1 Department of Molecular Biology and Biochemistry, Rutgers University,
Piscataway, NJ 08554, USA
2 Institute für Physiologische Chemie, Ruhr-Universität Bochum,
D-44801 Bochum, Germany
* Present address: Department of Molecular Biology, Princeton University,
Princeton, NJ 08854, USA
Present address: Union Biometrica, Somerville, MA 02143, USA
Author for correspondence (e-mail:
driscoll{at}mbcl.rutgers.edu)
Accepted 21 January 2003
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Summary |
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Key words: Zellweger syndrome model, Peroxin, Peroxisome biogenesis
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Introduction |
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The peroxisomal biogenesis disorders include Zellweger syndrome (ZS), which
is the most severe form, neonatal adrenoleukodystrophy (NALD), infantile
refsum disease (IRD) and rhizomelic type chondrodysplasia punctata (RCDP).
PBDs are inherited in an autosomal recessive fashion, and occur with
incidences between 1/25,000 and 1/50,000 births. The PBDs are characterized by
hepatic and renal dysfunction, developmental delay and neurological
abnormalities. Interestingly, ZS, NALD and IRD appear to represent a continuum
of related diseases that all show essentially the same biochemical
manifestations, including increased levels of very-long-chain fatty acids
(C24:0 and C26:0), bile acid intermediates and phytanic acid
(Moser, 1999), whereas RCDP
patients display a distinct phenotype.
The PBDs have been assigned to 12 genetic complementation groups with most
genes responsible for these disorders identified and implicated in the import
of peroxisomal proteins from the cytosol to the peroxisomal matrix. The first
mammalian peroxisome assembly gene was isolated using functional
complementation cloning with peroxisome-deficient CHO cells
(Tsukamoto et al., 1991). In
addition, several human PEX genes have been identified on the basis
of sequence comparison to yeast peroxisome biogenesis genes. The proteins they
encode are now all termed peroxins (Distel
et al., 1996
). In humans, at least 13 peroxins are required for
normal peroxisome biogenesis (Fujiki,
2000
; Gould and Valle,
2000
). The relationship between the biochemical abnormalities and
the resulting clinical and pathological manifestations of the PBDs can be
variable and is not well understood
(Moser, 1999
;
Wanders, 1999
).
The steps in peroxisome biogenesis have been elucidated on the basis of
work in both yeast and human cells
(Sacksteder and Gould, 2000).
Proteins destined for the peroxisomal matrix are translated in the cytosol and
are recognized by one of two intracellular receptors, Pex5p or Pex7p
(Hettema et al., 1999
;
Subramani et al., 2000
). These
receptors recognize proteins that contain either a C-terminal tripeptide
(peroxisomal targeting signal 1, PTS1) or an N-terminal nonapeptide
(peroxisomal targeting signal 2, PTS2), respectively. Pex5p contains
tetratricopeptide repeat (TPR) domains thought to mediate
proteinprotein interactions with the PTS1 signal of matrix proteins
(Gatto et al., 2000
). Defects
in PEX5 are associated with complementation group 2 of the PBDs
(Dodt et al., 1995
;
Wiemer et al., 1995
). Pex7p is
a member of the WD-40 protein family that contains 6WD repeats. Defects in
PEX7 result in rhizomelic chondrodysplasia punctata (complementation group 11)
(Braverman et al., 1997
).
After receptorcargo binding, the complex docks at the peroxisomal
membrane. Pex13p and Pex14p are membrane peroxins that probably serve as
docking targets for the PTS1 receptor Pex5p. There are reports suggesting that
Pex3p and Pex17p are also part of the docking event at the peroxisomal
membrane (Huhse et al., 1998;
Subramani et al., 2000
). The
receptorcargo complex is then translocated into the peroxisome by a
process that is poorly understood (Dammai
and Subramani, 2001
). The integral peroxisomal membrane proteins
Pex2p, Pex10p and Pex12p are all zinc-binding ring-finger proteins that act
downstream of the docking step, although their specific functions have not
been clearly defined.
Our interest is in understanding the functions of peroxisomes in
Caenorhabditis elegans and in establishing an invertebrate animal
model system for the PBDs. In both plants and yeasts, the ß-oxidation of
fatty acids occurs solely in the peroxisome. In humans, the oxidation of
short- and medium-chain fatty acids is accomplished in the mitochondria,
whereas the oxidation of very-long-chain fatty acids (C:24 or longer) is
carried out in the peroxisome. Bioinformatic analysis of the ß-oxidation
enzymes identified in the C. elegans genome predicts that, like
humans, the nematode has both mitochondrial and peroxisomal ß-oxidation
pathways (Gurvitz et al.,
2000), and suggests that the nematode should be a relevant system
in which to model the human peroxisomal disorders. More specifically,
establishing a peroxisomal biogenesis disease model in C. elegans
should allow genetic suppressor screens that might suggest novel intervention
strategies for the tragic PBDs.
The C. elegans genome encodes homologs of 11 of the 13 human peroxins. We found that inactivation of the C. elegans homologs of Pex5p, Pex6p, Pex12p, Pex13p and Pex19p by RNA-mediated interference (RNAi) produces an early larval developmental arrest before postembryonic cell division, indicating that peroxisome function is necessary for the normal development of C. elegans. We further show that the peroxisomal import machinery is disrupted when the C. elegans homolog of Pex5p, prx-5, is knocked down by RNAi strategies. Nematodes lacking peroxisomes exhibit a developmental block similar to starvation-arrested nematodes, although certain morphological features are different in starved nematodes. We discuss implications for roles that peroxisomes may play in postembryonic development, and the potential of this system to address human disease mechanisms.
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Materials and Methods |
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Strains and genetic analysis
C. elegans strains were maintained at 20°C as described
previously (Brenner, 1974). The
N2 Bristol strain was used for all experiments unless otherwise noted. Worms
were fed bacterial strain, OP50. The strain LT484 containing
Is[prnr-1GFP; rol-6(su1006)] was kindly provided
by the Padgett Lab (Waksman Institute, Rutgers University, Piscataway, NJ).
The unc-129::GFP strain was kindly provided by the Culotti lab
(University of Toronto, Ontario, Canada). The mig-2::GFP strain was
kindly provided by the Kenyon Lab (University of California, San Francisco,
CA).
Construction and analysis of GFPSKL reporter
We generated the peroxisome-targeted GFP construct
pHSP16/2GFPSKL by subcloning a C-terminal fragment
(MunI-XbaI) of a PTS1-tagged GFP from the vector
pcDNA3-PTS1GFP (gift of S. Gould, Johns Hopkins University, Baltimore, MD).
This construct has the amino acids PLHSKL added in frame to the C-terminus of
GFP. This C-terminal fragment of GFP was substituted into the C-terminal
region of GFP in the C. elegans vector pPD99.44 (gift of A. Fire,
Carnegie Institution of Washington, Baltimore, MD). This vector contains the
heat-shock promoter 16/2 driving expression of a GFP that contains five
artificial C. elegans introns. Germline transformation was performed
as previously described (Driscoll,
1995; Mello and Fire,
1995
). Plasmids for transformation were prepared using a Qiagen
miniprep kit and were injected at a concentration of 50-100 ng/µl. The
transformation marker pRF4 containing rol-6(su1006) was co-injected
at a concentration of 50 ng/µl. Transgenic rolling animals were selected
among the progeny. Transgenic lines were isolated and heat-shock experiments
were performed at 35°C for 4 hours for adult worms and for 1 hour for
younger worms. Worms were recovered for 1-2 hours before microscopy. Worms
were mounted on 2% agarose pads in M9 for fluorescence microscopy on a Zeiss
Axioplan 2 microscope.
RNA interference assays
Injection of dsRNA was carried out as previously described
(Fire et al., 1998). Briefly,
approximately 200 ng of double-stranded RNA (dsRNA) were injected into the
gonad of an L4-young adult worm. Injected worms were recovered for 8-16 hours
before being moved to individual plates to enable unaffected embryos to be
cleared from the hermaphrodite. The injected nematodes were separated to
individual plates and transferred at either 12 or 24 hour intervals to stage
development of their progeny. Development of the progeny was monitored daily
and compared with noninjected time-matched control worms. Arrested worms were
mounted in M9 buffer on 2% agarose pads for microscopy.
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Results |
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We heat-shocked adult transgenic animals for 4 hours and characterized the
GFP location after 2 hours of recovery. Peroxisomal structures have previously
been identified in the intestine by GFP tagging of catalase 2 (ctl-2)
and by immunochemical staining (Taub et
al., 1999; Togo et al.,
2000
). In addition to clear punctate signals in the intestinal
cells (Fig. 1A), we observed
numerous peroxisomes in the bands of hypodermal cells that run longitudinally
over the surface of the nematode (Fig.
1B). We also observed fluorescent GFP-containing particles in the
developing embryos (Fig. 1C).
Note that since this heat-shock promoter is not active in all cell types, our
results do not exclude the existence of peroxisomes in other tissues such as
neuronal cells. Our results and the results of other groups with targeting GFP
to the peroxisome prove that C. elegans contains functional
peroxisomal import machinery that is dependent on SKL
(Motley et al., 2000
;
Taub et al., 1999
). These
results extend characterization of the remarkable conservation of peroxisome
biogenesis to include the nematode C. elegans.
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C. elegans peroxin homologs
Table 1 lists the human
peroxins, their proposed functions and the C. elegans homologs, along
with the statistical significance of the match expressed as E values. Because
the gene classification of PEX has already been claimed in the C.
elegans field by Pachytene exit genes, the curator of
the C. elegans Genetic Consortium has supported the use of PRX to
define the peroxisome biogenesis genes in the nematode (J. Hodgkin, personal
communication). The homologs for PEX1 (prx-1/C11H1.6) and PEX6
(prx-6/F39G3.7) were also identified by Ghenea et al. and shown to be
expressed mainly in intestinal cells
(Ghenea et al., 2001). The
homolog for PEX5 (prx-5/C34C6.6) binds to the peroxisomal targeting
signal 1 in a yeast two-hybrid system, supporting its designation as the PEX5
homolog (Gurvitz et al.,
2000
). The C. elegans homologs for PEX2, PEX12, PEX13 and
PEX19 were also identified by Petriv et al.
(Petriv et al., 2002
). The
C. elegans homologs of PEX1, PEX5, PEX6, PEX12
(prx-12/F08B12.2) and PEX13 (prx-13/F32A5.6) show the
greatest sequence conservation, which is more than 50% similar over the entire
protein length of each when compared with the human proteins. The PEX2
(prx-2/ZK809.7), PEX3 (prx-3/C15H9.8), PEX14
(prx-14/R07H5.1) and PEX10 (prx-10/C34E10.4) homologs are
not as conserved, but are nevertheless statistically significant. The PEX11
(prx-11/C47B2.8) homolog was recently identified by Li et al.
(Li et al., 2002
).
|
The human peroxins for which neither C. elegans nor
Caenorhabditis briggsae homologs could be identified are PEX7 (the
PTS2 receptor) and PEX16, which is a peroxisomal membrane protein required for
membrane biogenesis (Eitzen et al.,
1995). It is not surprising that we did not identify a PEX-7
homolog, given that C. elegans orthologs of PTS-2-containing proteins
have no detectable PTS-2 signal, but instead have acquired a PTS-1 signal
(de Vet et al., 1998
;
Motley et al., 2000
). We also
searched the entire C. elegans genome for any predicted open reading
frames conaining a PTS-2 motif and found none. In addition, PEX5 is expressed
as a short form, lacking the PEX7 interaction motif that had been identified
in the long form of PEX5 from mammals
(Dodt et al., 2001
).
There are ten additional peroxins that have only been isolated in one or more yeast species and we did not identify homologs of any of these peroxins, even using less-stringent search parameters. Thus, this survey indicates another aspect in which C. elegans peroxisomes seem to be more similar to the human than to yeast peroxisomes.
C. elegans peroxins are required for normal progression to
postembryonic development
We used dsRNA interference to evaluate the biological requirements for five
putative C. elegans peroxin genes. We targeted the C.
elegans homologs of the PTS1 receptor Pex5p, ATPase Pex6p, peroxisomal
membrane proteins Pex12p and Pex13p, and a cytosolic protein required for
membrane protein import, Pex19p. We injected dsRNA into young adults and
scored the progeny of the injected animals. dsRNA interference directed
against these five C. elegans peroxins all resulted in the same
phenotype arrest at the first larval stage of development. This larval
stage normally lasts about 12 hours at 20°C
(Byerly et al., 1976). The
arrested worms were viable and mobile, moving on the plate and through the
food as would be normal for an L1 worm. The length of the larval arrest varied
from 2 to 8 days (Fig. 2A). The
variation in the length of arrest is probably a reflection of the efficiency
of dsRNA interference and probably relates to the quantity or stability of the
dsRNA introduced. Interestingly, arrested nematodes that recovered (most
likely a result of the eventual degradation of the dsRNA) resume development
that occurs at a normal rate and appears to have a wild-type outcome. This
suggests that absence of peroxisomal function can be tolerated at least for
several days in arrested L1 larvae.
|
Because we observed similar phenotypes in all RNAi experiments, we chose to analyze the PTS1 receptor homolog prx-5/C34C6.6 in more detail. Fig. 2B shows an arrested prx-5(RNAi) nematode 4 days after hatching, and illustrates how severely the development is arrested compared with a time-matched wild-type nematode. In addition, we observed decreased brood size in the worms injected with peroxin dsRNA. The brood size of prx-5(RNAi) worms was about half that of an unc-22(RNAi) control (Fig. 2C). Eggs that were laid, however, hatched efficiently.
Data from genome-wide RNAi feeding and soaking experiments support our
findings. The chromosome III RNAi screen undertaken by Gonczy and colleagues
included both the Pex10p homolog that we did not target, and the Pex19p
homolog, which we did test (Gonczy et al.,
2000). In the case of the Pex19p homolog, a similar phenotype,
arrested development, was reported and, in the case of the Pex10p homolog, a
Gro phenotype of slowed development was scored. Maeda and colleagues reported
that targeting of F39G3.7, the Pex6p homolog, causes a sick phenotype in their
large-scale RNAi screen (Maeda et al.,
2001
). In both cases, the broad scope of the screens permitted
examination of only gross phenotypes, which were not pursued in detail.
Recently, Petriv et al. found that RNAi inactivation of prx-5, prx-12,
prx-13 and prx-19 resulted in a developmental delay and a
greatly reduced percentage of adult progeny 3 days following injection of
dsRNA homologous to these genes (Petriv et
al., 2002
). The authors were not able to specify whether this was
due to delayed development or to an arrest in development. Our results allow
us to conclude that lack of any of these peroxins results in a developmental
arrest at the L1 stage. Furthermore, Petriv et al. did not find developmental
delay when targeting dsRNA to the PEX-6 homolog, prx-6
(Petriv et al., 2002
).
prx-5 is required for peroxisomal import
We injected prx-5 dsRNA into animals harboring the
pHSP16-2GFPSKL;pRF4 extragenic array. When we heat-shocked
the progeny of these animals we noted that the GFPSKL was no longer
localized in punctate structures in the cells that expressed GFP. Instead, the
animals had GFPSKL distributed through the cytoplasm and nucleus (see
Fig. 3A,B, which shows the
intestinal cells of injected vs. a noninjected control animal). In a few
animals we were able to detect two classes of cells those that show
punctate GFP localization and those that have diffuse and nuclear GFP probably
due to incomplete RNAi effects (see Fig.
3C). We conclude that prx-5 is required for peroxisomal
import of proteins containing the C-terminal targeting signal SKL.
Importantly, the phenotype of failure to import SKL proteins to the
peroxisomes is exactly the phenotype in human cell lines from patients with
peroxisomal disorders (Santos et al.,
1988).
|
prx-5(RNAi)-arrested nematodes do not initiate postembryonic
development
We injected prx-5 dsRNA into strain LT483 that contains an
integrated GFP reporter under control of the ribonucleotide reductase
(rnr-1) gene promoter. This promoter is active only during the S
phase of the cell cycle and expression of GFP can be considered a marker for
dividing nuclei. After an embryo hatches and begins postembryonic development,
a series of invariant cell divisions occur
(Sulston and Horvitz, 1977).
In the prnr-1GFP strain these cells in the L1 animal
become labeled by GFP (see Fig.
4E for an early L1 animal and
Fig. 4F for an older L1
animal). prnr-1GFP;prx-5(RNAi) animals display an
abnormal pattern of GFP expression with at most one or two cells in each
animal labeled by GFP (Fig.
4A,B). To determine whether these GFP-labeled cells marked by GFP
result from postembryonic cell divisions, rather than remnants of undergraded
GFP from embryonic development, we placed eggs onto plates without food to
induce a starvation arrest of the hatched animals. When C. elegans
embryos hatch in the absence of food they fail to initiate postembryonic
development (Johnson et al.,
1984
). Interestingly, when we compared the
prnr-1GFP;prx-5(RNAi) GFP expression pattern to
starvation-arrested prnr-1GFP animals we also observed
only one or two cells per animal that express GFP
(Fig. 4C,D). Thus, in both
prx-5(RNAi) and starvation-arrested animals, the nuclear divisions
that report progression into L1 development do not occur. Furthermore, we
could not detect Q-cell migration when prx-5 dsRNA was injected into
a pmig-2GFP, nor could we see any axonal migration when
prx-5 dsRNA was injected into an UNC-129::GFP strain
(Colavita et al., 1998
;
Honigberg and Kenyon, 2000
).
These are events that should occur during the L1 stage but could not be
detected in prx-5(RNAi) animals (data not shown).
|
We used Nomarski optics to further examine starvation-arrested animals. Starvation-arrested worms accumulate refractile structures in the intestinal cells and throughout their bodies (Fig. 4). Although we could detect some of these refractile objects in our prx-5(RNAi)-arrested worms, they were not nearly as abundant and did not appear to increase significantly in number as length of arrest increased (Fig. 4). Thus, although prx-5(RNAi) animals are blocked at a similar stage as starvation-arrested nematodes, and are unable to initiate postembryonic development, there are morphological differences between peroxisome-deficient arrest and starvation-arrest. We conclude that peroxisomal function plays a crucial role in C. elegans postembryonic development and, more specifically, the absence of peroxisome function causes a very early and broad block because all postembryonic cell divisions and cell migrations we monitored did not occur.
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Discussion |
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Each of the peroxins we targeted by RNAi should leave the animal without
functional peroxisomes and affect all biochemical pathways that would normally
occur in this organelle. It is not clear at this point whether L1 progression
is blocked by the presence of a general toxin or by the failure to produce a
required metabolite. However, the coincidence in phenotypes of starved L1s and
peroxisome-deficient animals may provide a clue as to the biochemical reason
for arrest. It is proposed that in nematode food there is a molecule that acts
as a signal for progression into L1 or for exit out of the dauer stage into
reproductive growth (Bargmann and Horvitz,
1991). It is possible that without peroxisomal function,
metabolism of this `signal' molecule or one of its byproducts is disrupted,
leading to a block in the pathway that signals the progression of
postembryonic development.
Starvation-arrested nematodes have not been extensively studied. However,
both RNA levels and enzymatic activities of the glyoxylate cycle enzymes are
induced in L1-starvation arrested nematodes, as well as in embryos and dauer
larvae (Khan and McFadden,
1982; Liu et al.,
1997
; O'Riordan and Burnell,
1990
). The glyoxylate cycle enables plants and micro-organisms to
synthesize carbohydrate from fatty acids (via acetyl-CoA) and the enzymes for
this pathway are found in specialized peroxisomes called glyoxysomes
(Cooper and Beevers, 1969
;
Parsons et al., 2001
). The
C. elegans enzymes of the glyoxylate cycle do not contain a PTS1 at
their C-terminus, suggesting either that they may use a different pathway for
import into peroxisomes or that the glyoxylate cycle is not localized in the
peroxisome. The glyoxylate cycle is nevertheless dependent on the peroxisomal
ß-oxidation of fatty acids for a source of acetyl-CoA. In
prx-5(RNAi) animals, the activity of the glyoxylate cycle is probably
reduced due to lack of substrate. Interstingly, a peroxisomal protein comatose
(CTS) in Arabidopsis is required for the transition from dormancy to
germination and vegetative growth (Footitt
et al., 2002
). The CTS protein is required for lipid mobilization
and transport of acyl CoAs into the peroxisome and is thus a major control
point for the switch between the opposing developmental programs of dormancy
and germination. It is possible that peroxisomal function is required in
C. elegans to effect a switch over to postembryonic development in an
analogous developmental control point as in Arabidopsis
development.
C. elegans as a model system for the PBDs
In both plants and yeasts the ß-oxidation of fatty acids is
accomplished solely in the peroxisome. In humans there is an additional
ß-oxidation compartment, the mitochondria. Bioinformatic analysis of
ß-oxidation enzymes in C. elegans have provided initial evidence
that nematodes are similar to humans in that some homologs of the
ß-oxidation enzymes contain mitochondrial signal sequences, whereas
others contain a peroxisomal targeting signal. This is an indication that both
cellular compartments can carry out the oxidation of fatty acids
(Gurvitz et al., 2000). The
striking similarity to humans suggests that C. elegans may be
particularly well suited to addressing problems in peroxisome biochemistry
relevant to the human PBDs.
The PBDs are characterized by global developmental delay and defects in
neuroblast migration during development
(Moser, 1999). The
well-documented developmental program of C. elegans, coupled with its
transparency and ease of culture, make the nematode an attractive organism in
which to study the pathogenesis of the PBDs. Although all cell migrations and
neuronal growth were blocked in the marker strains that we examined, possibly
looking at migrations that occur earlier in development may shed light onto
the cause of the developmental arrest. C. elegans is the only model
system which allows such facile observation of the nervous system in the
background of disruption of peroxisome biogenesis.
In addition to the biochemical similarities, we have shown at the cellular
level that disruption of prx-5 leads to the mislocalization of a
peroxisome-targeted GFP. Failure to localize peroxisome-targeted proteins is
exactly the cellular phenotype found in patients in complementation group 2 of
the PBDs (Dodt et al., 1995;
Wiemer et al., 1995
). These
patients have defects in pex-5. The severity of the developmental
arrest phenotype in C. elegans leads to the feasibility of
development of genetic suppressor screens to overcome or circumvent the
peroxisomal deficiency. Genetic suppressor analysis may shed new insight into
disease mechanisms of peroxisomal biogenesis disorders and also enable us to
learn about mechanisms of developmental arrest associated with peroxisomal
deficiency.
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Acknowledgments |
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