Download Assembly of AO and DHAS - Journal of Cell Science

RESEARCH ARTICLE
2863
Alcohol oxidase and dihydroxyacetone synthase, the
abundant peroxisomal proteins of methylotrophic
yeasts, assemble in different cellular compartments
Mary Q. Stewart, Renee D. Esposito, Jehangir Gowani and Joel M. Goodman
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041, USA
Author for correspondence (e-mail: [email protected])
Accepted 24 April 2001
Journal of Cell Science 114, 2863-2868 (2001) © The Company of Biologists Ltd
SUMMARY
Alcohol oxidase (AO) and dihydroxyacetone synthase
(DHAS) constitute the bulk of matrix proteins in
methylotrophic yeasts, model organisms for the study of
peroxisomal assembly. Both are homooligomers; AO is a
flavin-containing octamer, whereas DHAS is a thiamine
pyrophosphate-containing dimer. Experiments in recent
years have demonstrated that assembly of peroxisomal
oligomers can occur before import; indeed the absence of
chaperones within the peroxisomal matrix calls into
question the ability of this compartment to assemble
proteins at all. We have taken a direct pulse-chase
approach to monitor import and assembly of the two major
proteins of peroxisomes in Candida boidinii. Oligomers of
AO are not observed in the cytosol, consistent with the
proteins inability to undergo piggyback import. Indeed,
oligomerization of AO can be followed within the
peroxisomal matrix, directly demonstrating the capacity of
this compartment for protein assembly. By contrast, DHAS
quickly dimerizes in the cytosol before import. Binding and
import was slowed at 15°C; the effect on AO was more
dramatic. In conclusion, our data indicate that peroxisomes
assemble AO in the matrix, while DHAS undergoes
dimerization prior to import.
INTRODUCTION
(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
Peroxisomal protein import has been the subject of fruitful
research in the past several years, and as a result many steps
in this process have been identified (Hettema et al., 1999;
Kunau, 1998; Subramani et al., 2000). One of two peroxisomal
targeting signals, PTS1 and PTS2, allows proteins destined to
peroxisomes to bind to cytosolic receptors (Pex5p or Pex7p for
PTS1 and PTS2, respectively). The substrate-receptor pair then
binds to a docking complex (containing at least Pex13p,
Pex14p and Pex17p) on the peroxisomal membrane. Several
other membrane-associated proteins function downstream in a
way that is ill-defined at present to promote translocation of
the substrate. The medical significance of peroxisomal protein
import is demonstrated by the peroxisomal deficiency diseases
Zellweger syndrome and neonatal adrenoleukodystrophy, in
which there is a global failure of peroxisomal matrix protein
import (Fujiki, 1997).
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
Key words: Peroxisomes, Microbodies, Protein trafficking,
Membrane translocation, Chaperones
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JOURNAL OF CELL SCIENCE 114 (15)
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.
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 TL100 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).
MATERIALS AND METHODS
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).
Cell culture and generation of activated spheroplasts
Candida boidinii was cultured in mineral medium (Sahm and Wagner,
1972) supplemented with 1% MeOH and 0.5 M MgSO4. Addition of
0.5 M MgSO4 to the culture medium was found to yield more
reproducibility in labeling and expression of peroxisomal proteins
during metabolic labeling. All of the following steps to generate and
activate spheroplasts were performed at 30°C with pre-warmed
reagents and incubations in a rotary shaker. 100 ml of cell culture at
1 OD600/ml was harvested by centrifugation at 6,750 g for 10 minutes,
resuspended in 30 ml H2O, and repelleted. Cells were resuspended in
10 ml 0.1 M Tris-SO4 (pH 9.3) with 10 mM DTT, and incubated for
10 minutes. Cells were harvested and washed in 30 ml 0.5 M MgSO4.
The pellet was resuspended in 10 ml 20 mM KHPi pH 7.5 with 0.5
M MgSO4 and 1% MeOH, and then 15 µg of 100T Zymolyase was
added. Spheroplasting was allowed to occur for approximately 45-60
minutes until conversion to spheroplasts was complete, as monitored
microscopically by the ability of added H2O to lyse a large majority
of the cells. 20 ml 0.5 M MgSO4 was then added, the cells were
pelleted at 5,820 g for 10 minutes and then resupended in 10 ml
mineral medium with MeOH and MgSO4 (identical to culture
medium) and incubated at 30°C for 1 hour to activate prior to labeling.
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-[Nmorpholino]ethanesulfonic acid) pH 5.5, 0.2 mM AEBSF (4-(2aminoethyl)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
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 SDSPAGE gels.
RESULTS
Analysis of peroxisomal targeting intermediates required a
fractionation procedure to quickly, cleanly and reproducibly
separate cytosol from peroxisomal matrix and membrane in
high yield following pulse-chase. Such a method is outlined in
Fig. 1A. Following pulse-chase of activated spheroplasts, cells
were quickly lysed and subjected to a single centrifugation to
separate cytosol (and light membranes, the SUP fraction) from
peroxisomes (and other organelles, the PEL fraction). Fig. 1B
demonstrates that this step results in the release of 94% of a
cytosolic marker, formaldehyde dehydrogenase, a measure
of the high efficiency of spheroplast lysis. Integrity of
peroxisomes can be assessed in Fig. 1C, illustrating a protein
stain of the fractions after SDS-PAGE. Alcohol oxidase and
dihydroxyacetone synthase are the most abundant proteins in
the cell, an indication of the massive proliferation of
peroxisomes when cells are incubated in methanol medium
(Goodman et al., 1984; Sahm et al., 1975). Very few of these
matrix proteins are seen in the SUP fraction compared with the
PEL fraction. Densitometry of these bands reveals at least 80%
of these markers fractionate in the PEL (AO is generally above
90%), indicating that almost all peroxisomes are intact.
Fig. 1D, illustrating an immunoblot against the integral
peroxisomal membrane protein Pmp47 (Goodman et al., 1986),
shows that all of the peroxisomal membrane fractionates with
the PEL fractions.
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).
Assembly of AO and DHAS
2865
The data show that the matrix fraction contained virtually all
our data is that the dimeric form of DHAS is the major if not
the AO and DHAS from the pellet (Fig. 1C) and no
the only species that crosses the peroxisomal membrane.
peroxisomal membrane (Fig. 1D). In summary, there is little
Having shown that AO is imported as a monomer and DHAS
cross contamination between cytosol (in the SUP), matrix and
as a dimer, we next performed import experiments at lower
peroxisomal membrane.
temperatures to attempt to uncover mechanistic differences in
Using pulse-chase and this fractionation protocol, we
the import of the two substrates. At 15°C, the association of
followed the trafficking of newly synthesized AO and DHAS,
both AO and DHAS with peroxisomes revealed a complex
and the results up to 15 minutes of chase are shown in Fig.
triphasic kinetic pattern with AO exhibiting slower kinetics in
2A,B. Alcohol oxidase chases from the cytosol (SUP) to
all three phases. The slower import kinetics of AO were not
peroxisomes (PEL) as has been shown previously in a more
attributable to a decrease in the rate of octamerization in the
detailed analysis (Goodman et al., 1984). A similar pattern was
matrix (data not shown). Although AO appears to cross the
observed for DHAS, although the kinetics of association with
peroxisomal membrane more slowly than DHAS, our data do
peroxisomes was considerably faster (Goodman, 1985). The
not allow us to conclude that mechanistic differences exist
fractions of AO and DHAS found in the SUP could not be
between the import of these two substrates.
sedimented at 100,000 g, indicating that they are in the cytosol
In summary, we have been able to monitor simultaneously
(data not shown). During this time course both proteins
the import and assembly of endogenous peroxisomal 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.
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.
Fig. 1. Separation of peroxisomal subcompartments from activated
Parallel fractions from the same experiment were spheroplasts. (A) Outline of procedure. After metabolic labeling, activated
centrifuged in the sucrose gradients for longer times spheroplasts are subjected to osmotic lysis at pH 5.5. An organellar pellet (PEL)
to separate monomeric from dimeric DHAS (Fig. 4). and supernatant (SUP) are generated by centrifugating the lysate at 20,000 g.
In contrast to AO, DHAS clearly dimerizes in the (There are no prespins.) The pellet is resuspended in buffer at pH 9.0 to lyse
cytosol prior to import. Even at 0 minutes of chase peroxisomes, and then spun at 100,000 g, generating a supernatant containing
about half of DHAS is already a dimer; the ratio peroxisomal matrix (MAT) and a pellet containing peroxisomal membrane
(MEM). Analysis of markers in a typical experiment is shown in B-D.
increases further with longer chase. Dimeric DHAS is (B) Formaldehyde dehydrogenase, a cytosolic enzyme. There is little
seen in the matrix, even at the earliest time points. contamination of cytosol in the matrix. (C) Coomassie Blue staining indicates
Considering the efficient dimerization of DHAS in the the integrity of peroxisomes after cell lysis, and the subsequent release of
cytosol and the appearance of dimer in the matrix at matrix. (D) Immunoblot with anti-Pmp47, an integral peroxisomal membrane
the earliest time point, the simplest interpretation of protein.
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JOURNAL OF CELL SCIENCE 114 (15)
dimers assemble quickly in the cytosol before translocation
into the matrix.
DISCUSSION
Fig. 2. The import of AO and DHAS from cytosol into peroxisomal
matrix. Activated spheroplasts were labeled as described and chased
for the times indicated. Spheroplasts from each time point were
subjected to fractionation. (A) Fractions were immunoprecipitated
with antibody to the proteins indicated and subjected to SDS-PAGE
and fluorography. (B) Immunoprecipitates were quantified by
densitometry. Results were expressed as a ratio of the label in the
pellet compared with supernatant and pellet. (C) Sample buffer was
added directly to the PEL, MAT and MEM fractions before SDSPAGE and fluorography.
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
To examine the physiological significance of oligomeric import
and determine whether matrix is capable of forming oligomeric
proteins, we undertook a pulse-chase approach to directly
observe these processes. We chose AO and DHAS from C.
boidinii because each of these proteins comprise over 10% of
cell protein and are very easy to detect. Our pulse-chase results
depended on technical improvements over previous work in C.
boidinii (Goodman, 1985; Goodman et al., 1984). Cell growth
and spheroplasting conditions were adjusted to yield efficient
and reproducible radiolabeling and complete cell lysis while
keeping peroxisomes intact. This procedure obviated the need
to discard a low-speed pellet, which can contain most of the
cellular organelle mass (data not shown), thus skewing the
correct ratio of substrates in cytosol versus in organelles. The
subsequent lysis of the peroxisomal membrane by raising
the pH allowed a fast separation of cytosol, peroxisomal
membrane and matrix. This procedure was then coupled with
sucrose velocity sedimentation to determine the compartment
in which assembly of oligomers occurred.
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 wildtype cells catalyze this process in the cytosol of the mutants
Assembly of AO and DHAS
2867
Fig. 3. Alcohol oxidase is imported as a
monomer. The supernatant and matrix
fractions from pulse-chased spheroplasts
were subjected to velocity sedimentation.
Fractions of the sucrose gradients were
immunoprecipitated with anti-AO
antiserum. BSA and mature AO were used
as markers of the monomeric and octameric
species. Asterisk represents an AO
degradation product. Open arrowhead
indicates intermediate AO forms on
gradient.
Fig. 4. Dihydroxyacetone synthase is
imported as a dimer. Analysis similar to that
in Fig. 3 except the gradients were spun for a
longer time and fractions were subjected to
immunoprecipitation with anti-DHAS
antiserum. BSA and mature DHAS served as
markers of monomeric and dimeric species.
(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.
We thank Pamela A. Marshall and members of the Goodman lab
for many insightful discussions. Additional thanks to the laboratory
of Margaret Phillips. The authors gratefully acknowledge funding
from NIH grant GM-31859 and Robert A. Welch Foundation grant I1085 for this work.
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