Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041, USA
Author for correspondence (e-mail: Joel.Goodman{at}UTSouthwestern.edu )
Accepted April 24, 2001
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SUMMARY |
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Key words: Peroxisomes, Microbodies, Protein trafficking, Membrane translocation, Chaperones
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INTRODUCTION |
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While most organelles that undergo protein import accept only unfolded
substrates, several lines of evidence indicate that peroxisomes import folded
proteins and oligomers: (1) monomers of oligomeric peroxisomal proteins and
carriers that lack a PTS can be imported if coexpressed with partners
containing a PTS (Elgersma et al.,
1996; Glover et al.,
1994
; McNew and Goodman,
1994
); (2) microinjected
protein aggregates and even colloidal gold particles that contain a PTS can be
imported (Walton et al.,
1995
); (3) aminopterin does
not inhibit the import of dihydrofolate reductase into peroxisomes (Hausler et
al., 1996
); (4) mistargeting
of alanine:glyoxylate aminotransferase to mitochondria in the disease human
primary hyperoxaluria type I requires mutations that both generate a
mitochondrial targeting signal and disrupt dimerization (Leiper et al.,
1996
); (5) in vitro import of
protein dimers has been demonstrated (Brickner et al.,
1997
); and (6) the PTS
receptors Pex5p and Pex7p can be found within the peroxisomal matrix
suggesting that they accompany substrate into this compartment and then
recycle back to the cytosol (Dodt and Gould,
1996
; Szilard et al.,
1995
; Zhang and Lazarow,
1995
).
Recent evidence has shown that peroxisomes are not unique in their ability
to import folded proteins. The cvt pathway for the import of cytosolic
precursors into the yeast vacuole involves the engulfment of cytosolic
oligomers by membrane and fusion to the vacuole (Klionsky and Ohsumi,
1999). Thylakoid membranes of
plants can export proteins from the stroma through three different pathways,
one of them (the
pH pathway) accepts folded precursors as substrates
(Creighton et al., 1995
). A
similar mechanism of export, the twin-arginine translocation pathway, is found
in E. coli (Hunds et al.,
1998
).
While many experiments have clearly shown that peroxisomes can import oligomers, questions remain about the physiological importance of this mechanism. If a protein can be imported into peroxisomes as an oligomer, does it normally utilize that pathway? Is oligomeric import the exclusive pathway for peroxisomes? Since classical chaperones have not been found within the organelle, do peroxisomes have the capacity to assemble monomers into active oligomers, and do they ever do so?
To answer these questions we used a pulse-chase approach in the yeast
Candida boidinii to ascertain the relationship between peroxisomal
import and oligomerization. Methylotrophic yeasts such as C.
boidinii, when cultured on methanol, contain large peroxisomes that
consist almost exclusively of two oligomeric proteins, alcohol oxidase (AO),
an octamer, and dihydroxyacetone synthase (DHAS), a dimer (Goodman,
1985; Veenhuis and Harder,
1987
). We now report that AO
is imported into peroxisomes over several minutes as a monomer, consistent
with a previous finding that AO lacking a PTS could not be imported with
subunits containing a PTS (Waterham et al.,
1997
). Octamerization of AO is
demonstrated to occur in the peroxisomal matrix. By contrast, DHAS quickly
dimerizes in the cytosol prior to its import.
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MATERIALS AND METHODS |
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Metabolic labeling and organelle fractionation
Activated spheroplasts were radiolabeled for 5 minutes with 30 µCi
35S-methionine (New England Nuclear Life Science Products, Boston,
MA) per 500 µl spheroplasts. Unlabeled methionine/cysteine, a solution of
250 mM each, was then added to a final concentration of 10 mM each for the
chase, and cells were allowed to incubate for the times indicated. At each
time point of chase, 200 µl of cell suspension was quickly pipetted into 1
ml of pre-chilled 1 M sorbitol on ice. (For the temperature shift experiments,
the 15 degree shift was performed in a water bath using small volumes of cell
suspension (less than 1 ml) and in thin plastic tubes to ensure rapid
temperature equilibration.) Cells were harvested by centrifugation at 5,000
g spin for 5 minutes at 4°C in a microfuge. They were then
resuspended in 200 µl 1 M SMA (1 M sorbitol, 5 mM MES
(2-[N-morpholino]ethanesulfonic acid) pH 5.5, 0.2 mM AEBSF
(4-(2-aminoethyl)benzenesulfonyl fluoride), then lysed by the addition of 800
µl 0.25 M (referring to sorbitol concentration) SMA. The lysate was
vortexed three times for 10 seconds with a 5 minute incubation on ice in
between each vortexing. Separation of cytosol (SUP) and organellar pellet
(PEL) was performed by centrifugation at 20,000 g (in a
microfuge) for 15 minutes at 4°C. For experiments in which the PEL was
further fractionated, a duplicate PEL fraction was resuspended in 500 µl 30
mM Tris-HCl pH 9.0 with 5 mM EDTA to lyse peroxisomes contained therein.
Separation of membrane (MEM) and matrix (MAT) fractions was then performed by
a single centrifugation at 100,000 g for 15 minutes at 4°C
in a Beckman TL-100 rotor. Immunoprecipitation of AO and DHAS was performed on
each fraction using antibodies to the corresponding proteins (see Reagents and
other methods). Radiolabel was visualized by fluorography and the dried gel
was exposed to BioMax single emulsion film (Eastman Kodak, Rochester, NY).
Analysis of oligomeric state of AO and DHAS
The oligomeric state of AO contained in SUP and MAT fractions was
determined by velocity sedimentation in sucrose gradients and
immunoprecipitation of gradient fractions as previously reported (Goodman et
al., 1984). A similar
procedure was performed to establish the oligomeric state of DHAS except
gradients were centrifuged for 12 hours instead of 3.5 hours. Bovine serum
albumin (BSA), mature DHAS and AO were used as molecular mass standards in the
gradients and visualized by Coomassie Blue staining of SDS-PAGE gels.
Reagents and other methods
Polyclonal antibodies for AO (Goodman et al.,
1984) and DHAS (Goodman,
1985
), and a monoclonal
antibody against Pmp47 (Goodman et al.,
1986
) used in immunoblots,
have been previously described. Immunoblots were performed with Enhanced
Chemiluminescence reagents as directed by the manufacturer (Amersham Life
Science, Arlingon Heights, IL). SDS-PAGE (Laemmli,
1970
) was performed with 9%
gels and a separating gel of pH 9.2. Formaldehyde dehydrogenase was assayed
enzymatically (Egli et al.,
1980
).
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RESULTS |
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To release matrix from the PEL fraction, the pH was raised to 9.0 (Goodman
et al., 1984), and the sample
was subjected to high speed centrifugation
(Fig. 1A). Virtually all the AO
and DHAS bands remained in the supernatant (MAT,
Fig. 1C), while Pmp47 was
recovered only in the pellet (MEM, Fig.
1D). The data show that the matrix fraction contained virtually
all the AO and DHAS from the pellet (Fig.
1C) and no peroxisomal membrane
(Fig. 1D). In summary, there is
little cross contamination between cytosol (in the SUP), matrix and
peroxisomal membrane.
Using pulse-chase and this fractionation protocol, we followed the
trafficking of newly synthesized AO and DHAS, and the results up to 15 minutes
of chase are shown in Fig.
2A,B. Alcohol oxidase chases from the cytosol (SUP) to peroxisomes
(PEL) as has been shown previously in a more detailed analysis (Goodman et
al., 1984). A similar pattern
was observed for DHAS, although the kinetics of association with peroxisomes
was considerably faster (Goodman,
1985
). The fractions of AO and
DHAS found in the SUP could not be sedimented at 100,000 g,
indicating that they are in the cytosol (data not shown). During this time
course both proteins appeared in the peroxisomal matrix. We always saw a lag
in appearance of both labeled proteins between the PEL and in the MAT
(Fig. 2A). Very little AO or
DHAS was seen in the MEM fraction at any time point. This loss suggested that
the immunoprecipitation from the membrane fraction was ineffective. When the
fractions were visualized directly without immunoprecipitation
(Fig. 2C), there was clearly a
significant amount of newly synthesized AO and DHAS associated with the
membrane fraction at early time points. In fact, more AO precursor is observed
in the membrane than the matrix at 0 and 1 minutes.
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The oligomeric state of AO in the cytosol and matrix, as determined by velocity sedimentation, as a function of time after synthesis, is shown in Fig. 3. AO is a monomer in the cytosol at all time points examined, suggesting that oligomerization does not happen in this compartment. As AO appears in the matrix (1 minute chase), it is mostly monomer. Most of the monomer that we detect in the matrix is very sensitive to proteolysis during sample processing, much more so than monomers in the cytosol, and is clipped to a stable intermediate (indicated by asterisks in Fig. 3). We have been unable to fully prevent this degradation with protease inhibitors. At longer chase times, octamer accumulates in the matrix, while monomers still enter peroxisomes (15 minutes). In some experiments AO of an intermediate size is seen (15 minute point, open arrowhead, more apparent at lower temperatures), which may indicate dimeric or tetrameric forms. This experiment demonstrates that the peroxisomal matrix has the capacity to oligomerize AO.
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Parallel fractions from the same experiment were centrifuged in the sucrose gradients for longer times to separate monomeric from dimeric DHAS (Fig. 4). In contrast to AO, DHAS clearly dimerizes in the cytosol prior to import. Even at 0 minutes of chase about half of DHAS is already a dimer; the ratio increases further with longer chase. Dimeric DHAS is seen in the matrix, even at the earliest time points. Considering the efficient dimerization of DHAS in the cytosol and the appearance of dimer in the matrix at the earliest time point, the simplest interpretation of our data is that the dimeric form of DHAS is the major if not the only species that crosses the peroxisomal membrane.
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Having shown that AO is imported as a monomer and DHAS as a dimer, we next performed import experiments at lower temperatures to attempt to uncover mechanistic differences in the import of the two substrates. At 15°C, the association of both AO and DHAS with peroxisomes revealed a complex triphasic kinetic pattern with AO exhibiting slower kinetics in all three phases. The slower import kinetics of AO were not attributable to a decrease in the rate of octamerization in the matrix (data not shown). Although AO appears to cross the peroxisomal membrane more slowly than DHAS, our data do not allow us to conclude that mechanistic differences exist between the import of these two substrates.
In summary, we have been able to monitor simultaneously the import and assembly of endogenous peroxisomal proteins in C. boidinii. Our data show that AO enters peroxisomes as monomers and that the peroxisomal matrix has the capacity to assemble octamers from this precursor. By contrast, DHAS dimers assemble quickly in the cytosol before translocation into the matrix.
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DISCUSSION |
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These observations are novel in two ways. First, this is the only direct
demonstration to our knowledge that the peroxisomal matrix can support the
formation of quaternary structure. Our results suggest that the import of
microinjected AO octamers into peroxisomes does not reflect the physiological
import mechanism for these substrates (Walton,
1996). Once AO monomers cross
the membrane, an intermediate can be detected that migrates between monomer
and octamer in sucrose velocity gradients. It is not clear whether this
oligomeric form represents homomers or heteromers. Why does oligomerization of
AO occur in the matrix and not the cytosol? There are three possibilities,
which are not mutually exclusive: (1) conditions within the matrix are
favorable for spontaneous assembly of AO octamers (an optimal pH or specific
ion concentration, for example); (2) proteins within the matrix catalyze
assembly into octamers; or (3) molecular chaperones within the cytosol prevent
assembly. Chaperones may not be required for octamer formation since monomers
that have been dissociated from holoenzyme by treatment with glycerol can
spontaneously reassemble into active enzyme (Boteva et al.,
1999
). However, dissociation is
irreversible once monomers are sufficiently denatured to release cofactor.
Does the monomeric form that is imported into peroxisomes contain FAD?
Riboflavin starvation in the yeast Hansenula polymorpha leads to
inefficient import of AO, suggesting that binding to FAD normally precedes
import (Evers et al., 1994
).
Nevertheless, data is also available to suggest involvement of chaperones in
the assembly process. Cytosolic hsp70 is known to keep proteins unfolded and
competent for import into mitochondria and endoplasmic reticulum (Deshaies et
al., 1988
) and could play a
similar role in preventing AO assembly in the cytosol. There is also evidence
for the involvement of matrix chaperones: mutants in H. polymorpha
that fail to generate peroxisomes assemble AO in the cytosol, as if folding
factors found in the matrix in wild-type cells catalyze this process in the
cytosol of the mutants (Tan et al.,
1995
). We have attempted to
form oligomers by incubating metabolically pulse-labeled monomers with
concentrated matrix but have not yet been successful since we have been unable
to control the action of a protease activity to which monomeric AO is
particularly sensitive (data not shown). This activity is apparent in Figs
3, 5.
Second, this report is the first study in which the extent to which a
protein oligomerizes prior to peroxisomal import has been determined. Our data
show that DHAS extensively and perhaps completely dimerizes prior to import
since very little if any monomeric DHAS is detected in the peroxisomal matrix.
These data suggest that oligomeric import is the normal mechanism for
translocation of this substrate, unlike AO. Since our results are based on
kinetic analysis alone, we cannot rule out the possibility that DHAS binds to
the membrane as a dimer, quickly dissociates into monomers, and reassembles
into dimers on the matrix side of the membrane following translocation. This
pathway appears very unlikely. The only known chaperones that have the ability
to dissociate and unfold proteins and protein aggregates are those in the
Hsp100 family, and studies in vivo and in vitro have shown that this process
requires several minutes (Glover and Lindquist,
1998); we see no such lag in
our import reactions, we do not detect a buildup of DHAS in the membrane prior
to import, and the amount of monomers at any time in the matrix is negligible.
Certainly the simplest interpretation of our data is that the dimeric form
itself is imported.
The experiments at low temperature were an attempt to kinetically dissect the import process of AO and DHAS. Import is slowed for both substrates but especially for AO. Further experiments in a more genetically manipulatable system may allow us to pinpoint the rate limiting step of the import process at a molecular level.
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ACKNOWLEDGMENTS |
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