Hindawi Publishing Corporation
International Journal of Cell Biology
Volume 2012, Article ID 512721, 18 pages
Pexophagy: TheSelectiveDegradation of Peroxisomes
Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0322, USA
Correspondence should be addressed to Suresh Subramani, firstname.lastname@example.org
Received 16 October 2011; Accepted 23 November 2011
Academic Editor: Masaaki Komatsu
Copyright © 2012 Andreas Till et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Peroxisomes are single-membrane-bounded organelles present in the majority of eukaryotic cells. Despite the existence of great
diversity among different species, cell types, and under different environmental conditions, peroxisomes contain enzymes involved
in β-oxidation of fatty acids and the generation, as well as detoxification, of hydrogen peroxide. The exigency of all eukaryotic cells
to quickly adapt to different environmental factors requires the ability to precisely and efficiently control peroxisome number
and functionality. Peroxisome homeostasis is achieved by the counterbalance between organelle biogenesis and degradation.
The selective degradation of superfluous or damaged peroxisomes is facilitated by several tightly regulated pathways. The most
prominent peroxisome degradation system uses components of the general autophagy core machinery and is therefore referred to
as “pexophagy.” In this paper we focus on recent developments in pexophagy and provide an overview of current knowledge and
future challenges in the field. We compare different modes of pexophagy and mention shared and distinct features of pexophagy
in yeast model systems, mammalian cells, and other organisms.
Peroxisomes were initially described as “microbodies” in a
Ph.D. thesis on the cellular morphology of rodent kidneys
 and were characterized as novel eukaryotic organelles
by De Duve and Baudhuin in the 1960s . Biochemical
analysis of isolated peroxisomes from rat liver resulted in the
identification of several enzymes involved in hydrogen per-
oxide generation and detoxification and thus led to the term
“peroxisome” for this new organelle. Almost 50 years later,
despite significant insights regarding peroxisome function,
This is partly based on the fact that peroxisomes display
an unusually high variability in function, morphology, and
biochemical features. For example, the presence of enzymes
involved in the glyoxylate cyclehasresulted in the denotation
“glyoxysomes” for some plant peroxisomes , while the
same organelle is dubbed “glycosome” in trypanosomatids
because it houses glycolytic enzymes [4, 5]. Exemplifying
the remarkable specialization of peroxisomal enzymes is
the protein luciferase and proteins required for synthesis of
penicillin. Luciferase is responsible for the bioluminescent
characteristic of the firefly Photinus pyralis [6, 7] and the
enzymatic cascade involved in penicillin production derives
from the fungus Penicillium chrysogenum and its relatives [8,
9]. In vertebrates, peroxisomes harbor the enzymatic path-
ways for synthesis of specialized ether phospholipids vital for
integrity of the central nervous system .
In contrast to these specializations, most peroxisomes
acyl-CoA derivatives, as well as for the production and deg-
radation of hydrogen peroxide and other reactive oxygen
isome subtypes is best illustrated by the ubiquitous presence
of orthologs of a specific set of PEX genes, encoding per-
oxins, involved in peroxisome biogenesis, maintenance, and
division. Additional commonalities are that all peroxisomal
proteins are encoded in the nucleus, translated in the cytosol
and imported into the peroxisomes by a highly conserved set
of localization signals (called peroxisomal targeting signals
or PTSs) and corresponding receptors and transporters [11,
12]. Figure 1 summarizes shared and unique metabolic and
enzymatic functions of peroxisomes.
debate . Their presence in all main eukaryotic taxa and
the mentioned similarities argue for a singular evolutiona-
ry origin in a common ancestor of eukaryotic cells, most
likely as a consequence of an increase in oxygen levels in
2 International Journal of Cell Biology
Fatty acid oxidation
Sealing septal pores
Ether lipid synthesis
vertebrate nervous system
Acyl chain shortening
in bile acid and
Figure 1: Overview of peroxisome functions in different organisms and tissues. Peroxisomes display a great variety in metabolic pathways
as defined by their respective enzymatic content. Most eukaryotes share peroxisomal enzymes for fatty-acyl-CoA metabolism (α- and β-
matrix of various organisms or tissues are shown.
the archaic atmosphere. While it was initially hypothesized
that peroxisomes evolved in the course of events related to
endosymbiosis, similar to mitochondria and plastids [14–
16], research in the past decade has provided conclusive
evidence that peroxisomes are not remnants of endosymbi-
otic microorganisms but have evolved from specialization of
distinct parts of the endoplasmatic reticulum (ER) [17, 18].
Peroxisomes (unlike mitochondria and chloroplasts) have a
single membrane, do not possess their own genome, and
require several peroxisomal membrane proteins (PMPs) that
transit via the ER before reaching their final destination in
the peroxisomal membrane [19–22]. In the light of these
findings it is generally believed that peroxisomes represent
brane system, rather than examples of endosymbiotic events.
The vital importance of peroxisomes in higher eukary-
otes is documented by the dramatic effects of peroxisome
dysfunction on human health. Peroxisomal disorders (PDs)
are subdivided into two major groups: “single peroxisomal
enzyme/transporter deficiencies” (PEDs) and “Peroxisomal
Biogenesis Disorders” (PBDs). PEDs are caused by a func-
tional defect in one peroxisomal pathway and include met-
abolic syndromes such as acatalasia, Acyl-CoA deficiency
and X-linked adrenoleukodystrophy . PBDs are caused
by mutations affecting a set of at least 12 human genes,
which function in peroxisome biogenesis and assembly (PEX
genes), resulting in manifestation of numerous pathological
developmental disorders that display numerous other symp-
toms including skeletal and craniofacial dysmorphism, liver
dysfunction, and retinopathy. These diseases are caused by
complete or partial loss of peroxisome functionality and
include the Zellweger syndrome spectrum disorders (e.g.,
Zellweger syndrome, neonatal adrenoleukodystrophy, and
infantile Refsum’s disease) as well as rhizomelic chondrodys-
plasia punctata [26, 27].
The severity of these defects emphasizes the pivotal role
neuronal cells. In line with these observations, peroxisomes
serve an important function in the central nervous system
for the formation and maintenance of the myelin sheath and
for the preservation of long-term axonal integrity [10, 28,
29]. In addition, recent reports point to a specific role of
peroxisomal metabolism as a determinant of the cellular ag-
ing process, with peroxisome-derived ROS being triggers of
antiaging pathways (at low concentrations), but also being
decisive accelerators of aging by damage accumulation (at
high concentrations) [30, 31]. This is further underscored
by the finding that aging human fibroblasts accumulate per-
oxisomes with impaired protein import capacity, leading to
ROS accumulation and exacerbation of the aging process
. Homeostasis in peroxisome number and functionality
for the physiological aging process.
Due to their importance for a variety of metabolic functions,
peroxisome number is tightly controlled by environmen-
tal conditions. Yeasts (e.g., Hansenula polymorpha, Pichia
pastoris, and Saccharomyces cerevisiae), are capable of uti-
lizing different carbon sources and increasing peroxisome
number and biomass when grown in these media requiring
peroxisomal metabolism (Figure 2). Conversely, the shift of
International Journal of Cell Biology3
Figure 2: Comparison of peroxisome number and morphology in different eukaryotic cells and under different proliferation conditions.
(a) Upper panel: Human HeLa cells expressing the peroxisomal marker, RFP-SKL, under basal growth conditions. Lower panel: S. cerevisiae
cells expressing RFP-SKL after peroxisome induction in oleate medium. The relative number of peroxisomes per cell differs greatly between
panel: large, clustered methanol-induced peroxisomes; lower panel: small, unclustered oleate-induced peroxisomes. Note the difference in
size and appearance of peroxisomes induced by different carbon sources. Size marker = 2μm.
these cells from peroxisome induction conditions to carbon
sources wherein peroxisomes are unnecessary triggers the
observations of peroxisome induction and removal have
resulted in utilization of yeasts as model organisms to study
peroxisome biogenesis, and turnover, which have led to the
identification of several genes and mechanisms controlling
peroxisome homeostasis [11, 33–47]. In rodents, the admin-
istration of phthalate esters or hypolipidemic drugs such as
fibrates results in upregulation of peroxisomal proteins and a
ess is dependent on members of a special class of nuclear
receptors, called “peroxisome proliferator-activated recep-
tors (PPARs)” [49, 50]. However, this effect does not repre-
sent a general conserved mechanism since PPAR agonists fail
to induce peroxisome proliferation in human cells [51, 52].
In contrast, it has been demonstrated that drugs, such as
4-phenylbutyrate (4-PBA), that act as chemical chelators
and/or affect histone deacetylase (HDAC) activity can act as
nonclassical peroxisome proliferators independent of PPAR
activity in human cells [53, 54].
Here we present model systems to study peroxisome
turnover and outline mechanisms that contribute to peroxi-
some homeostasis by regulating the selective degradation of
of peroxisomes in the vacuolar/lysosomal compartment, a
process mediated by components of the general autophagy
core machinery and usually referred to as pexophagy.
3.Methylotrophic Yeastsas Model
The large peroxisome clusters of methylotrophic yeasts (e.g.,
P. pastoris and H. polymorpha), as well as the experimental
ease of manipulation of peroxisome number, volume, and
content by media shifts in a genetically tractable organism,
have facilitated studies on pexophagy. These yeasts, when
grown in media containing methanol as the sole carbon
source, rapidly proliferate their peroxisomes, which can oc-
cupy up to 40% of the cell volume. This makes fluorescence
imaging of tagged proteins involved in pexophagy, as well
as biochemical analysis of peroxisomal markers, much easier
to monitor than in mammalian or other yeast systems. The
Duve and Baudhuin  when they observed the appearance
of peroxisomes within the lysosomes of mammalian cells,
thus documenting the earliest description of pexophagy.
Since then, much has been learnt from studies on pexophagy
conducted in methylotrophic yeasts.
4.Modes of Pexophagy: Micropexophagy
All organisms from yeast to humans possess basal and indu-
cible macroautophagy. During macroautophagy (referred to
here as “autophagy”), a double membrane originates from a
site known as the phagophore assembly site (PAS) to engulf
cargo into a double-membrane vesicle known as the autoph-
agosome, which upon fusion with a lysosome (or vacuole
in yeast cells), releases into the lysosomal/vacuolar lumen
an autophagic body comprised of a single membrane sur-
rounding the cytosolic cargo. Once in the lysosomal lumen,
the membrane and other macromolecular contents of the
autophagic body are degraded by hydrolases to their con-
ess, from the assembly of the PAS, to the engulfment of cargo
into autophagosomes, fusion of autophagosomes with the
lysosome/vacuole and subsequent degradation of the cargo,
is orchestrated by the hierarchical recruitment of autophagy-
related (Atg) proteins . An alternative process called
4International Journal of Cell Biology
Figure 3: Similarities and differences between selective autophagy pathways. Various selective autophagy pathways share similar molecular
mechanisms. They require a receptor that interacts with the cargo, recruits a scaffold protein (Atg11) that organizes the core autophagic
machinery at the PAS, and mediates recruitment of Atg8, which initiates phagophore elongation from the PAS. In the Cvt pathway (a) Atg19
and Atg34 are the receptors for the cargo proteins aminopeptidase I (Ape1) and alpha-mannosidase, respectively. These receptors bind
to Atg11 at the Cvt-specific PAS to initiate membrane expansion of the phagophore. (b) The mitophagy-specific phagophore membrane
expansion from the PAS is initiated by Atg32, a mitochondrial outer membrane protein. Atg32 also interacts with Atg11 and Atg8. (c) The
pexophagy receptor, Atg30, is localized at the peroxisome membrane, via interaction with the PMPs, Pex3, and Pex14. It is phosphorylated
upon induction of pexophagy resulting in interaction of Atg30 with core autophagic machinery components, Atg11 and Atg17. In the case
of pexophagy, the direct Atg8 interaction partner is still unknown.
microautophagy also exists, in which the lysosome/vacuole
membrane invaginates to engulf cytosolic cargo directly to
degrade and recycle it [81, 82].
In contrast to the nonselective nature of cargo engulfed
by macroautophagy and microautophagy, other autophagy-
related pathways capture cargo selectively from the cytosol.
These include oligomeric proteins delivered to the vacuole
(ribophagy), and subcellular organelles such as peroxisomes
(pexophagy), mitochondria (mitophagy), parts of the ER
(ER-phagy), and segments of the nucleus (micronucleopha-
gy) . In most of these selective processes, the phago-
phore membrane, originating from specific PAS structures
required for each form of selective autophagy (e.g., Cvt- or
pexophagy-specific PAS, Figures 3(a) and 3(c)), engulfs the
specific cargo and delivers it to the lysosome/vacuole for
degradation. The source of the phagophore membrane is a
widely debated topic within the field of autophagy, with the
focus primarily on how Atg9 (ATG9L1 in mammals), which
is recruited to the PAS . Atg9 is thought to be involved
directly or indirectly in trafficking membrane and/or lipid
components during phagophore expansion from the PAS.
To date, 35 autophagy-related (ATG) genes involved in
several autophagy-related pathways have been discovered.
Macropexophagy and micropexophagy (described next) are
both used in P. pastoris for selective peroxisome degradation
[87, 88]. As these represent specialized types of autophagy,
International Journal of Cell Biology5
it should not be surprising that they require many of the
core genes also used for autophagy as well as specific genes
in addition (see Table 1) [38, 89]. Many yeast mutants with
pexophagy defects provide insights into the mechanism of
the two pexophagy modes (see Table 1).
Micropexophagy occurs when a cluster of peroxisomes
is directly engulfed by vacuolar sequestering membranes
(VSMs) that extend from a septated vacuole, and a double-
membrane structure called the micropexophagy-specific
membrane apparatus (MIPA) . The MIPA extends from
oxisomes and fuses with the VSMs to completely sequester
the targeted peroxisomes from the cytosol and to ultimately
deliver the pexophagic body into the vacuole lumen to be
the phagophore membrane originating from the pexophagy-
specific PAS to form a double membrane-bounded pexopha-
gosome (Figure 4(a)), before the outer membrane fuses with
the vacuole membrane in a process resembling macroau-
tophagy . In P. pastoris, the choice between induction
of either micro- or macropexophagy is determined by ATP
levels in the cell . High levels of ATP induce micropex-
ophagy while lower levels activate macropexophagy. One
explanation for this observation may be that the massive
vacuolar rearrangement during micropexophagy and forma-
tion of the MIPA may be a more energy-intensive process
more energy in the form of ATP from the cell.
5.NutrientConditions That InducePexophagy
In S. cerevisiae, pexophagy is induced by transferring cells
from growth media containing oleate as a carbon source to
glucose-containing medium without a nitrogen source .
In P. pastoris, peroxisomes can be induced when cells are
grown in media containing methanol, oleate, or amines.
Transferring cells grown in methanol to ethanol or from ole-
ate or methylamine to glucose without nitrogen induces ma-
cropexophagy (Figures 4(b) and 4(c)) . Shifting cells
from methanol medium to glucose induces micropexophagy
(Figures 4(b) and 4(c)) . Intriguingly, the two modes of
pexophagy can be triggered by different experimental con-
ditions in different yeasts. In H. polymorpha, macropex-
ophagy, rather than micropexophagy, is induced when cells
are shifted from methanol medium to glucose .
Interestingly, it was shown that simultaneous treatment
of H. polymorpha with both nitrogen limitation and excess
glucose conditions results in concomitant induction of both
microautophagy and macropexophagy, thus exemplifying
the fact that selective (i.e. pexophagy) and nonselective (au-
tophagy) pathways can be initiated in parallel .
6.Regulation of Yeast Pexophagy
It has long been realized that not only surplus, but also
damaged components or potentially toxic structures within
the cytosol of eukaryotic cells can be selectively removed
by autophagy. Using ectopic expression of a temperature-
sensitive degron-Pex3 fusion, it was recently shown in H.
polymorpha that damage to peroxisomes by abruptly remov-
ing the essential PMP, Pex3, causes pexophagy to occur .
This conditional selective degradation was apparent even
when cells were placed in conditions that would normally
require peroxisome biogenesis for cell growth. In methanol-
excess conditions the authors saw a transient increase of ROS
in wild-type cells that corresponds with the degradation of
the peroxisome matrix protein, alcohol oxidase, as well as
PMPs, Pex3, and Pex14, suggesting the possible physiological
significance of pexophagy. However, it is unclear at present if
there is a similar requirement of Pex3 removal from the per-
oxisome membrane for pexophagy in other methylotrophic
yeasts such as P. pastoris, where Pex3 is actually essential to
recruit the pexophagy receptor, Atg30, to the peroxisome,
before the organelle is targeted for pexophagy.
The signaling events that regulate the specific removal
of cellular components are still poorly understood. The
emerging role of intracellular signaling pathways controlling
pexophagy was shown by our group and has since been rep-
licated and refined further. Using the degradation of a perox-
isomal marker to investigate the role protein kinases play in
kinase (MAPK) and several other upstream components of
this signaling pathway were shown to be required for pex-
ophagy, but not for pexophagosome formation, suggesting a
block at the step of pexophagosome targeting or pexophago-
some-vacuole fusion .
autophagy has been extended by the recent discovery that
Slt2 also plays a role in mitophagy , along with another
MAPK (Hog1) (Figure 5) [95, 96]. Slt2 is crucial for
recruiting mitochondria to the PAS, a step required for the
specific packaging of cargo into autophagosomes. Interest-
ingly, mitophagy in mammalian cells is activated by ERK2,
another MAPK . Thus, the differential involvement of
MAPK pathways represents a central process in controlling
diverse selective autophagy pathways .
Other signaling pathways have also been shown to have
a direct role in pexophagy [35, 77, 98]. The phosphoinos-
itide, phosphatidylinositol-3-phosphate (PtdIns3P), as well
as the sole phosphatidylinositol 3-kinase, Vps34, that gen-
erates PtdIns3P in yeast, are required for all autophagy-
related pathways, including pexophagy [36, 99]. In addition,
phosphatidylinositol-4-phosphate (PtdIns4P), as well as the
kinase that is responsible for PtdIns4P generation (Pik1) and
Atg26, a sterol-glucosyltransferase that binds PtdIns4P via
its GRAM domain, are necessary for micropexophagy in P.
7.GeneralThemes of Selective
Since all autophagy-related pathways share common com-
ponents required for PAS assembly, elongation of the pha-
gophore membrane around cargo, vesicle formation, fusion
and vacuolar degradation, the key decision point in any
selective autophagy pathway is the mechanism by which the
6 International Journal of Cell Biology
of pexophagy is indicated by check marks. Genes denoted in bold font are (by current knowledge) exclusively involved in pexophagy, but not
in other autophagy pathways. Genes denoted in regular font represent components of the core machinery involved in different autophagy
lack of conclusive evidence. Table adapted from Sakai et al. .
Gene Description of molecular events
ATG1Serine/threonine kinase required for PAS formation[42, 56, 57]
ATG2 Peripheral membrane protein required for Atg9 recycling
ATG3 E2-like ubiquitin ligase that catalyzes lipidation of Atg8
Protease that processes Atg8 as prerequisite for conjugation
with phosphatidylethanolamine (PE)
ATG6 Subunit of PI3K complexes I and II
E1-(ubiquitin activating enzyme)-like protein involved in
conjugation of Atg12-Atg5 and Atg8-PE conjugates
Ubiquitin-like protein that is anchored to the expanded
phagophore membrane in its processed and lipidated form,
involved in phagophore membrane expansion
Transmembrane protein cycling between the PAS and a
Coiled-coil adaptor protein that interacts with the core
machinery and known receptors for selective autophagy
[33, 60, 61]
ATG16 Essential component of the Atg12-Atg5-Atg16 complex(?)
ATG17 Scaffold protein that is responsible for PAS organization
PtdIns3P-binding protein whose localization is dependent
Atg9 and PtdIns-3P; recruits Atg2 and needed for Atg9
WD40 protein with phosphoinositide binding domain that is
involved in pexophagosome formation
Sorting nexin protein involved in fusion events with the
Coiled-coil protein that co-localizes with Atg11 at the PAS,
required for macropexophagy
Sterol glucosyltransferase that plays a role in phagophore
Coiled-coil protein required for peroxisome sequestration
during micropexophagy and vacuole fusion of
pexophagosomes in macropexophagy
Pexophagy receptor that interacts with peroxins, Pex3 and
Pex14, and adaptor proteins, Atg11 and Atg17
Localizes to the perinuclear structure; regulates MIPA
formation and interacts with Atg28 and Atg17
[44, 46, 68]
GCN1-4 Involved in general amino acid control
Sec protein required for MIPA and proper pexophagosome
PEP4 Vacuolar protease
PMP peroxin required for peroxisome biogenesis and for
recruitment of pexophagy receptor
PMP peroxin required for peroxisome biogenesis and for
recruitment of pexophagy receptor
[34, 71, 72]
PtdIns-4-kinase required for MIPA formation
Subunit of phosphofructokinase complex
TUP1 Transcriptional repressor
International Journal of Cell Biology7
Table 1: Continued.
Gene Description of molecular events
N-myristoylated armadillo-repeat protein of the vacuolar
membrane, required for VSM formation
SNARE protein that is involved in vacuolar fusion events
with the phagophore membrane
VPS15Regulatory subunit of PI3K
VPS34Phosphatidylinositol-3-kinase (PI3K) [36, 78]
YPT7Rab GTPase involved in phagophore membrane fusion[71, 79]
core autophagy machinery is redirected to degrade primarily
selective cargo. The study of these selectivity factors for
applicable, we describe how these events are relevant to other
selective autophagy pathways.
(1) Every selective autophagy pathway studied to date
requires a specific cargo receptor. Examples of these
include Atg30 for pexophagy , Atg19 and Atg34
for the Cvt pathway [100–102], and Atg32 for mito-
phagy [103, 104].
(2) These cargo receptors typically have a tripartite role
in (a) cargo binding, (b) interaction with Atg11, a
ways to create the specific PAS structures from which
the phagophore membrane will expand [100, 103,
104], and (c) interaction with Atg8, via an Atg8-in-
pansion [100, 103, 104]. The receptors Atg19 and
Atg32, required for the Cvt and mitophagy pathways
in yeast, have all these properties, but as of now, only
the first two roles have been attributed to Atg30 dur-
ing pexophagy .
(3) The selective autophagy receptors are often synthe-
sized even under conditions wherein the cargoes are
not degraded, but receptor activation often relies on
protein modifications, such as phosphorylation or
ubiquitination [34, 96, 106].
(4) Some of the pexophagy-mediating factors, such as
Atg11 and the sterol glucosyltransferase Atg26 that
binds PtdIns4P ,arerequiredinanabsolutefash-
ion for the degradation of large cargoes, but are par-
tially dispensable when the cargo size is small .
We predict that since the phagophore membrane has
to engulf cargoes of varying sizes from individual
cytosolic proteins to organelles, bacteria and viruses,
analogous factors will be required for selective auto-
phagy of other large cargoes.
(5) Specialized membrane structures, such as the MIPA,
are needed for micropexophagy, and not for macro-
pexophagy. Indeed, the protein Atg35 is needed for
MIPAformationduring micropexophagy, butnot for
pexophagosome formation during macropexophagy
(6) Generally the receptors are degraded in the vacuole
along with the cargo.
8.The Pexophagy-Specific PAS
Like autophagy, pexophagy is also initiated at a specific PAS
(Figure 3(c)) that is distinct from other types of PAS for se-
lective autophagy (Figures 3(a) and 3(b)). The autophagy-
specific PAS is organized by Atg11, Atg17, Atg29, and Atg31,
but Atg11 is dispensable . The Cvt-specific PAS requires
whereas the mitophagy-specific PAS uses Atg11 and Atg32
[103, 104] (Figure 3(b)). The pexophagy-specific PAS is or-
ganized by Atg11, Atg17, and Atg30 [34, 107].
rylation by a hitherto unknown kinase occurs and facilitates
direct physical interaction with Atg11 . The two proteins
colocalize at the PAS, and Atg30 also directly interacts with
Atg17. The roles of Atg11 and Atg17 are as scaffolds at
the PAS that recruit other proteins, such as constituents of
the core autophagy machinery described next. Surprisingly,
there is a size requirement of the scaffolding proteins. For
degradation of small peroxisomes, Atg11 and Atg17 are only
partially required, but are essential for degradation of large
peroxisomes in nitrogen-starvation conditions .
The assembly of a specific PAS is followed by the recruit-
including, but not limited to Atg1, Atg2, Atg5, Atg8, Atg9,
Atg12, Atg13, Atg16, Atg18, Atg23, Atg24, Atg25, Atg27,
Atg28, Atg35, and the PtdIns3-kinase (PI3K) complex. These
proteins typically assemble in a complex hierarchy ,
such as our demonstration that the recruitment of PtdIns-
3-Kinase to the PAS precedes Atg8 recruitment .
9.Elongationof the Phagophore Membrane
The protein Atg35 is a micropexophagy-specific protein re-
but not for pexophagosome formation, giving the first evi-
dence that the formation of the MIPA could be genetically
distinct from the formation of the pexophagosome in ma-
Oku et al.  discovered that Atg26, a sterol glucosyl-
transferase that synthesizes sterol glucoside, is essential for
8International Journal of Cell Biology
Mkk1 + Mkk2
Figure 4: Micropexophagy and macropexophagy. (a) Micropexophagy differs from macropexophagy in vacuole dynamics and formation
of the MIPA instead of the pexophagosome. A pexophagy-specific PAS, required for both forms of pexophagy, is characterized by its
by vacuole remodeling to form cup-like vacuolar sequestration membranes (VSMs) and a lid-like cover called the MIPA (micropexophagy-
followed by its fusion with the vacuole for degradation and recycling. Pexophagy signaling is dependent on Mitogen-activated protein kinase
(MAPK) pathways (Mid2-Slt2 cascade), but may also be triggered by internal (unknown) factors, including signals related to the status of,
or metabolic need for, (e.g., damaged or superfluous) peroxisomes. (b) The upper panel depicts a single P. pastoris cell that has undergone
peroxisome induction (in methanol) and has then been switched to micropexophagy conditions (glucose). The vacuole (red, FM 4–64) is
shown surrounding the targeted peroxisome cluster (blue, BFP-SKL). The MIPA (green, GFP-Atg8) forms a lid over the cup-like VSMs. The
lower panel illustrates pexophagosome formation around a single peroxisome under macropexophagy conditions (ethanol). (c) S. cerevisiae
cell labeled with GFP-tagged thiolase (a peroxisome matrix marker) and vacuole marker (FM 4–64, red) shows proliferated peroxisomes
under nutrient-rich conditions (in oleate, top panel). When the cells are switched to glucose without nitrogen, peroxisomes are targeted to
the vacuole by macropexophagy and GFP accumulates in the vacuole (lower panel).
International Journal of Cell Biology9
Figure 5: Signal transduction cascades regulating selective auto-
phagy in yeast. Mitogen-activated protein kinase (MAPK) cascades
contribute to differential regulation of selective autophagy path-
ways. As recently shown, the Slt2 and Hog1 signal transduction
pathways regulate both mitophagy and pexophagy [37, 96]. Besides
the obvious role of environmental factors such as nutritional con-
ditions, details of other upstream events are poorly understood.
pexophagy, but not autophagy in P. pastoris. They showed
that the protein is associated with the MIPA during micro-
pexophagy, and that a single amino acid substitution within
the GRAM domain (domain found in glycosyltransferases,
Rab-like GTPase activators, and myotubularins) of the pro-
tein abolished this association . However, it was found
that although Atg26 is required for utilization of decane in
Y. lipolytica, it was unnecessary for pexophagy in this yeast,
showing that sterol glucosyltransferase play different func-
tional roles in the two yeasts .
initiates de novo membrane synthesis that is required for
pexophagy. PtdIns4P, generated primarily by the PtdIns-4-
kinase, Pik1, recruits Atg26 via its GRAM domain , and
the sterol glucosyltransferase activity of Atg26 at the nucle-
ation complex is necessary for the elongation of the mem-
In both S. cerevisiae and P. pastoris, the only integral
membrane protein of the autophagy machinery, Atg9, cycles
between a peripheral compartment comprising a reservoir
of Atg9 and the PAS, or PAS-like structures. The shuttling
mechanism has been studied in both organisms but the
process is better understood in S. cerevisiae and is therefore
described next, before the role of this protein in pexophagy
In S. cerevisiae, Atg9 colocalizes at the PAS but is not
present on completed autophagosomes, suggesting it must
be recycled during autophagosome formation. It cycles be-
tween a peripheral compartment and the PAS . The an-
terograde trafficking of Atg9 from the peripheral compart-
ment to the PAS requires Atg11, Atg23, and Atg27 .
Atg9 retrieval from the PAS is regulated by the Atg1-
Atg13 signaling complex and requires Atg2, Atg18, and the
PtdIns3P generated by the Atg14-containing PtdIns-3-kinase
complex . However, only Atg2, Atg18, and PtdIns3P are
necessary for Atg9 recycling, while the Atg1-Atg13 complex
and Atg1 kinase activity, but not Atg2, Atg18, and PtdIns3P,
are necessary for Atg23 cycling to and from the PAS .
The subcellular movement of Atg9 in S. cerevisiae re-
quires interaction with the actin cytoskeleton as has been
shown by the sensitivity of relocation of Atg9 to the inhibitor
Latrunculin A, as well as by the phenotype displayed by con-
ditional mutants of actin and the actin-related protein Arp2
The proteins Atg11 and/or Atg17 are necessary for Atg9
recruitment to the PAS [84, 113]. Also required at the PAS
is PtdIns3P, generated by the Vps34 (PtdIns-3-kinase) com-
plex, to recruit PtdIns3P-binding proteins (e.g., Atg18 and
Atg24), which then recruit yet other proteins, such as Atg2,
to the PAS .
P. pastoris Atg9 (PpAtg9) is necessary for the formation
of the VSM, assembly of the MIPA, and for pexophagosome
to the PAS from a peripheral compartment, perhaps sup-
plying the membrane to the PAS and elongating the phago-
phore membrane to form the VSM, MIPA, and pexophago-
some . PpAtg9 shuttles from a peripheral compartment
near the ER/mitochondria to unique perivacuolar structures
(PVS; PAS-like structures) that contain Atg11, but not Atg2
or Atg8. Atg9 then traffics from the vacuole surface to the
VSMs that engulf peroxisomes for degradation . Move-
ment of the PpAtg9 from the peripheral compartment to the
3-kinase). PpAtg2 and PpAtg7 are essential for PpAtg9 traf-
ficking from the PVS to the vacuole and sequestering mem-
branes, whereas trafficking of PpAtg9 proceeds independent
of PpAtg1, PpAtg18, and PpVac8. How exactly PpAtg9 con-
tributes to the formation of the MIPA and pexophagosome
formation is less clear.
In P. pastoris, expression of dominant-negative forms
(Sar1-T34N and Sar1-H79G) of the ER protein Sar1, impairs
Atg8 lipidation and MIPA formation, but not the formation
of the VSMs or the trafficking of Atg11 and Atg9 to these
VSMs during micropexophagy . During macropexopha-
gy, the expression of Sar1-T34N inhibited the formation
of the pexophagosome, whereas Sar1-H79G suppressed the
delivery of the peroxisome from the pexophagosome to the
vacuole. In this case, the pexophagosome contained Atg8 in
wild-type cells, but in cells expressing Sar1-H79G these or-
ganelles contain both Atg8 and endoplasmic reticulum com-
ponents, suggesting a defect in retrieval of components back
to the ER, prior to pexophagosome/vacuole fusion.
The protein Atg25 has been described in H. polymorpha
to be required for macropexophagy. It interacts with Atg11
and colocalizeswith it atthe PAS.In its absence,peroxisomes
are constitutively degraded by nonselective microautophagy,
a process that in wild-type H. polymorpha is only observed
under nitrogen starvation conditions, suggesting that non-
selective microautophagy is deregulated in H. polymorpha
atg25Δ cells .
10International Journal of Cell Biology
10.Requirementof Specific Proteinsduring
the FinalStages of Pexophagy
Atg24, a molecule with a PtdIns3P-binding module (PX do-
main), is required for micropexophagy and macropexopha-
gy, but not for general autophagy in P. pastoris and S.
cerevisiae . CFP-tagged PpAtg24 localizes to the vertex
and boundary region of the pexophagosome-vacuole fusion
complex during macropexophagy. Depletion of PpAtg24
blocked macropexophagy after pexophagosome formation
PpAtg24 is involved in the regulation of membrane fusion
at the vacuolar surface during pexophagy via binding to
PtdIns3P and could potentially be involved in pexophago-
some fusion with the vacuole . During micropexophagy,
Ppatg24Δ cells form the MIPA and exhibit aberrantly sep-
uolar fusion, but engulfment of peroxisomes is also impaired
In contrast to yeast models, which have greatly contributed
to the mechanistic understanding of pexophagy as outlined
above, the molecular details of mammalian pexophagy are
ferences between mammalian and yeast peroxisomes. While
in yeasts the number of peroxisomes varies between 1–20
dependent on the species and growth conditions (see above),
thousands of peroxisomes (Figure 2(a)) . Induction of
peroxisome proliferation in rodents by phthalate esters [e.g.,
di-(2-ethylhexyl)phthalate; DEHP)], hypolipidemic drugs
(e.g., fibrates) or nonclassical peroxisome proliferators (e.g.,
4-PBA) results in a 2-3-fold increase of peroxisomal mass,
which is a significantly smaller effect compared to the effects
observed in yeasts. Consequently, quantitative analyses of
peroxisome turnover in mammalian systems are limited by
the detection method applied. Mammalian peroxisomes dif-
fer from those of yeast cells not only in number and in-
duction mechanisms, but also by their modes of selective
degradation. At least three independent degradation systems
have been described: the Lon protease system, 15-lipoxygen-
ase (15-LOX)-mediated autolysis and lysosomal degrada-
conditional knockout mice it is estimated that up to 20–30%
mediated mechanisms and 15-LOX-mediated autolysis of
peroxisomes, whereas the remaining 70–80% are destroyed
by autophagic mechanisms .
The peroxisomal isoform of the Lon protease is an
ATP-dependent protease with chaperone-like activity that is
involved in degradation of misfolded and unassembled per-
oxisomal proteins. Lon protease is upregulated in rats under
peroxisome proliferation conditions (e.g., administration of
DEHP) and further increases its levels after withdrawal of
the inducing drug while peroxisomal enzymes are quickly
degraded . Subsequently, Lon protease activity catalyz-
es the breakdown of proteins resident in the peroxisomal
Figure 6: Peroxisome degradation pathways in mammalian cells.
Surplus peroxisomes or their contents (e.g., peroxisomal matrix
protease-mediated proteolysis, 15-lipoxygenase (15-LOX)-mediat-
ed cytosolic degradation (autolysis), and pexophagy (autophagy-
mediated lysosomal degradation). Current studies suggest that the
majority of peroxisomes are degraded by pexophagy (indicated by
matrix, indicating that it contributes to the reduction of
peroxisome mass, if not quantity. Interestingly, the yeast
ortholog of the Lon protease (encoded by the PLN gene in
H. polymorpha) appears to be essentially involved in perox-
isome quality control mechanisms, with only about a 25%
contribution to reduction of peroxisome numbers, which
increased only slightly from 2.6/cell to 3.3/cell in the absence
of Pln, whereas the peroxisome number increased to 5.4/cell
in the absence of the ATG1 gene required for all forms of
autophagy . Assuming that the Lon protease has similar
roles in yeast and mammals, it is conceivable that relative to
autophagic mechanisms, it plays a relatively modest role (in
the range of 25%) in reducing peroxisome number.
The cytosolic enzyme, 15-LOX, can associate with per-
oxisomal membranes leading to localized membrane disrup-
tion . Structural breakdown subsequently exposes the
peroxisomal content to cytosolic proteases resulting in its
rapid degradation. This pathway appears to be initiated in
parallel to pexophagy after drug-mediated accumulation of
peroxisomes and accounts for removal of a limited fraction
of excess peroxisomes.
While the abovementioned pathways contribute partially
to peroxisome homeostasis under certain cellular conditions
and other data argue for a role of the proteasome system by
undefined mechanisms , the vast majority of selective
peroxisome degradation is mediated by autophagosomal-
lysosomal processes resembling yeast macropexophagy. As
mentioned, early reports from the 1970s already noted the
selective lysosomal degradation of mitochondria and perox-
isomes during the diurnal cycle in rat inner organs ,
but it was only shown later that the autophagy machinery is
specifically involved in degradation of surplus peroxisomes
in mouse liver . This was demonstrated by compar-
ing abundance and degradation efficiency of peroxisomes
after treatment with phthalate ester for 2 weeks and chase
International Journal of Cell Biology11
after drug removal one week later in wild-type and autopha-
gy-deficient Atg7−/−mice. The salient findings of this study
emphasize mechanistic similarities to the above-mentioned
tions that require peroxisomal enzymes (e.g., oleate/metha-
lead to peroxisome proliferation, followed by pexophagic
degradation when the organelles are no longer required or
can be used as a resource for alternative pathways. This bio-
logical theme of a metabolic switch involving adaptation to
changing external factors and thereby triggering pexophagy
is also reminiscent of organelle remodeling in pathogenic
fungi and parasitic protozoa as will be outlined in the next
A detailed functional analysis of peroxisome degradation
using an in vitro cell culture system showed for the first
time that peroxisomes are preferentially degraded over cy-
tosolic proteins under starvation/recultivation conditions
. This study used Chinese Hamster Ovary (CHO) cells
to describe autophagy-mediated peroxisome turnover when
switching culture conditions from starvation in Hank’s solu-
tion to reconstitution in nutrient-rich medium. The authors
show convincingly that the peroxisomal membrane protein,
Pex14, is bound by autophagosome-anchored LC3-II (i.e.,
the processed and lipidated form of LC3) under starvation
conditions. Pex14 is an essential component of the peroxiso-
proteins into the peroxisomal matrix. It is noteworthy that
the dual role of Pex14 for both peroxisome assembly and
selective degradation has also been shown for yeast systems
. Moreover, the study by Hara-Kuge and Fujiki points
to an involvement of the cytoskeleton in this process by
demonstrating the requirement of intact microtubules for
of the competitive nature of the processes involved, binding
Pex5, proved to be mutually exclusive. This might point
to a general mechanism that ensures functional segregation
of metabolically active and degradation-prone organelles.
Although this study uses an unusual experimental setup by
applying starvation followed by recultivation in rich me-
dium, it opens the avenue for future studies addressing the
question of how exactly PMPs contribute to physical inter-
actions with the autophagy machinery. In line with these
observations, a recent study describes the role of a Rab7-
effector protein, FYCO1 (FYVE and coiled-coil domain-con-
taining 1), as the physical link between LC3 family members,
PtdIns3P and microtubule plus end-directed transport [122,
123], but the exact role of this mechanism for pexophagy in
particular has not been addressed yet.
The dynamics of peroxisome turnover in mammalian
cells under normal cultivation conditions have nicely been
addressed in a recent publication . Using HaloTag-
fate of peroxisomes in cultured CHO cells and mouse fibro-
blasts, the authors show that mammalian peroxisomes have
a half-life of approximately 2 days under normal cultivation
conditions and that peroxisomes of different age display a
different capacity to import newly synthesized proteins. In
addition, this study shows that even under normal growth
conditions, pexophagy contributes to the majority of turn-
over of this organelle as demonstrated by sensitivity to 3-
methyl adenine (3-MA, an autophagy inhibitor) treatment.
These findings emphasize the dual role of autophagy-related
pathways: while autophagy principally serves to ensure nu-
trient recycling under starvation conditions, the same ma-
chinery fulfills the purpose of a quality control and homeo-
stasis mechanism even in the presence of all nutrients.
Because the autophagy machinery in mammalian cells
targets ubiquitinated protein aggregates, experiments were
designed to address whether monoubiquitination of perox-
isomal proteins could cause the autophagic clearance of per-
oxisomes . Using overexpression of PMPs, Pmp34 and
Pex3, fused on the cytosolic side to a ubiquitin variant genet-
ically tailored to block polyubiquitination, it was found that
exposure of a single ubiquitin moiety on the cytosolic face of
the peroxisomal membrane was sufficient to trigger turnover
of this organelle. Specificity of this affect was demonstrated
by analyzing sensitivity to protein topology and to the auto-
phagy inhibitor, 3-MA, thus confirming the requirement of
the autophagy machinery in degradation of the ubiquitin-
labeled peroxisomes. Moreover, the study showed that the
ubiquitin-binding autophagy adaptor, p62, is involved in
selective degradation of peroxisomes under the chosen con-
ditions. Although this study is primarily based on overex-
pression of ectopic proteins and the artificial placement of
a ubiquitin tag on the peroxisomal membrane and does not
identify the physiological target of this process, it has some
interesting implications. The general requirement of p62 for
in the absence of ectopic ubiquitin tagging, since knock-
down of p62 significantly increased endogenous peroxisome
numbers under the experimental conditions. Furthermore,
of all mammalian autophagy adaptors identified so far (e.g.,
p62/SQSTM1, NDP52, and NBR1), only p62 has as yet been
shown to be involved in selective degradation of peroxiso-
mes. Since these adaptors partly share mechanistic features
such as bridging ubiquitinated cargo (e.g., cytoinvasive bac-
teria in the case of NDP52) to LC3 family members to link
with the autophagy machinery, it is unclear to date how
cargo selectivity is facilitated in mammals. An interesting
finding on this theme comes from the field of xenophagy,
the selective degradation of cytosolic pathogens (reviewed
elsewhere in this special issue): As shown recently, the two
ubiquitin-binding autophagy adaptors p62 and NDP52 are
recruited independently to cytoinvasive Salmonella sp. and
pathogens . The authors argue that two individual
adaptor complexes are required for effective xenophagy of
microdomains associated with bacteria. With respect to pex-
ophagy, it has not been analyzed whether or not different
adaptor proteins are involved in selective degradation of per-
oxisomes and what their respective contribution is. Answers
to this type of question will be informative not only for
pexophagy, but for the whole field of selective autophagy
pathways. A hypothetical mechanistic model of mammalian
pexophagy is illustrated in Figure 7.
12International Journal of Cell Biology
Figure 7: Hypothetical mechanistic model of pexophagy in mammalian cells. Processed and lipidated LC3 (LC3-II) is integrated into the
expanding phagophore membrane (PM) and also may be involved in facilitating directed movement of the PM structure by interacting
with microtubules (MT) via the RAB7 effector FYCO1 and motor protein Kinesin. Targeting of peroxisomes may either be accomplished by
p62-mediated detection of ubiquitin (UB) motifs on still unknown peroxisomal membrane (or membrane associated) proteins (X) or by
direct binding of LC3 to PEX14, a process which is discussed to compete with the binding of PEX5 to PEX14 (dotted arrow). See text for
A very interesting and unexpected perspective originates
from recent studies in the field of parasitology and infec-
tion biology showing that pexophagy is required for the
phytopathogenicity of the cucumber anthracnose fungus,
Colletotrichum orbiculare [127, 128]. This plant pathogen
forms a specific structure termed the appressorium, which
is required for penetration of the host epidermal cells
in the course of infection. The authors used a random
insertional mutagenesis screen to identify fungal genes that
contribute to pathogenicity. They identified the C. orbiculare
ortholog of P. pastoris ATG26 to be essential for host cell
infection. PpATG26 is a well-characterized pexophagy gene
encoding a sterol glucosyltransferase, which is essential for
pexophagy in methylotrophic yeasts [46, 92]. In the case
of C. orbiculare, the pathogen undergoes morphological
changes reminiscent of pexophagy during development of
its appressoria as indicated by vacuolar localization of
peroxisomes and the requirement for the central autophagy
protein, Atg8. While appressoria could still be formed
in the atg26 deletion mutant, the infection process was
significantly delayed. Moreover, deletion of atg8 completely
abolished appressoria formation, suggesting an essential
role of the autophagy machinery during infection. As the
authors show, nonselective general autophagy is essential for
early morphogenesis during pathogen development, while
Atg26-dependent selective pexophagy is essential for later
stages of direct host-pathogen infection steps. The authors
conclude that Atg26-mediated pexophagy might be involved
in maturation of the infection structures by providing
molecular building blocks through organelle recycling.
Another report points to the role of pexophagy during
developmental and environmental changes in the parasitic
protozoan, Trypanosoma brucei. This human pathogen,
which causes sleeping sickness and Chagas disease, harbors
essential enzymes of glycolysis in its peroxisomal structures,
which are therefore referred to as “glycosomes.” The para-
sitic life cycle of this pathogen, which comprises different
host, requires adaptation of its metabolism to the changing
environment. The necessary dynamic remodeling of glyco-
somal structures is facilitated by fusion of glycosomes with
acidic lysosomes through autophagy-related mechanisms
resembling pexophagy . As shown recently, the acidic
pH of the lysosomal compartment is responsible for inacti-
without affecting its protein level . In addition, the pH
change renders the enzyme sensitive to metabolic feedback
regulation by both its substrate and product (ATP and ADP,
resp.) and to modulation by other glycosomal metabolites,
Thus, pexophagy appears to allow for a novel mechanism of
regulating enzymatic activity by facilitating pH-dependent
structural changes and concomitant feedback responses.
These data point to an unexpected role of pexophagy as a
during development and adaptation.
requires adjustment of peroxisome number for survival
after phagocytosis by immune cells . The authors
used fluorescent fusion proteins of transcription factors and
peroxisomal enzymes to assess the metabolic status of the
engulfed parasite. Using this approach, they showed that the
number initially, most likely to fight phagocyte-induced
oxidative stress. However, prolonged phagocytosis resulted
in carbon starvation and a pexophagy-mediated decrease of
by the dramatic loss in parasite survival during phagocytosis
when the selective autophagy gene, CgATG11, or the general
autophagy gene, CgATG17, were knocked out. The authors
conclude that autophagy-related mechanisms, including
pexophagy, represent important survival mechanisms for
Candida after engulfment by phagocytes, pointing to the
pivotal role of these pathways for providing essential cellular
International Journal of Cell Biology13
Kawaguchi et al. (2011) recently reported on a possible
physiological role of pexophagy in yeast. This was achieved
by exploring the relationship between the methylotrophic
yeast Candida boidinii and the phyllosphere of growing
Arabidopsis thaliana leaves . The authors developed
a methanol sensing assay in live C. boidinii cells using a
PTS1-tagged fluorescent protein expressed from a methanol-
inducible promoter, whereby an increase in environmental
methanol concentrations resulted in enhanced fluorescence
levels. They then used this assay to measure local methanol
concentrations at the phyllosphere of growing A. thaliana
leaves and showed that methanol concentrations at the phyl-
losphere change throughout the day corresponding to the
light-dark cycle, whereby methanol concentration increased
in the dark period, compared to the light period. In addition,
they showed that autophagy as well as pexophagy are both
required for yeast growth and survival at the phyllosphere,
as autophagy and pexophagy mutants exhibited impaired
proliferation on growing A. thaliana leaves. These results
reveal interesting mechanisms used by methylotrophic yeast
to survive at the phyllosphere, and how both autophagy and
pexophagy are used to adapt to changes in environmental
methanol dynamics, providing insight into plant-microbe
The common conclusion of the studies mentioned above
is that pexophagy represents an important mechanism for
survival and development under changing environmental
conditions. Peroxisomes represent highly dynamic struc-
tures: Their biomass can easily be increased when peroxiso-
mal functions are needed for specialized metabolic pathways
or breakdown of damaging ROS, but they are quickly recy-
cled when conditions change and they are not essential, so
molecular building blocks and energy resources can be pro-
vided for alternative cellular functions.
Taken together, these studies point to a pivotal role of
tant plant and human pathogens.
to unraveling the molecular mechanisms contributing to
pexophagy in various organisms, several aspects still remain
to be resolved. The physiological role of pexophagy in model
organisms such as yeast cells is still a matter of debate.
With few exceptions, knockout of genes specifically involved
in yeast pexophagy does not necessarily result in reduced
may rather be associated with quick adaptation to changing
environmental conditions and thus may only emerge under
under peroxisome proliferation conditions. In line with this
view,pexophagy in other organismsappearsto playa rolefor
removal and recycling of unwanted or nonessential peroxi-
somes under condition when the cell is in need of molecular
building blocks for alternative pathways, for example, for
vital morphogenesis and development. In addition, the role
of peroxisome turnover and linked changes in cellular redox
state with cellular aging processes is increasingly recognized
and warrants further investigation.
Although recent advances have pointed to a formerly
unrecognized role of specific signal transduction pathways
for the regulation of pexophagy (and mitophagy), the mo-
lecular framework of this process still remains to be eluci-
Which are the pivotal targets of the protein kinase activity
in this context, and how do these contribute to pexophagy
regulation? Is there a molecular link between mitophagy and
pexophagy? Moreover, it is not known if this mechanism
is restricted to yeasts or if other organisms share the same
regulatory circuits and if they have functional homologs of
all the selectivity factors. Future work will therefore focus
on elucidating the underlying conserved (or distinct) mech-
The identity and mode of action of autophagy adaptors
for yeast pexophagy is one major aspect of current research
efforts. While Atg30 has been identified as an Atg11-binding
pexophagy adaptor, the identity of the (proposed) Atg8-
binding partner remains unresolved. In addition, while the
requirement of phosphorylation for adaptor protein binding
to Atg11 is well established [34, 96], it has not been ad-
dressed yet to what extent other posttranslational modifica-
tions, such as ubiquitination and/or alternative processing,
Indeed, phosphorylation events in close proximity to Atg8-
interaction motifs (AIMs) or LC3-interacting regions (LIRs)
in the mammalian adaptor protein optineurin (OPTN) have
been suggested as an important regulatory mechanism in
xenophagy . Unraveling the corresponding mechanism
in yeast and mammalian pexophagy therefore represents an
The origin of membrane material for the autophago-
some/phagophore membrane is still an unanswered ques-
tion. Several current models argue for a contribution of the
ER to provide membrane lipids and structural components.
Sar1, an ER protein required for the secretory pathway, has
been shown to have a role in pexophagosome formation, but
the data do not unambiguously show that the pexophago-
some membrane derives from the ER . In addition, we
have previously shown that Atg17 trafficks from the periph-
eral ER and colocalizes with Atg35, which regulates MIPA
formation  but there is not a decisive mechanism of
membrane trafficking as of yet.
In addition, the subcellular sorting mechanisms, which
would be required to facilitate this process of membrane re-
cruitment, are largely unknown yet recent advances towards
membrane expansion and the requirement of SNAREs for
autophagosome formation provide some insights .
fusion events are orchestrated during pexophagy.
The role of cytoskeleton components for pexophagy is
not yet fully understood. While pexophagy in yeast cells re-
quires the actin skeleton , it appears that pexophagy
in mammalian cells is dependent on tubulin-mediated in-
teraction of LC3 family members with peroxisomal mem-
brane proteins such as Pex14 . Another form of se-
lective autophagy, xenophagy of intracellular Listeria and
14International Journal of Cell Biology
Salmonella, relies on components of the actin skeleton, a
process mediated by the increasingly characterized class of
septin proteins [135, 136]. Further work is needed to deci-
pher the contribution of cytoskeleton elements and septins
for different selective autophagy pathways.
While present studies have focused on experimental sys-
tems wherein pexophagy is induced by peroxisome prolifer-
ation followed by different starvation conditions, it will be a
challenging task to analyze shared and distinct mechanisms
for the degradation of damaged peroxisomes. Recent exper-
iments have provided inroads to examine damage-induced
pexophagy by destabilization of peroxisome membrane pro-
teins . Whether or not this interesting finding relates
to physiological processes, and if the same mechanism is
conserved in other yeasts and higher eukaryotes, remains to
the infection cycle of important viral pathogens (e.g., HIV,
influenza, and rotavirus) on the one hand, and the contri-
bution of peroxisomes to viral detection and innate immune
responses on the other hand [137, 138], it will be of utmost
importance to define the role of pexophagy in the context of
these important human pathogens.
Future work will shed light on these and other unan-
swered questions addressing the molecular basis of peroxi-
to further our understanding of selective autophagy in gen-
AIM: Atg8-Interaction Motif
CHO: Chinese Hamster Ovary cells
Cvt: Cytosol-to-Vacuole Targeting
DEHP: Di-(2-ethylhexyl) phthalate
FYCO1: FYVE and coiled-coil domain-containing 1
GRAM: Domain found in glycosyltransferases, Rab-like
GTPase activators and myotubularins
MAPK: Mitogen Activated Protein Kinase
MIPA: Micropexophagy-specific membrane Apparatus
PAS:Phagophore Assembly Site
PBD: Peroxisomal Biogenesis Disorders
PD: Peroxisomal Disorders
PED: Peroxisomal Enzyme/Transporter Deficiencies
PM: Phagophore Membrane
PMP: Peroxisomal Membrane Protein
PPAR: Peroxisome-Proliferator Activated Receptor
PTS: Peroxisomal Targeting Signal
TbHK1: Trypanosoma brucei peroxisomal Hexokinase
VSM: Vacuole Sequestering Membrane.
Reactive Oxygen Species
Conflict of Interests
The authors declare no competing financial interests.
The authors would like to thank lab members Jean-Claude
Farr´ e, Taras Nazarko, and Katharine Ozeki for critically
reviewing this paper. This work was supported by Grants
GM069373 (to S. Subramani), a DFG (German Research
Foundation) fellowship (Ti-640 1-1, to A. Till) and a Seed
Grant from the San Diego Center of Systems Biology
(SDCSB, to A. Till and S. Subramani). S. F. Burnett was
supported by NSF GK-12 STEM Fellows in Education
Grant 0742551 to Maarten Chrispeels and by funds from a
Chancellors Associates Chair to S. Subramani.
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