAACGCCAGCTCCTCCACAGGCAGCTGCAGCTGCTCCTCCGCGGCAC-3#) for G122A, and DMRev (5#-CCGGCTCGAGTCACAACGCCAGCTCCTCCACAGGCAGCTGCAGCTGCTCCGCGGCAGCGAAC-3#) for G120122A. The His6-tagged Gly-120 truncated
mutant, named G120, was similarly obtained by PCR using 5#-CCGGCTCGAGTCAACCGAACGTGTTCTCGC-3# as the reverse primer. ATG8For was
used as the forward primer in PCR to obtain all ATG8 mutant versions. All
ATG8 mutants (G120A, G122A, G120122A, and G120) were purified as
described for wild-type ATG8.
The ATG4 coding region from S. cerevisiae was amplified by PCR from yeast
genomic DNA using the following primers: ATG4For (5#-CCGGCATATGCAGAGGTGGCTACAACTGTG-3#) and ATG4Rev (5#-CCGGCTCGAGCTAGCATTTTTCATCAATAG-3#), designed to contain a NdeI or XhoI site
(underlined), respectively. Cloning and protein expression were performed
as described above for Chlamydomonas ATG8. Prior to purification, His6tagged ATG4 was solubilized with 4 M urea due to the presence of the
recombinant protein in inclusion bodies. Purification was performed as
described for CrATG8.
Chlamydomonas cells from liquid cultures were collected by centrifugation
(4,000g, 5 min), washed once in 50 mM Tris-HCl (pH 7.5) buffer, and
resuspended in a minimal volume of the same solution. Cells were lysed by
two cycles of slow freezing to 280°C followed by thawing to room temperature. The soluble cell extract was separated from the insoluble fraction by
centrifugation (15,000g, 15 min) in a microcentrifuge at 4°C. Yeast extracts
from logarithmically growing cells were prepared in lysis buffer (50 mM TrisHCl, pH 7.5, and 0.1% Triton X-100) by vortexing them 10 times for 1 min each
time with glass beads (Sigma-Aldrich). Crude extracts were cleared by
centrifugation at 15,000g for 15 min at 4°C. Proteins were quantified with
the Coomassie Brilliant Blue dye-binding method as described by the manufacturer (Bio-Rad).
In Vitro ATG4 Cleavage Assays
Recombinant wild-type CrATG8 and the different Gly-to-Ala mutant
versions (5 mg) were incubated with different amounts of purified ScATG4
in 100 mL of Tris-buffered saline (TBS) containing 1 mM EDTA and 1 mM 1,4dithiothreitol. The reaction mixtures were incubated at 30°C for different
times and stopped by the addition of Laemmli sample buffer followed by
5 min of boiling. Proteins were resolved on a 15% SDS-PAGE gel and stained
with Coomassie Brilliant Blue. For the detection of ATG4 activity in Chlamydomonas, cell-free extracts were prepared as described above and incubated
with 10 ng of the different recombinant ATG8 proteins in TBS buffer for
45 min. To determine which protease group Chlamydomonas ATG4 belongs to,
proteolytic assays were carried out in the presence of the protease inhibitor
iodoacetamide (3 mM) or PMSF (5 mM). The reactions were stopped by the
addition of Laemmli sample buffer followed by 5 min of boiling. Proteins were
resolved on a 15% SDS-PAGE gel and immunoblotted with the anti-CrATG8
antibody.
Antibody Production and Immunoblot Analysis
The anti-CrATG8 polyclonal antibody was produced by injecting the
recombinant wild-type CrATG8 protein into a rabbit using standard immunization protocols. Polyclonal serum was affinity purified by coupling the
recombinant protein CrATG8 (12 mg) to a solid resin (Aminolink Plus
Immobilization kit; Thermo Scientific) according to the manufacturer’s instructions. For immunoblot analyses, total protein extracts that had been
subjected to SDS-PAGE were transferred to nitrocellulose membranes (BioRad). Anti-CrATG8 and secondary antibodies were diluted 1:2,000 and
1:10,000, respectively, in phosphate-buffered saline containing 0.1% Tween
20 (Sigma-Aldrich) and 5% milk powder. The ECL-Plus immunoblotting
detection system (GE Healthcare) was used to detect the proteins with
horseradish peroxidase-conjugated anti-rabbit secondary antibodies. The
anti-FKBP12 antibody has been described previously (Crespo et al., 2005).
The anti-pAPI antibody was obtained from the laboratory of D.J. Klionsky
(University of Michigan). Both antibodies were used at 1:2,000 dilution. The
anti-D1 and anti-vacuolar membrane, anti-BiP, and anti-FOX1 antibodies were
purchased from Agrisera and diluted to 1:20,000, 1:10,000, 1:2,000, and
1:10,000, respectively.
Subcellular Fractionation and Solubilization of CrATG8
For fractionation assays, Chlamydomonas whole-cell extract was prepared
from wild-type cells as described above. After freezing and thawing, samples
were centrifuged at 500g for 5 min to remove cell debris. The supernatant was
centrifuged at 15,000g for 15 min to generate the soluble extract and the
low-speed membrane fraction. The microsomal fraction was obtained by
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Autophagy Regulation in Chlamydomonas
centrifuging the soluble extract in an 80-Ti rotor in a Beckman Coulter
ultracentrifuge at 100,000g for 2 h at 4°C. For CrATG8 solubilization, the
membrane pellet fraction was resuspended in extraction buffer plus 1%
deoxycholate and incubated on ice for 1 h. Samples were then centrifuged at
100,000g for 2 h to separate soluble from insoluble material. PlpD treatment
was performed by incubating the membrane fraction at 37°C for 1 h with
Streptomyces chromofuscus PlpD (Sigma), and reactions were stopped by the
addition of SDS-PAGE sample buffer. The different fractions were processed
and subjected to western-blot analysis with the anti-CrATG8 antibody. Suc
density gradient analysis was performed as described previously (Diaz-Troya
et al., 2008a).
Fluorescence Microscopy
Wild-type Chlamydomonas cells were fixed and stained for IF microscopy as
described previously (Cole et al., 1998; Diaz-Troya et al., 2008a). The primary
antibody used was rabbit polyclonal anti-CrATG8 (1:500). For signal detection, a fluorescein isothiocyanate-labeled goat anti-rabbit antibody (1:500;
Sigma) was used. Preparations were photographed on a DM6000B microscope
(Leica) with an ORCA-ER camera (Hamamatsu) and processed with the Leica
Application Suite Advanced Fluorescence software package (Leica). Deconvolution analysis of images was performed with the same software. For the
comparative analysis of the fluorescein isothiocyanate signal from different
samples, the same acquisition time was fixed except for tunicamycin-treated
cells, where it was reduced to 50% to avoid signal saturation.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. IF localization of CrATG8.
ACKNOWLEDGMENT
We thank Daniel J. Klionsky for the generous gift of wild-type and atg8
yeast strains and the anti-pAPI antibody.
Received December 21, 2009; accepted January 24, 2010; published January 27,
2010.
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