T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 171, No. 4, November 21, 2005 603–614
The Rockefeller University Press$8.00
p62/SQSTM1 forms protein aggregates degraded
by autophagy and has a protective effect on
huntingtin-induced cell death
and Terje Johansen
Biochemistry Department, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway
Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
utophagic degradation of ubiquitinated protein
aggregates is important for cell survival, but it is
not known how the autophagic machinery rec-
ognizes such aggregates. In this study, we report that po-
lymerization of the polyubiquitin-binding protein p62/
SQSTM1 yields protein bodies that either reside free in
the cytosol and nucleus or occur within autophagosomes
and lysosomal structures. Inhibition of autophagy led to
an increase in the size and number of p62 bodies and
p62 protein levels. The autophagic marker light chain 3
(LC3) colocalized with p62 bodies and coimmunoprecipi-
tated with p62, suggesting that these two proteins par-
ticipate in the same complexes. The depletion of p62 in-
hibited recruitment of LC3 to autophagosomes under
starvation conditions. Strikingly, p62 and LC3 formed a
shell surrounding aggregates of mutant huntingtin. Reduc-
tion of p62 protein levels or interference with p62 func-
tion significantly increased cell death that was induced by
the expression of mutant huntingtin. We suggest that p62
may, via LC3, be involved in linking polyubiquitinated
protein aggregates to the autophagy machinery.
Several reports have described p62, which is also named se-
questosome 1 (SQSTM1), as a common component of protein
aggregates that are found in protein aggregation diseases af-
fecting both the brain and the liver. These include Lewy bodies
in Parkinsons disease, neurofibrillary tangles in Alzheimer’s
disease, and huntingtin aggregates (Kuusisto et al., 2001a,
2002; Zatloukal et al., 2002; Nagaoka et al., 2004). In the liver
Mallory bodies, hyaline bodies in hepatocellular carcinoma and
1 antitrypsin aggregates contain p62 (Zatloukal et al., 2002).
All of these aggregates contain polyubiquitinated proteins. The
440–amino acid–long p62 protein has an NH
and Bem1p (PB1) domain followed by a ZZ type zinc finger
domain, a PEST region containing putative phosphorylation
sites, and a COOH-terminal ubiquitin-associated
main (Geetha and Wooten, 2002). The latter domain binds
ubiquitin noncovalently (Vadlamudi et al., 1996). This raises
the possibility that p62 could be recruited to ubiquitinated pro-
tein aggregates as a result of its ability to bind polyubiquitin via
the UBA domain (Donaldson et al., 2003). The NH
PB1 domain is used both for the polymerization of p62 and for
binding to other proteins containing PB1 domains (Gong et al.,
1999; Sanz et al., 1999, 2000; Avila et al., 2002; Cariou et al.,
2002; Lamark et al., 2003).
The p62 protein level increases after oxygen radical
stress. Both mRNA and protein levels increase, suggesting an
induced transcription of the gene (Ishii et al., 1997). The tran-
scription factor Nrf2 is activated after oxidative stress, and in-
duction of p62 is severely inhibited in cells from Nrf2 knockout
mice (Ishii et al., 2000). Inhibition of proteasomal activity also
causes induction of p62 (Ishii et al., 1997; Kuusisto et al.,
2001b; Thompson et al., 2003). Interestingly, p62 was recently
identified as a protein that is induced as a response to the ex-
pression of mutant huntingtin (Nagaoka et al., 2004). Hunting-
ton’s disease is a late onset progressive autosomal dominant
neurodegenerative disorder caused by the expression of mutant
forms of the huntingtin (Htt) protein containing a polyglu-
tamine expansion encoded by CAG repeats in exon 1 of the
gene (Vonsattel and DiFiglia, 1998). The disease
causes selective neuronal cell death in the striatum. Cells ex-
pressing the mutant form of huntingtin display both diffuse and
Correspondence to Terje Johansen: email@example.com
Abbreviations used in this paper: EEA1, early endosome antigen 1;
light chain 3; PB1, Phox and Bem1p; siRNA, small interfering RNA; UBA;
The online version of this article contains supplemental material.
JCB • VOLUME 171 • NUMBER 4 • 2005604
aggregated localization of the protein. The mutant protein has
cytotoxic properties, and aggregation seems to be a mechanism
for cell survival (Arrasate et al., 2004). Protein inclusions
formed by aggregate-prone proteins with polyglutamine and
polyalanine expansions are degraded by macroautophagy
(hereafter referred to as autophagy; Kegel et al., 2000; Raviku-
mar et al., 2002, 2004), which is a bulk degradation pathway in
which a double or multimembrane-bound structure called the
autophagosome forms to sequester cytoplasm. Subsequently,
the autophagosome fuses with the lysosome, and its content
and internal membranes are degraded as it recycles the macro-
molecules (Levine and Klionsky, 2004; Yoshimori, 2004).
Most long-lived proteins and some organelles are degraded by
autophagy, and autophagy, in addition to cellular homeostasis,
has also been implicated in cellular differentiation, tissue re-
modelling, growth control, bacterial and viral infections, cell
defense, adaptation to adverse environments, neurodegen-
erative diseases, cardiomyopathies, apoptosis, and cancer
(Cuervo, 2004). Among the autophagosomal marker proteins
are Atg8 in yeast and light chain 3 (LC3) in mammals (Kabeya
et al., 2000). After synthesis, LC3 is cleaved at its COOH ter-
minus to produce the cytosolic LC3-I form. LC3-I is converted
to LC3-II, which is tightly associated with the autophagosomal
membrane probably via conjugation to phosphatidylethanola-
mine (Kabeya et al., 2000, 2004).
In this study, we report that the polyubiquitin-binding
and homopolymerizing p62 protein may, via LC3, be in-
volved in linking polyubiquitinated protein aggregates to the
autophagic machinery, facilitating the clearance of such aggre-
gates and, thereby, contributing to reduced toxicity of mutant
The p62 bodies in the cytoplasm of HeLa
cells are ubiquitin-containing protein
The p62 protein has been reported to be present in several types
of cytoplasmic inclusions in neurodegenerative diseases and
other protein aggregation diseases (Zatloukal et al., 2002). In a
previous study, we noted that endogenous as well as ectopi-
cally expressed p62 was present in numerous round bodies in
the perinuclear area of human HeLa cells (Lamark et al., 2003).
By studying this further using both a monoclonal and poly-
clonal antibody, the p62 protein was found to be located in the
cytoplasm, with weak staining in the nucleus in
cells (Fig. 1 A). In the cytoplasm, the protein is enriched in
round bodies with a diameter ranging from the detection limit
of the confocal microscope (0.1
seemed to be of two distinct types: a high number of faint, 0.1–
m-diameter bodies and 5–10 times more intense bodies
ranging from 0.5 to 1
m in diameter. Interestingly,
the cell population contained large p62 bodies that were 1–2
in diameter. In
15% of the cells, fluorescence intensities in the
cytoplasm and nucleus were similar. Nuclear p62 was enriched
in speckles or bodies, with a minority of the cells (
playing a nuclear accumulation of p62, and 0.5–1-
85% of the
m) to 2
m. These structures
p62 bodies were stained 5–10 times more intensely than the
diffuse nuclear p62. In broad terms, this pattern of p62 local-
ization was observed in a wide range of cell lines, including
HeLa, HEK293, MDCK II, U2OS, A431, HT1080, TERT fi-
broblasts, NIH3T3 fibroblasts, and several human neuroblas-
containing protein aggregates. (A) HeLa cells were fixed and stained for
p62. The different patterns of p62 distribution were scored in ?200 cells
as cytoplasmic with small bodies, cytoplasmic with large and intense bod-
ies, equal nuclear and cytoplasmic staining, and nuclear enriched. The
percentage of cells in the respective groups is indicated. (B) Expression
level of GFP-p62 in HeLa cells stably expressing GFP-p62 (S–GFP-p62)
compared with the level of endogenous p62 in the parent HeLa cells.
(C) Cells stably expressing GFP-p62 display a cytoplasmic punctuate lo-
calization of GFP-tagged p62 similar to endogenous p62. Video micros-
copy of live cells demonstrated that the small, faint bodies (thin arrows)
displayed directed migration, whereas the majority of the larger, intense
bodies (thick arrows) were nonmigratory (Videos 1 and 2, available at
http://www.jcb.org/cgi/content/full/jcb.200507002/DC1). A still im-
age from Video 1 is shown here. (D) Bodies containing either endogenous
p62 or transiently or stably transfected GFP-p62 all contain polyubiquitin.
HeLa cells were fixed and stained with p62 and polyubiquitin (clone FK1)
mAbs directly coupled with AlexaFluor555 (red) and AlexaFluor488
(green), respectively. Cells expressing GFP-p62 were only stained for
polyubiquitin (red). Bars, 20 ?m.
The p62 bodies in the cytoplasm of HeLa cells are ubiquitin-
62 IN AUTOPHAGIC PROTEIN DEGRADATION • BJØRKØY ET AL. 605
toma cell lines. Although the reason for heterogeneous p62 dis-
tribution is not known, it is evident that p62 bodies in one form
or another are widespread among various cell types.
We generated a HeLa cell line (stable [S] GFP-p62) that
stably expressed a moderate level of the GFP-p62 fusion pro-
tein at a level that was about fourfold higher than in the parent
cell line (Fig. 1 B). The S–GFP-p62 cell line displayed all four
distributions depicted in Fig. 1 A, but the population of large,
round bodies was increased from 5 to 40%. Video confocal mi-
croscopy of the GFP-tagged p62 fusion protein also suggested
that the p62 bodies could be divided into two populations (Fig.
1 C and Videos 1 and 2, available at http://www.jcb.org/cgi/
content/full/jcb.200507002/DC1): a high number of faint, mo-
bile bodies (most of them smaller than 0.3
m) with low or no mobility. Notably, these larger
and more intensely labeled structures were equally fluorescent
throughout the whole structure, suggesting that they are not
vesicles. These bodies seemed to grow by fusion, and their
size and intensity increased with increasing expression levels.
Altogether, these results suggest that p62 bodies represent two
different populations differing in size, intensity, and mobility.
Importantly, as far as we could observe by confocal fluores-
cence microscopy, all p62 bodies contained ubiquitin (Fig. 1 D).
m) and larger bod-
This is consistent with the ability of p62 to bind to polyubiq-
uitin via its UBA domain.
Both the PB1 and UBA domains are
needed for p62 to form cytoplasmic
To map the domains of p62 involved in the formation of cyto-
plasmic bodies, different GFP-p62 cDNA constructs were tran-
siently transfected into NIH3T3 cells, and the fusion proteins
were analyzed by confocal fluorescence microscopy (Fig. 2 A).
NIH3T3 cells were used because they have a low level of en-
dogenous p62, but similar data were obtained with HeLa cells
(not depicted). All deletion constructs lacking the PB1 domain
(i.e., p62(124–440), p62(256–440), and p62(385–440)) have a
completely diffuse distribution in NIH3T3 cells (Fig. 2 A and
not depicted). This is also the case with the D69A mutant,
which abrogates PB1 domain–mediated polymerization of p62
(Fig. 2 A; Lamark et al., 2003). This strongly indicates that the
PB1 domain–mediated polymerization of p62 is essential for
the formation of cytoplasmic bodies.
Given its ability to bind to polyubiquitin, the UBA domain
may be needed for the formation of cytoplasmic bodies. In sup-
port of this, we found that a construct lacking the UBA domain
needed for p62 to form cytoplasmic bodies. (A) The
indicated deletion constructs were fused COOH ter-
minal to GFP and expressed in NIH3T3 fibroblasts.
Asterisks indicate point mutations in the PB1 (D69A)
and UBA (I431A) domains, respectively. (B) S–GFP-
p62 cells were transiently transfected with the indi-
cated myc-tagged p62 constructs, fixed, and stained
for the myc tag. Bars, 20 ?m.
Both the PB1 and UBA domains are
JCB • VOLUME 171 • NUMBER 4 • 2005 606
(p62(1–385)) and the I431A mutant, which affects the folding of
the UBA domain, both showed a diffuse localization in trans-
fected NIH3T3 cells (Fig. 2 A). The isolated PB1 domain of
p62(1–122) formed large bodies (Fig. 2 A), as did a construct en-
compassing amino acids 1–256 (not depicted). However, we
consistently observed that the structures formed by these trun-
cated p62 constructs were distinct, with a less regular shape
than the round bodies formed by full-length p62. In contrast,
a construct containing only the PB1 and UBA domains
123–385) formed bodies that were indistinguishable from
those formed by full-length p62. These data suggest that both the
PB1 and UBA domains are necessary and sufficient for the lo-
calization of full-length p62 into the ubiquitin-containing bodies.
In vivo, the length of p62 polymers may be reduced as a
result of the binding of p62 to other PB1 domain–interacting
partners. In line with this, overexpression of p62 D69A (Fig.
2 B) or p62 R21A (not depicted) counteracted the formation
of GFP-p62 bodies in S–GFP-p62 cells. These PB1 domain
mutants presumably compete with wild-type p62 for binding to
a growing chain of p62 molecules acting as chain terminators.
However, overexpression of the p62
p62(1–385) also prevented the formation of cytoplasmic dots
in S–GFP-p62 cells (Fig. 2 B). In contrast to PB1 mutants, de-
letion of the UBA domain had no effect on the ability of p62 to
interact with itself or to form polymers in vitro (unpublished
data). Only the PB1 domain is needed for polymerization of
p62 in vitro. Therefore, the role of the UBA domain may be to
cross-link p62 polymers, presumably by interacting with poly-
UBA construct myc-
p62 bodies are found both as membrane-
free protein aggregates (sequestosomes)
and as membrane-confined
autophagosomal and lysosomal structures
To characterize the two populations of p62 bodies, we per-
formed a series of colocalization experiments in both fixed and
live cells. We looked at both endogenous p62 and ectopically
expressed GFP-p62 or epitope-tagged p62. There was no colo-
protein aggregates and as membrane-confined autophago-
somal and lysosomal structures. (A) Localization of bodies
containing endogenous p62 or transiently expressed GFP-
p62 relative to EEA1-positive early endosomes. Endogenous
p62 were stained green using p62 antibodies directly la-
beled with AlexaFluor488, and EEA1 was stained red with
EEA1 mAbs directly labeled with AlexaFluor555. Alterna-
tively, transiently expressed GFP-p62 was expressed in HeLa
cells, and EEA1 was stained red with EEA1 mAb (bottom).
The boxed area is shown to the right at a higher magnifica-
tion. (B) Colocalization of GFP-p62 and CD63 (stained red
using a mAb) in HeLa cells stably expressing GFP-p62. (C)
Immunoelectron micrograph of S–GFP-p62 stained with a
GFP pAb (10-nm gold particle, arrows) and monoclonal
CD63 (15-nm gold particle, arrowheads). (D) Rapid deter-
gent extraction of GFP-p62 from LysoTracker-positive acidic
organelles. HeLa cells transiently expressing GFP-p62 were
labeled with LysoTracker for 60 min. The detergent extrac-
tions were imaged in a time series with 15-s time intervals
after adding 1% Triton X-100. The boxed area is shown in
the bottom two images at a higher magnification before and
after detergent extraction. Bars (A, B, and D), 20 ?m.
p62 bodies are found both as membrane-free
62 IN AUTOPHAGIC PROTEIN DEGRADATION • BJØRKØY ET AL. 607
calization of early endosomal markers with p62 bodies. Early
endosome antigen 1 (EEA1) did not colocalize with endoge-
nous p62 or ectopically expressed GFP-p62 (Fig. 3 A). In line
with this, there was no colocalization between p62 and EGF re-
ceptor (fixed cells) or fluorescent-labeled EGF (live cells) at
time points up to 15 min of internalization (unpublished data).
However, at 30 and 60 min, there was a minor fraction of the
smaller p62 bodies that colocalized with EGF receptor or its
ligand (unpublished data). Moreover, there was no association
of GFP-p62 with the recycling compartment that was visual-
ized by internalization of fluorescently labeled transferrin (un-
published data). Thus, these data suggested that the weakly
stained smaller p62 bodies could be late endosomes or lyso-
somes. CD63 is often used as a marker for the late endosomes/
lysosomes and is highly enriched in multivesicular endosomes
(Escola et al., 1998). In S–GFP-p62 cells, immunostaining with
an anti-CD63 antibody revealed colocalization with a fraction
of the smaller GFP-p62 structures (Fig. 3 B). The large p62
bodies were consistently negative for CD63. By performing
immuno-EM with antibodies against CD63 and GFP, vesicles
containing both CD63 and GFP-p62 were identified, although
CD63 and GFP-p62 were also found in separate compartments
within the same vesicular structures (Fig. 3 C).
To further analyze whether p62 partly localizes to the
late endosome/lysosomal compartment, we took advantage of
the fluorescent dye LysoTracker, which displays intense flu-
orescence when it faces the acidic environment in the late en-
dosomal/lysosomal compartment. As shown in Fig. 3 D,
LysoTracker labeled a subfraction of the p62 bodies, and these
structures were rapidly lost by detergent extraction, whereas
the intense bodies were detergent resistant. As a control, we
did the same experiment with GFP-tagged p40phox, which,
similar to p62, contains a PB1 domain but is located in endo-
somes. As expected, p40phox was extracted from the endo-
somes at the same time as the LysoTracker fluorescence was
lost (Fig. S1, available at http://www.jcb.org/cgi/content/full/
We were not able to detect endogenous p62 by immuno-
EM. However, by performing immuno-EM on S–GFP-p62
cells, we observed both membrane-free protein aggregates (se-
questosomes) and membrane-surrounded p62 bodies (Fig. 4 A).
To further characterize the sequestosomes on the ultrastructural
level, we performed correlative immunofluorescence and EM.
Proteasomal inhibitors such as PSI induce a prominent increase
in the amount of p62 protein in cells (Kuusisto et al., 2001b;
Thompson et al., 2003). We took advantage of this to increase
the size of endogenous p62 bodies in HeLa cells to facilitate
EM/immunofluorescence studies. The results show that the
large and intensely fluorescent p62 structures are membrane-
free protein aggregates that are not related to endocytic vesicles
(Fig. 4 B). The aggregates had a filamentous appearance and
seemed to exclude any cytosolic material. Both in the stably
transfected cell line and in transiently transfected HeLa cells,
p62 was found within double membrane structures that are in-
dicative of autophagosomes (Fig. 4 C). Autophagic structures
and autolysosomes represented the dominant fraction of p62
bodies in S–GFP-p62 cells.
p62 bodies are degraded by autophagy
The presence of GFP-p62 within autophagosomes suggests that
p62 bodies are degraded by autophagy. Pulse-chase experi-
ments in which cells were added to
dicate a half-life of p62 in HeLa cells of
the radiolabeled protein was lost after 24 h (Fig. 5 A). The pro-
S-labeled methionine in-
6 h, and almost all of
solic aggregates/sequestosomes. (A) Immuno-EM of GFP-
p62. S–GFP-p62 cells were labeled with rabbit anti-GFP
(Abcam) followed by protein A–gold (15 nm). We observed
labeling in membrane-free cytosolic structures and sequesto-
somes (arrows) as well as in endosomes (Endo). (B) Correlative
immunofluorescence/EM of HeLa cells treated with 10 ?M PSI
for 5 h displaying typical sequestosomes. The insets show
two magnifications of the sequestosome, which is labeled 1.
(C) Representative image of a p62-containing autophago-
some. HeLa cells transfected with GFP-p62 were immunogold
labeled as in A. Note the cisternal-like membrane (arrow-
heads) surrounding the GFP-positive material. The arrow indi-
cates a fused endosome.
p62 is found both in autophagosomes and in cyto-
JCB • VOLUME 171 • NUMBER 4 • 2005608
tein synthesis inhibitor cycloheximide is known to cause a
drastic reduction in autophagy-induced protein degradation
(Lawrence and Brown, 1993; Abeliovich et al., 2000). Consis-
tently, the level of p62 protein was stable in cells treated with
cycloheximide for as long as 24 h (Fig. 5 A).
To further explore the relationship between autophagy
and p62 degradation, cells were treated with rapamycin, which
is an inducer of autophagy (Noda and Ohsumi, 1998), or ba-
filomycin A1. Bafilomycin A1 is an inhibitor of the vacuolar
ATPase, which blocks the fusion of autophagosomes with lyso-
somes, leading to an accumulation of autophagosomal struc-
tures (Yamamoto et al., 1998). Rapamycin treatment caused a
slight decrease of endogenous p62, whereas treatment with ba-
filomycin A1 for 18 h resulted in an accumulation of endoge-
nous p62 in HeLa cells (Fig. 5 B). It also resulted in an increase
of both endogenous and GFP-tagged p62 in S–GFP-p62 cells
(Fig. 5 B). LC3 is involved during the late steps of autophagy
after the isolation membrane has formed (Klionsky, 2005). The
unmodified LC3-I form is cytosolic, whereas LC3-II is pre-
sumably covalently attached to phosphatidylethanolamine at its
COOH terminus and is tightly bound to autophagosomal mem-
branes, serving as an important marker for autophagy (Kabeya
et al., 2000, 2004; Mizushima, 2004). A Western blot with an
antibody against LC3 and the highest affinity for the LC3-II
form (Kabeya et al., 2000) revealed a strong induction of LC3-II
upon treatment of HeLa cells and S–GFP-p62 cells with bafilo-
mycin A1 (Fig. 5 B). Interestingly, the background level of
LC3-II is higher in S–GFP-p62 cells than in the parent HeLa
cells, suggesting a higher autophagic activity in these cells
(Fig. 5 B). Consistent with the Western blot data (Fig. 5 B), im-
munostaining revealed an extensive accumulation of small p62
m) in HeLa cells that were treated with ba-
filomycin A1 (Fig. 5 C). Similarly, GFP-p62–containing dots
accumulated in S–GFP-p62 cells upon treatment with bafilo-
mycin A1 (Fig. 5 D). Treatment of cells with 3-methyladenine,
which inhibits the sequestration step during autophagy, also re-
sulted in an accumulation of p62 in cytoplasmic dots, although
the effect was less pronounced than that of bafilomycin A1
(Fig. 5 D).
Presently, there are no antibodies available that allow im-
munostaining of endogenous LC3 in mammalian cells, and
overexpressed myc-LC3 could only be detected using anti-myc
antibodies. After transient transfection, myc-LC3 was present
in a large fraction of the cytoplasmic bodies formed by endoge-
nous p62 in HeLa cells (not depicted), and it also strongly colo-
calized with GFP-p62 in S–GFP-p62 cells (Fig. 5 E). Generally,
it was difficult to find myc-LC3–positive dots that did not also
contain p62. It should be noted that in HeLa cells, autophago-
somes are small and are commonly visualized as dots by fluo-
rescence microscopy (Mizushima, 2004). In cells treated with
bafilomycin A1, there was an extensive accumulation of cyto-
plasmic bodies containing both p62 and myc-LC3 (unpub-
lished data). Coexpression of GFP-LC3 with p62 fused to a
novel, very bright, red fluorescent protein, tdTomato (Shaner et
al., 2004), enabled the visualization of p62-LC3–positive bod-
ies in living cells. Video confocal microscopy of HeLa cells
coexpressing tdTomato-p62 and GFP-LC3 showed that many
of the punctuate structures containing p62 and LC3 had a high
mobility (Video 3, available at http://www.jcb.org/cgi/content/
LC3 is associated with the isolation membrane during
its formation and remains on the membrane after a spherical
autophagosome has formed. Transiently overexpressed GFP-
LC3 has, therefore, been shown to be a very good marker for
p62 degradation using pulse-chase labeling with 35S-methionine and im-
munoblotting after cycloheximide (CHX) treatment. HeLa cells were pulsed
with 35S-methionine and incubated for the indicated times in nonradioactive
medium. After immunopurification, the amount of radioactive p62 was
determined by autoradiography, and the total amount of p62 was deter-
mined by an immunoblot of the same membrane. The p62 level in total
cellular lysates after different times of 10 ?g/ml cycloheximide treatment
determined by immunoblotting (bottom). (B) The levels of p62 and LC3-II
change with autophagic activity. HeLa or S–GFP-p62 cells were either left
untreated or rapamycin (10 ?g/ml) or bafilomycin A1 (Baf. A1; 10 ?g/ml)
was added for 18 h. Immunoblots were sequentially probed using LC3,
p62, and actin antibodies. (C and D) The amount of p62 located to cyto-
plasmic bodies increases upon inhibition of autophagy. HeLa cells or S–GFP-
p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h,
the cells were fixed, and p62 was either stained red using a p62 mAb (C)
or imaged directly (D). Nuclei were visualized using the Draq5 DNA
stain. The settings for imaging were identical for the treated cells and the
untreated control. (E) The majority of S–GFP-p62 cytoplasmic bodies are
stained with antibodies recognizing transiently expressed LC3. S–GFP-
p62 cells were transiently transfected with myc-LC3. Myc-LC3 was stained
red using an anti-myc tag mAb. The boxed area indicates the part of the
cell that is shown to the left at a higher magnification. Bars, 5 ?m.
p62 bodies are degraded by autophagy. (A) Comparison of
62 IN AUTOPHAGIC PROTEIN DEGRADATION • BJØRKØY ET AL.609
autophagy, as its localization changes from diffuse to a punctu-
ate or dotted pattern when autophagy is induced (Mizushima,
2004). GFP-LC3 dots represent isolation membranes and au-
tophagosomes (Mizushima, 2004). Amino acid starvation in-
duces autophagy and results in a transient increase in the number
of autophagosomes. In line with this, we found that the fraction
of HeLa cells with GFP-LC3 in punctuated structures increased
from 27 to 53% after amino acid starvation in Hanks medium
for 60 min. Almost all of the LC3-positive bodies stained posi-
tive for endogenous p62 (Fig. 6 A). Interestingly, the redistribu-
tion of overexpressed LC3 into punctuated structures appeared
to depend on the presence of p62. In cells transfected twice
with small interfering RNA (siRNA) to deplete endogenous
p62, very few cells contained punctuated GFP-LC3 structures
(Fig. 6 A). In contrast, cooverexpression of HA-p62 strongly
increased the frequency of cells with punctuated GFP-LC3
(Fig. 6 A). Cooverexpression of the D69A mutant, which in-
hibits PB1 domain–mediated polymerization, resulting in a dif-
fuse localization of p62 (Lamark et al., 2003), also resulted in a
diffuse localization of GFP-LC3 (Fig. 6 A). Consistent with a
direct or indirect association between GFP-LC3 and p62, both
endogenous p62 and overexpressed HA-p62 coimmunopre-
cipitated with GFP-LC3 from HeLa cell extracts (Fig. 6 B).
When the D69A mutant was overexpressed, less p62 was
coimmunoprecipitated because polymers of p62 were not
formed. However, when a p62 mutant lacking the UBA do-
main was coexpressed with GFP-LC3, both endogenous p62
UBA were efficiently coimmunoprecipitated (Fig. 6 B).
Together, the aforementioned results suggest a close associa-
tion between LC3 and p62 bodies and that a large fraction of
p62 bodies are degraded by autophagy.
p62 forms a shell around huntingtin
The p62 protein is associated with protein aggregates in a num-
ber of aggregation diseases (Kuusisto et al., 2001a; Zatloukal et
al., 2002; Nagaoka et al., 2004). However, the role of p62 in
the handling of such aggregates is unknown. To start analyzing
the possible functional role of p62, we chose an established
model in which we expressed an NH
(amino acids 1–171) of huntingtin containing a 68–amino acid
polyglutamine expansion (N-HttQ68) that was found to be caus-
ative for disease development (Saudou et al., 1998). Expression
of Flag-tagged N-HttQ68 caused the formation of cytoplasmic
30% of the expressing cells, with a strong
colocalization of p62 and N-HttQ68 in these aggregated struc-
tures (Fig. 7 A). These structures also contained ubiquitin
(Fig. S3, available at http://www.jcb.org/cgi/content/full/
jcb.200507002/DC1). In the rest of the cells, the N-HttQ68
protein was diffusely distributed in the cytosol. The cells con-
taining huntingtin aggregates also seemed to express high lev-
els of p62, whereas the cells containing diffuse huntingtin did
not show an increase in immunostained p62. Confocal micros-
copy at higher resolution revealed that p62 apparently formed a
shell surrounding GFP–N-HttQ68–containing aggregates (Fig.
7 B). However, a possibility could be that antibodies may not
penetrate to the core of the aggregate, giving an illusion of a
p62-containing shell. Thus, to test this more rigorously, we ex-
pressed DsRed2-tagged p62 together with GFP–N-HttQ68 and
observed live cells in the confocal microscope. With this
strategy, it was clear that DsRed-p62 formed a shell surround-
ing the huntingtin aggregate (Fig. 7 C). The DsRed2 protein
alone did not show any association with the huntingtin aggre-
gates (Fig. S2, available at http://www.jcb.org/cgi/content/full/
structures in HeLa cells. (A) HeLa cells transiently transfected with GFP-LC3
alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A
were either left in normal medium or starved for amino acids for 1 h. The
cells were fixed, and p62 was stained using a p62 mAb. More than 200
randomly selected cells for each condition were scored for the cytoplasmic
pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-
LC3–positive cells with punctuate localization are indicated to the right. (B)
Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a
UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from
HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cel-
lular extracts after cotransfecting the indicated constructs. The cell cultures
were either left untreated or starved for amino acids for 1 h as indicated.
Copurified endogenous or ectopically expressed myc-tagged p62 constructs
were detected using the p62 mAb. Bars, 20 ?m.
p62 is required for the formation of GFP-LC3–positive punctuated
JCB • VOLUME 171 • NUMBER 4 • 2005610
jcb.200507002/DC1). Similarly, when p62 was fused to the
nonaggregating, very brightly fluorescing tdTomato, many
structures with p62 surrounding GFP–N-HttQ68 aggregates
were observed (Fig. 7 D). Stably expressed GFP-tagged p62
could also be seen to enclose aggregates of transiently ex-
pressed Flag-HttQ68 (not depicted). It has previously been
shown that huntingtin aggregates are degraded by autophagy
(Ravikumar et al., 2002, 2004). Interestingly, we found that
myc-LC3 protein localized to structures containing GFP-p62
and Flag–N-HttQ68 in S–GFP-p62 cells (Fig. 7 E, top). By co-
expressing Flag–N-HttQ68 and tdTomato-LC3, we were able
to show that LC3 was part of the shell surrounding the huntingtin
aggregates (Fig. 7 E, bottom). We speculate that a p62/LC3-
containing shell around huntingtin aggregates might serve to
mark these for autophagic degradation.
p62 shows a protective effect against cell
death that is induced by overexpression
of polyglutamine-expanded huntingtin
Expression of N-HttQ68 caused the induction of apoptosis,
which was scored as GFP-positive, rounded, and detached
cells that displayed condensed or fragmented nuclei (Fig. 8 A).
Expression of wild-type N-HttQ17 gave the same low level of
apoptosis as the GFP control (not depicted). The percentage of
HttQ68 that induced cell death after 48 h of expression varied
from experiment to experiment, ranging from 17 to 46% of the
GFP-HttQ68–transfected HeLa cells with a mean of 29% in
five independent experiments. We consistently observed that
reduction of endogenous p62 levels by expression of p62 anti-
sense RNA increased apoptosis of GFP–N-HttQ68–expressing
cells. There was also a less pronounced but significant increase
of apoptosis in control cells expressing GFP alone (Fig. 8 B).
Also, expressing siRNA against p62 caused a reduced level of
endogenous p62 and a corresponding increase in apoptosis after
expression of mutant huntingtin (unpublished data). Interestingly,
coexpression of a deletion construct of p62 lacking the UBA
UBA) gave a potent increase in the frequency of
huntingtin-expressing apoptotic cells (Fig. 8 B). These findings
were observed in both HeLa cells and the neuroblastoma cell
line SHSY-5Y. Because the depletion of p62 by antisense RNA
or siRNA and interference with the formation of p62 bodies by
UBA overexpression increased huntingtin-induced cell
death, we reasoned that increasing p62 levels by overexpressing
p62 might have a protective effect. We could indeed observe a
small protective effect in every experiment we performed (Fig.
8 B), but HeLa cells already have a reasonably high level
of p62 expression, and mere overexpression could simply di-
rect p62 into sequestosomes. Altogether, these results indicate
that p62 protects against huntingtin-induced cell death.
Proteins are degraded via two main pathways in eukaryotic
cells. Short-lived proteins are degraded by the proteasome,
whereas long-lived proteins are degraded by autophagy. Protein
aggregates that form during oxidative stress and other conditions,
leading to misfolding and aggregation of proteins such as poly-
HeLa cells transiently expressing a Flag-tagged NH2-terminal fragment
(amino acids 1–171) of huntingtin containing a 68-polyglutamine expan-
sion, Flag–N-HttQ68. After 48 h, the cells were fixed, and Flag–
N-HttQ68 was stained green using a Flag mAb. Endogenous p62 was
stained red using the p62 pAb. (B) Endogenous p62 forms a shell around
aggregated GFP–N-HttQ68. Transiently transfected HeLa cells were fixed
48 h after transfection, and endogenous p62 was detected using a p62
mAb (red). (C and D) The p62 shell surrounding GFP–N-HttQ68 was de-
tected independently of the use of antibodies. (C) GFP–N-HttQ68 and
DsRed-tagged p62 were cotransfected into HeLa cells, and images of live
cells were obtained after 48 h of expression. The left panel shows one
confocal plane of a representative GFP–N-HttQ68 and DsRed-p62 dou-
ble-positive structure, and the right panel shows the Z-stack of planes with
side views of the aggregate at the side and at the top. N, nucleus. (A–C)
Nuclei were detected using Draq5. (D) GFP–N-HttQ68 and tdTomato-
tagged p62 were cotransfected into HeLa cells, and images of live cells
were obtained after 24 h of expression. (E) Transiently expressed Flag–
N-HttQ68 and myc-LC3 colocalize in GFP-p62–positive structures.
S–GFP-p62 cells were fixed 48 h after transfection, Flag–N-HttQ68 was
stained red using a Flag tag mAb, and myc-LC3 was stained far red
(blue) using a chicken myc tag pAb (top). The bottom panel shows a live
cell image of a GFP–N-HttQ68 aggregate surrounded by a shell contain-
ing tdTomato-LC3. Bars, 5 ?m.
p62 forms a shell around huntingtin protein aggregates. (A)
62 IN AUTOPHAGIC PROTEIN DEGRADATION • BJØRKØY ET AL. 611
glutamine and polyalanine expansion mutations, are degraded
by autophagy (Ravikumar et al., 2002, 2004). Autophagy is
generally thought of as a nonspecific bulk degradation mecha-
nism. It is presently unclear whether there is a specific recogni-
tion or targeting of polyubiquitinated protein aggregates by the
Our data suggest that p62 may link polyubiquitinated pro-
teins to the autophagic machinery. This function seems depen-
dent on both the polymerization of p62 via the NH
domain and polyubiquitin binding via the COOH-terminal UBA
domain of p62. Both endogenous and ectopically expressed p62
could be copurified with the autophagy marker LC3. Both p62
and LC3 colocalized with mutant huntingtin aggregates. Such
aggregates were recently shown to be degraded by autophagy
(Ravikumar et al., 2002, 2004). Very recently, studies of condi-
tional knockout mice of Atg7 demonstrated that autophagy is
needed for clearance of ubiquitin-positive aggregates (Komatsu
et al., 2005). We found that p62 formed a shell surrounding hun-
tingtin aggregates. Cell death induced by the expression of aggre-
gation-prone mutant huntingtin was increased both in HeLa and
SHSY-5Y neuroblastoma cells after antisense RNA–mediated
depletion of p62 levels or by interfering with p62 function by ex-
pressing a p62 deletion mutant lacking the UBA domain.
We found that p62 was located in two different types of
bodies in the cytosol. The first type of structure appears as
large protein aggregates (sequestosomes) that are not sur-
rounded by a membrane and have very low mobility in living
cells. However, the majority of p62 bodies in S–GFP-p62 cells
were generally smaller structures with a much higher mobility
that colocalized poorly with early endosomal markers but
strongly with coexpressed myc- or GFP-tagged LC3. The find-
ing that a high number of p62 bodies colocalized with
LysoTracker and the lysosomal marker CD63 is consistent
with p62 being localized to autophagosomes. By detergent
extraction of live cells, we observed two populations of p62
bodies. LysoTracker-positive p62 bodies were rapidly lost af-
ter extraction, whereas LysoTracker-negative structures were
not dissolved by 1% Triton X-100. This is consistent with p62
being partly located to membrane-enclosed autophagosomes
and partly in cytosolic sequestosomes. Induction of autophagy
by amino acid starvation led to a clear increase in the number
of GFP-LC3–labeled autophagosomes, and all of these were
positive for endogenous p62. The idea that a large fraction of
p62 bodies are autophagosomes was also supported by the ex-
tensive accumulation of p62-LC3–positive structures observed
in cells upon treatment with bafilomycin A1. Bafilomycin A1
inhibits the autophagosome–lysosome fusion step leading to
accumulation of autophagosomal vacuoles. EM experiments
confirmed that p62 is found both in autophagosomes and in
sequestosomes and that p62 is colocalized with CD63 in
cytoplasmic membrane-enclosed autophagosomal structures.
Interestingly, we found p62 and LC3 to be components of the
same protein complex by coimmunoprecipitation. It should be
noted that coexpression of p62 with GFP-LC3 did not increase
the total amount of p62 that was copurified with LC3. This sug-
gests that the interaction might not be direct but may depend on
a limiting third cellular factor. This notion is consistent with
our failure to detect any interaction between GST-p62 and in
vitro translated LC3 in a GST pull-down assay (unpublished
data). Furthermore, the p62 D69A mutant that inhibits poly-
merization of p62 was very inefficiently coimmunoprecipitated
with GFP-LC3, suggesting that polymeric p62 is important for
interaction with LC3. Our data suggest that p62 is needed for
the accumulation of GFP-LC3 in dots in HeLa cells in response
to amino acid starvation. No GFP-LC3 dots were formed in re-
sponse to amino acid starvation in cells depleted for endogenous
p62 after transfection with siRNA. Similarly, no GFP-LC3 dots
were formed in cells coexpressing mutants of p62, resulting in
diffuse localization of endogenous p62. These results indicate
that p62 polymerization is important for autophagosome for-
mation in HeLa cells.
Previous studies have established p62 as a stress response
protein induced by oxidative stress (Ishii et al., 1997, 2000).
The protein has also been identified as a common component
in protein aggregates that was found in a wide range of protein
aggregation diseases (Zatloukal et al., 2002). Recently, expres-
sion of mutant huntingtin was shown to induce p62 (Nagaoka
et al., 2004). Interestingly, reactive oxygen species are pro-
duced in response to proteasomal inhibition (Ling et al., 2003),
and aggregation-prone mutant proteins with expanded poly-
glutamine stretches inhibit proteasomal activity (Bence et al.,
2001). Thus, the induction of p62 in aggregation diseases
might also be caused by reactive oxygen species. Prostaglandin
J2 is known to induce oxidative stress by causing decreases in
glutathione, glutathione peroxidase, and mitochondrial mem-
death induced by mutant huntingtin. (A) GFP-positive cells were scored as
live or apoptotic by the appearance of nuclear Draq5 staining. After 48 h
of expression, GFP-positive cells attached to the surface with a normal nu-
clear appearance (open arrowheads) and were scored as live, whereas
rounded, partially detached cells with a condensed or fragmented nuclei
(arrows) were scored as dead. More than 200 GFP-positive cells in ran-
domly selected microscope views were scored. Bars, 20 ?m. (B) The effect
of coexpressing either a p62 antisense construct or a deletion construct of
p62 lacking the UBA domain on N-HttQ68–induced cell death in HeLa
and SHSY-5Y neuroblastoma cells.
Interfering with p62 protein level or function increases cell
JCB • VOLUME 171 • NUMBER 4 • 2005612
brane potential as well as increases in the production of pro-
tein-bound lipid peroxidation products (Kondo et al., 2001).
In human neuroblastoma cells, p62 is needed for the sequestra-
tion of ubiquitinated proteins into bodies in response to treat-
ment with the inflammatory agent prostaglandin J2 (Wang and
Figueiredo-Pereira, 2005). Our present results provide a molec-
ular mechanism of how p62 might recognize ubiquitinated pro-
tein bodies and present these to the autophagic machinery.
Mutant huntingtin is found to be both diffuse in some
cells and aggregated in others. It seems clear that aggregation is
a mechanism for cell survival (Arrasate et al., 2004). In line
with this, Steffan et al. (2004) found that ubiquitination of mu-
tant huntingtin is an important way to detoxify the protein,
whereas sumoylation of the same residues prevents aggrega-
tion and leads to cell death. Autophagy is important for the
clearance of huntingtin aggregates, and induced autophagy
leads to increased cell survival of cells expressing mutant hun-
tingtin (Ravikumar et al., 2002, 2004). Similar to Nagaoka et
al. (2004), we found that mutant huntingtin could also form
aggregates in the absence of p62. Thus, we believe that the
protective role of p62 may be to recruit autophagosomal com-
ponents to the polyubiquitinylated protein aggregates rather
than to help or facilitate the formation of these aggregates.
Given the essential role of autophagy in preventing protein
aggregate–induced neurodegeneration, p62 and proteins with
related functions could be attractive targets for the develop-
ment of neuroprotective drugs.
Materials and methods
Subconfluent HeLa and SHSY-5Y neuroblastoma cells were transfected
using LipofectAMINE Plus (Invitrogen). Stable transfectants were selected
using G418, and GFP-positive colonies were isolated and subcloned
twice using minimal dilution. A siGENOME SMARTpool SQSTM1 siRNA
oligonucleotide mixture against p62 (Dharmacon) was transfected twice,
with a 24-h interval at 100 nM using LipofectAMINE Plus. siRNA (Dhar-
macon) against human tissue factor was used as an unrelated control.
Knockdown of p62 by siRNA was verified by immunoblotting.
Antibodies and reagents
The following antibodies were used: anti-p62 and EEA1 mAbs (BD Trans-
duction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb
(clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Stud-
ies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Ab-
cam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz
Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 anti-
body (gift from T. Yoshimori, National Institute of Genetics, Shizuoka,
Japan; Kabeya et al., 2000). All fluorescent Alexa-labeled secondary
antibodies, LysoTracker, fluorescent-labeled dextran, and EGF as well as
a Zenon kit for direct Alexa labeling of mAbs were obtained from Invitro-
gen. Bafilomycin A1, epoximicin, 3-methyladenine, and rapamycin were
all purchased from Sigma-Aldrich. Proteasome inhibitor I (PSI) was ob-
tained from Calbiochem. Draq5 was obtained from Biostatus Ltd. Redivue
S-methionine was obtained from GE Healthcare.
The following vectors have been described previously (Lamark et al.,
2003): pCW7-mycUbi (a gift from J. Lukas, Danish Cancer Society,
Copenhagen, Denmark; Thullberg et al., 2000); pcDNA3-myc-LC3 and
pEGFP-C1-LC3 (Simonsen et al., 2004); the Gateway entry clones pENTR-
p62 and pENTR-p40phox; the Gateway destination vectors pDestEGFP-
C1 and pDestmyc; and the Gateway expression vectors pDestHA-p62,
pDestmyc-p62, pDestHA-p62 D69A, pDestmyc-p62 D69A, pDestEGFP-
p62 D69A, and pEGFP-p62. pENTR-p62 I431A was constructed by the
mutagenesis of pENTR-p62 using the QuikChange Site-Directed Mutagen-
esis Kit (Stratagene). The p62 deletion constructs pENTR-p62
pENTR-p62(387–440), pENTR-p62(1–122), pENTR-p62(124–440), and
pENTR-p62(1–385) were made by subcloning of the indicated p62
fragments from pENTR-p62 into Gateway entry vectors (pENTR1A or
pENTR3C; Invitrogen). pENTR-Htt(1–171)68Q-Flag was made by the sub-
cloning of human huntingtin(1–171)68Q-FLAG cDNA from plasmid
pcDNA1-Htt(1–171)68Q-Flag (obtained from U. Moens, University of
Tromsø, Tromsø, Norway; Saudou et al., 1998) into pENTR3C. The mam-
malian tdTomato fusion expression vector ptdTomato-C1 was made by ex-
changing the EGFP gene of pEGFP-C1 (CLONTECH Laboratories, Inc.)
with tdTomato from the bacterial expression vector pRSET-B-tdTomato
(obtained from R. Tsien, University of California, San Diego, San Diego,
CA; Shaner et al., 2004). ptdTomato-LC3 was made by inserting a 400-bp
EcoRI–BamHI fragment from pEGFP-C1-LC3 into ptdTomato-C1 cut with
the same enzymes. The Gateway destination vector pDest-tdTomato-C1
was made by exchanging the EGFP gene of pDestEGFP-C1 with tdTo-
mato, and Gateway destination vector pDestDsRED2-C1 was constructed
by the insertion of Gateway cassette B (Gateway vector conversion sys-
tem; Invitrogen) into pDsRED2-C1 (CLONTECH Laboratories, Inc.). The
Gateway expression vectors pDestDsRED2-p62, pDest-tdTomato-p62,
pDestEGFP-p62(1–122), pDestEGFP-p62(387–440), pDestEGFP-p62
386, pDestEGFP-p62(1–385), pDestmyc-p62(1–385), pDestEGFP-p62
(124–440), pDestEGFP-p62 I431A, pDestEGFP-Htt(1–171)68Q-Flag, and
pDestEGFP-p40phox were made using Gateway recombination reactions
(Invitrogen). The antisense p62 construct pAS-p62-c was made by religa-
tion of pAS-p62 (Lamark et al., 2003) after digestion with EcoRV. All con-
structs were verified by DNA sequencing (BigDye; Applied Biosystems).
Oligonucleotides for mutagenesis, PCR, and DNA sequencing reactions
were obtained from Eurogentec.
Confocal microscopy analyses
The cell cultures were directly examined under the microscope or fixed in
4% PFA and stained as previously described (Lamark et al., 2003). Live
cells were imaged at 37
C in Hanks medium containing 10% serum,
whereas fixed cells were imaged at RT in PBS. Images were collected us-
ing a microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.) equipped
with a 40
1.2W C-Apochroma objective and a confocal module
(LSM510 META; Carl Zeiss MicroImaging, Inc.) and using the LSM5 soft-
ware version 3.2 (Carl Zeiss MicroImaging, Inc.). Images were processed
using Photoshop (Adobe). Z-stack video images were acquired using an
imager (Ultraview RS Live Cell; PerkinElmer) on the Axiovert microscope
with a 63
NA 1.4 plan-Apochromat objective. In total, 8–10 confocal
m planes covering the whole height of the cells were superimposed for
each time point. The video was acquired with Imaging Suite software
(PerkinElmer) and compressed using Premiere Pro software (Adobe).
Two-color live cell videos were obtained using the LSM510 META confo-
cal module imaging a single confocal plane. Videos were compressed
using QuickTime Pro.
Cells for immuno-EM were fixed and embedded as described previously
(Peters et al., 1991). Small blocks were cut and infused with 2.3 M sucrose
for 1 h, mounted on silver pins, and frozen in liquid nitrogen. Ultrathin cryo-
sections were cut at
C on an ultramicrotome (Ultracut; Leica) and
collected with a 1:1 mixture of 2% methyl cellulose and 2.3 M sucrose.
Sections were transferred to formvar/carbon-coated grids and labeled
with primary antibodies followed by a bridging secondary antibody and
protein A–gold conjugates essentially as described previously (Slot et al.,
1991). For double-labeling experiments, we included a blocking step be-
tween the first protein A–gold and the second primary antibody (15-min
incubation in 0.1% glutaraldehyde and 0.1 M PBS). After embedding in
2% methyl cellulose/0.4% uranyl acetate, we observed sections at 80 kV
in an electron microscope (CM10; Philips).
Correlative immunofluorescence/EM was performed on HeLa cells
that were grown on gridded coverslips (Eppendorf). Cells were treated
with PSI for 3 h, fixed in 3% PFA/PBS (30 min), permeabilized with
0.05% saponin/PBS, and labeled with mouse anti-p62 (1:2,000) fol-
lowed by donkey anti–mouse Cy2 (1:500). After embedding in moviol,
the samples were observed on a microscope (LSM510 META; Carl Zeiss
MicroImaging, Inc.), and the localization of interesting cells was re-
corded. For EM observation, the same coverslips were then fixed with 2%
glutaraldehyde in 0.1 M phosphate buffer and postfixed with 2% OsO
and 1.5% KFeCN in 0.1 M phosphate buffer. Thereafter, the coverslips
were stained en bloc with 4% uranyl acetate for 60 min, dehydrated in
ethanol, and embedded in Epon. After polymerization, the coverslips
were removed with 40% hydrofluoric acid, and the flat specimens were
62 IN AUTOPHAGIC PROTEIN DEGRADATION • BJØRKØY ET AL.613
glued onto Epon stubs. The block was thereafter trimmed down to the re-
gions observed on the gridded coverslips in the fluorescence microscope
and sectioned parallel to the substratum at 50–70-nm section thickness.
The sections were poststained with lead citrate (2 min). Electron micro-
graphs were taken and overlaid with the confocal images in Photoshop.
Immunoblot and immunoprecipitations
All expression constructs were controlled by the immunoblotting of total
cell extracts that was made after stable transfection or 24 h after transient
transfection as described previously (Lamark et al., 2003). Cells were la-
beled with 0.14
Ci/ml S-methionine by a 30-min pulse in methionine-
free medium, washed in PBS, and incubated for different times in normal
medium. Endogenous p62 was immunoprecipitated using the p62 mAb
from total cell lysates from
S-methionine cells as described previously
(Lamark et al., 2003). GFP-LC3 was immunoprecipitated from total cellu-
lar extracts using anti-GFP antibody.
Online supplemental material
Video 1 shows the migration of S–GFP-p62 expressed in HeLa cells over
2 min. In total, 130 image stacks were collected with a time interval of 1 s.
Video 2 shows the migration of S–GFP-p62 expressed in HeLa cells over
1 h. 60 image stacks were collected with a time interval of 60 s. Video 3
shows dynamic movements of pDest-tdTomato-LC3 and GFP-p62 tran-
siently coexpressed in HeLa cells. One confocal plane was imaged for 3
min with a time interval of 4 s. Videos 1, 2, and 3 are displayed as 15, 6,
and 10 frames per second, respectively. Fig. S1 shows rapid detergent
extraction of both GFPp40phox- and LysoTracker-positive vesicles in HeLa
cells. Fig. S2 shows a lack of association between DsRed fluorescent pro-
tein and a GFP–N-HttQ68 aggregate. Fig. S3 shows the accumulation of
endogenous p62 and polyubiquitin in mutant huntingtin aggregates. Online
supplemental material is available at http://www.jcb.org/cgi/content/
We thank Hege Avsnes Dale and Endy Spriet at the Molecular Imaging Cen-
tre in Bergen for help with video images from the live cell imager. We are
grateful to R. Tsien for the gift of pRSET-B-tdTomato, J. Lukas for pCW7-mycUbi,
U. Moens and
F. Saudou for pcDNA1-Htt(1–171)68Q-Flag, and T. Yoshimori
for LC3 antibodies.
This work was supported by grants to T. Johansen from the Norwegian
Research Council, the Norwegian Cancer Society, the Aakre Foundation,
Simon Fougner Hartmanns Familiefond, and the Blix Foundation. A. Brech is the
recipient of a career fellowship from The National Programme for Research in
Functional Genomics in Norway of the Norwegian Research Council.
Submitted: 1 July 2005
Accepted: 12 October 2005
Abeliovich, H., W.A. Dunn Jr., J. Kim, and D.J. Klionsky. 2000. Dissection of
autophagosome biogenesis into distinct nucleation and expansion steps.
J. Cell Biol. 151:1025–1034.
Arrasate, M., S. Mitra, E.S. Schweitzer, M.R. Segal, and S. Finkbeiner. 2004.
Inclusion body formation reduces levels of mutant huntingtin and the
risk of neuronal death. Nature. 431:805–810.
Avila, A., N. Silverman, M.T. Diaz-Meco, and J. Moscat. 2002. The Drosophila
atypical protein kinase C-ref(2)p complex constitutes a conserved mod-
ule for signaling in the toll pathway. Mol. Cell. Biol. 22:8787–8795.
Bence, N.F., R.M. Sampat, and R.R. Kopito. 2001. Impairment of the ubiquitin-
proteasome system by protein aggregation. Science. 292:1552–1555.
Cariou, B., D. Perdereau, K. Cailliau, E. Browaeys-Poly, V. Bereziat, M. Vas-
seur-Cognet, J. Girard, and A.F. Burnol. 2002. The adapter protein ZIP
binds Grb14 and regulates its inhibitory action on insulin signaling by re-
cruiting protein kinase Czeta. Mol. Cell. Biol. 22:6959–6970.
Cuervo, A.M. 2004. Autophagy: in sickness and in health. Trends Cell Biol.
Donaldson, K.M., W. Li, K.A. Ching, S. Batalov, C.C. Tsai, and C.A. Joazeiro.
2003. Ubiquitin-mediated sequestration of normal cellular proteins into
polyglutamine aggregates. Proc. Natl. Acad. Sci. USA. 100:8892–8897.
Escola, J.M., M.J. Kleijmeer, W. Stoorvogel, J.M. Griffith, O. Yoshie, and H.J.
Geuze. 1998. Selective enrichment of tetraspan proteins on the internal
vesicles of multivesicular endosomes and on exosomes secreted by hu-
man B-lymphocytes. J. Biol. Chem. 273:20121–20127.
Geetha, T., and M.W. Wooten. 2002. Structure and functional properties of the
ubiquitin binding protein p62. FEBS Lett. 512:19–24.
Gong, J., J. Xu, M. Bezanilla, R. van Huizen, R. Derin, and M. Li. 1999. Differ-
ential stimulation of PKC phosphorylation of potassium channels by
ZIP1 and ZIP2. Science. 285:1565–1569.
Ishii, T., T. Yanagawa, K. Yuki, T. Kawane, H. Yoshida, and S. Bannai. 1997.
Low micromolar levels of hydrogen peroxide and proteasome inhibitors
induce the 60-kDa A170 stress protein in murine peritoneal macrophages.
Biochem. Biophys. Res. Commun. 232:33–37.
Ishii, T., K. Itoh, S. Takahashi, H. Sato, T. Yanagawa, Y. Katoh, S. Bannai, and
M. Yamamoto. 2000. Transcription factor Nrf2 coordinately regulates a
group of oxidative stress-inducible genes in macrophages. J. Biol. Chem.
Kabeya, Y., N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E.
Kominami, Y. Ohsumi, and T. Yoshimori. 2000. LC3, a mammalian ho-
mologue of yeast Apg8p, is localized in autophagosome membranes af-
ter processing. EMBO J. 19:5720–5728.
Kabeya, Y., N. Mizushima, A. Yamamoto, S. Oshitani-Okamoto, Y. Ohsumi,
and T. Yoshimori. 2004. LC3, GABARAP and GATE16 localize to au-
tophagosomal membrane depending on form-II formation. J. Cell Sci.
Kegel, K.B., M. Kim, E. Sapp, C. McIntyre, J.G. Castano, N. Aronin, and M. Di-
Figlia. 2000. Huntingtin expression stimulates endosomal-lysosomal ac-
tivity, endosome tubulation, and autophagy. J. Neurosci. 20:7268–7278.
Klionsky, D.J. 2005. The molecular machinery of autophagy: unanswered
questions. J. Cell Sci. 118:7–18.
Komatsu, M., S. Waguri, T. Ueno, J. Iwata, S. Murata, I. Tanida, J. Ezaki, N.
Mizushima, Y. Ohsumi, Y. Uchiyama, et al. 2005. Impairment of starva-
tion-induced and constitutive autophagy in Atg7-deficient mice. J. Cell
Kondo, M., T. Oya-Ito, T. Kumagai, T. Osawa, and K. Uchida. 2001. Cyclopen-
tenone prostaglandins as potential inducers of intracellular oxidative
stress. J. Biol. Chem. 276:12076–12083.
Kuusisto, E., A. Salminen, and I. Alafuzoff. 2001a. Ubiquitin-binding protein
p62 is present in neuronal and glial inclusions in human tauopathies and
synucleinopathies. Neuroreport. 12:2085–2090.
Kuusisto, E., T. Suuronen, and A. Salminen. 2001b. Ubiquitin-binding protein
p62 expression is induced during apoptosis and proteasomal inhibition in
neuronal cells. Biochem. Biophys. Res. Commun. 280:223–228.
Kuusisto, E., A. Salminen, and I. Alafuzoff. 2002. Early accumulation of p62 in
neurofibrillary tangles in Alzheimer’s disease: possible role in tangle
formation. Neuropathol. Appl. Neurobiol. 28:228–237.
Lamark, T., M. Perander, H. Outzen, K. Kristiansen, A. Overvatn, E.
Michaelsen, G. Bjorkoy, and T. Johansen. 2003. Interaction codes within
the family of mammalian Phox and Bem1p domain-containing proteins.
J. Biol. Chem. 278:34568–34581.
Lawrence, B.P., and W.J. Brown. 1993. Inhibition of protein synthesis separates
autophagic sequestration from the delivery of lysosomal enzymes. J. Cell
Levine, B., and D.J. Klionsky. 2004. Development by self-digestion: molecular
mechanisms and biological functions of autophagy. Dev. Cell. 6:463–477.
Ling, Y.H., L. Liebes, Y. Zou, and R. Perez-Soler. 2003. Reactive oxygen spe-
cies generation and mitochondrial dysfunction in the apoptotic response
to Bortezomib, a novel proteasome inhibitor, in human H460 non-small
cell lung cancer cells. J. Biol. Chem. 278:33714–33723.
Mizushima, N. 2004. Methods for monitoring autophagy. Int. J. Biochem. Cell
Nagaoka, U., K. Kim, N.R. Jana, H. Doi, M. Maruyama, K. Mitsui, F. Oyama, and
N. Nukina. 2004. Increased expression of p62 in expanded polyglutamine-
expressing cells and its association with polyglutamine inclusions. J.
Noda, T., and Y. Ohsumi. 1998. Tor, a phosphatidylinositol kinase homologue,
controls autophagy in yeast. J. Biol. Chem. 273:3963–3966.
Peters, P.J., J.J. Neefjes, V. Oorschot, H.L. Ploegh, and H.J. Geuze. 1991.
Segregation of MHC class II molecules from MHC class I molecules in
the Golgi complex for transport to lysosomal compartments. Nature.
Ravikumar, B., R. Duden, and D.C. Rubinsztein. 2002. Aggregate-prone pro-
teins with polyglutamine and polyalanine expansions are degraded by
autophagy. Hum. Mol. Genet. 11:1107–1117.
Ravikumar, B., C. Vacher, Z. Berger, J.E. Davies, S. Luo, L.G. Oroz, F. Scar-
avilli, D.F. Easton, R. Duden, C.J. O’Kane, and D.C. Rubinsztein. 2004.
Inhibition of mTOR induces autophagy and reduces toxicity of poly-
glutamine expansions in fly and mouse models of Huntington disease.
Nat. Genet. 36:585–595.
Sanz, L., P. Sanchez, M.J. Lallena, M.T. Diaz-Meco, and J. Moscat. 1999.
The interaction of p62 with RIP links the atypical PKCs to NF-kappaB
activation. EMBO J. 18:3044–3053.
Sanz, L., M.T. Diaz-Meco, H. Nakano, and J. Moscat. 2000. The atypical PKC-
JCB • VOLUME 171 • NUMBER 4 • 2005 614 Download full-text
interacting protein p62 channels NF-kappaB activation by the IL-1-
TRAF6 pathway. EMBO J. 19:1576–1586.
Saudou, F., S. Finkbeiner, D. Devys, and M.E. Greenberg. 1998. Huntingtin acts
in the nucleus to induce apoptosis but death does not correlate with the
formation of intranuclear inclusions. Cell. 95:55–66.
Shaner, N.C., R.E. Campbell, P.A. Steinbach, B.N. Giepmans, A.E. Palmer, and
R.Y. Tsien. 2004. Improved monomeric red, orange and yellow fluores-
cent proteins derived from Discosoma sp. red fluorescent protein. Nat.
Simonsen, A., H.C. Birkeland, D.J. Gillooly, N. Mizushima, A. Kuma, T.
Yoshimori, T. Slagsvold, A. Brech, and H. Stenmark. 2004. Alfy, a
novel FYVE-domain-containing protein associated with protein granules
and autophagic membranes. J. Cell Sci. 117:4239–4251.
Slot, J.W., H.J. Geuze, S. Gigengack, G.E. Lienhard, and D.E. James. 1991. Im-
muno-localization of the insulin regulatable glucose transporter in brown
adipose tissue of the rat. J. Cell Biol. 113:123–135.
Steffan, J.S., N. Agrawal, J. Pallos, E. Rockabrand, L.C. Trotman, N. Slepko,
K. Illes, T. Lukacsovich, Y.Z. Zhu, E. Cattaneo, et al. 2004. SUMO
modification of Huntingtin and Huntington’s disease pathology. Science.
Thompson, H.G., J.W. Harris, B.J. Wold, F. Lin, and J.P. Brody. 2003. p62
overexpression in breast tumors and regulation by prostate-derived Ets
factor in breast cancer cells. Oncogene. 22:2322–2333.
Thullberg, M., J. Bartek, and J. Lukas. 2000. Ubiquitin/proteasome-mediated
degradation of p19INK4d determines its periodic expression during the
cell cycle. Oncogene. 19:2870–2876.
Vadlamudi, R.K., I. Joung, J.L. Strominger, and J. Shin. 1996. p62, a phosphoty-
rosine-independent ligand of the SH2 domain of p56lck, belongs to a new
class of ubiquitin-binding proteins. J. Biol. Chem. 271:20235–20237.
Vonsattel, J.P., and M. DiFiglia. 1998. Huntington disease. J. Neuropathol. Exp.
Wang, Z., and M.E. Figueiredo-Pereira. 2005. Inhibition of sequestosome 1/p62
up-regulation prevents aggregation of ubiquitinated proteins induced by
prostaglandin J2 without reducing its neurotoxicity. Mol. Cell. Neurosci.
Yamamoto, A., Y. Tagawa, T. Yoshimori, Y. Moriyama, R. Masaki, and Y.
Tashiro. 1998. Bafilomycin A1 prevents maturation of autophagic vacu-
oles by inhibiting fusion between autophagosomes and lysosomes in rat
hepatoma cell line, H-4-II-E cells. Cell Struct. Funct. 23:33–42.
Yoshimori, T. 2004. Autophagy: a regulated bulk degradation process inside
cells. Biochem. Biophys. Res. Commun. 313:453–458.
Zatloukal, K., C. Stumptner, A. Fuchsbichler, H. Heid, M. Schnoelzer, L. Kenner,
R. Kleinert, M. Prinz, A. Aguzzi, and H. Denk. 2002. p62 Is a common
component of cytoplasmic inclusions in protein aggregation diseases.
Am. J. Pathol. 160:255–263.