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INTRODUCTION |
In eukaryotic cells most organellar proteins are synthesized in
the cytosol and sorted to their target compartments by unique signals.
These signals are recognized by specific receptor proteins, which are
either located in the cytosol (e.g. signal recognition particle for the endoplasmic reticulum (1)) or bound to the membrane of
the organelle (e.g. TOM proteins at the mitochondrial outer
membrane (2)).
Proteins destined for the peroxisomal matrix are encoded by nuclear
genes and synthesized in the cytosol. Specific topogenic signals have
been detected at the extreme C terminus (the PTS1) or in the N terminus
of the protein (termed PTS2) (3). It is now widely accepted that the
PTS1 and PTS2 are recognized in the cytosol by their specific receptor
molecules, Pex5p and Pex7p, and that this interaction in fact initiates
the actual protein import process. Both pathways converge at the
docking site, consisting of at least Pex13p, Pex14p, and Pex17p.
Maintenance of the correct stoichiometry of the components of the
docking complex seems to be important since relatively minor
alterations in the level of Pex14p already affect normal import (4).
Another peroxin complex, consisting of the two zinc-binding proteins
Pex10p and Pex12p, most likely binds Pex5p at a later stage after
docking (5, 6). It can be envisaged that both complexes reflect an
import cascade with different affinities for the receptor-cargo
complex. These interactions could lead to the dissociation of the cargo from the receptor, and the hand over of the cargo molecule to the
translocation machinery. Alternatively, the receptor-cargo complex may be completely translocated across the peroxisomal membrane,
followed by dissociation of the complex in the peroxisomal lumen (7).
Indeed, in Hansenula polymorpha we have obtained evidence
for this pathway in which Pex4p, an ubiquitin-conjugating enzyme, plays
a role (8).
In yeast, deletion of the PEX5 gene results in a general
block in PTS1 matrix protein import. However, at PTS2 protein-inducing conditions (e.g. oleate, in case of H. polymorpha
primary amines), small peroxisomes were observed in these mutants since
both the PTS2 import and the insertion of peroxisomal membrane proteins are independent of Pex5p function (9, 10). When H. polymorpha pex5 mutant cells were grown in carbon-limited chemostats on
glucose/choline, conditions that strongly induce peroxisome
proliferation, a few small peroxisomes were observed. These organelles
contained the PTS2 protein amine oxidase
(AMO,1 induced by choline),
whereas the PTS1 proteins alcohol oxidase, dihydroxyacetone synthase,
and catalase were mislocalized in the cytosol (11). However, under
AMO-repressing conditions during growth of cells on methanol/ammonium
sulfate, peroxisomes are fully absent in pex5 cells (12).
Here we analyzed whether such cells contained peroxisomal membrane
structures and normal peroxisomal membrane proteins. We have used the
integral peroxisomal membrane protein Pex10p, carrying a C-terminal Myc
tag (Pex10p.myc) as a marker protein for the detection of these
structures. Also, we studied whether peroxisome development upon
induction of AMO protein initiates from such structures or whether
alternative mechanism existed. The results of these studies are the
focus of this paper.
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MATERIALS AND METHODS |
Organisms and Growth Conditions--
The H. polymorpha strains used in this study are listed in Table
I. The cells were cultivated in mineral
media (13) containing 0.5% (w/v) glucose, 0.5% (v/v) ethanol, or a
mixture of 0.15% (v/v) glycerol and 0.2% (v/v) methanol as carbon
sources supplemented with 0.25% (w/v) ammonium sulfate, 0.25% (w/v)
ethylamine, or 0.25% (w/v) methylamine as respective nitrogen sources
at 37 °C. For the selection of mutants, solid YND media were used,
containing 2% (w/v) yeast nitrogen base (Difco) and 1.5% (w/v)
agar.
Construction of H. polymorpha WT and pex5 Strains, Producing
Pex10p.myc--
A leucine-auxotrophic PEX5 deletion strain,
suitable for transformation with ScLEU2-based plasmids (14),
was constructed as follows. By PCR, a BamHI site was
introduced downstream from the 5'-untranslated region of
PEX5, using the primer 5'-GGG GGA TCC ATT GAT
GGT TTG TGC TCA AG-3' (BamHI site underlined). A 2.2-kb Bgl2 (sticky ends filled-in using Klenow
enzyme)-BamHI DNA fragment, containing the HpURA3
gene (15), was inserted between the engineered BamHI site
and the genomic NruI site at position +1504 in the PEX5
open reading frame. By using BsiWI and
NaeI, a PEX5 disruption cassette was obtained,
containing the HpURA3 gene flanked by 0.2-kb PEX5-5'-untranslated region and 0.3-kb PEX5 open
reading frame and 3'-untranslated region, which was used to transform
H. polymorpha NCYC495 leu
ura
. One transformant was selected
which was uracil prototrophic, methanol utilization-deficient and
showed the expected genomic changes after Southern blot analysis.
The chimeric gene, encoding Pex10p.myc, was constructed by PCR-mediated
amplification of the PEX10 gene of pET4 (Table I) using the
following primers (oligonucleotides were obtained from Life
Technologies, Inc.): upstream 5'-AGA GGA TCC ATG TTT AAG CTT TTG TC-3' (BamHI-site underlined) and downstream 5'-AGA
GAA TTC TTA CAA GTC TTC CTC AGA AAT AAG CTT CTG
CTC TCG TAG AGG CAA CAG CTG-3' (EcoRI site underlined
and the nucleotides coding for the Myc epitope (16) are depicted in
italics). The PCR product was ligated as a 0.94-kb
BamHI-EcoRI fragment in pBluescript II
KS+, digested with BamHI and EcoRI.
The resulting plasmid, pFAS01, was digested with BamHI and
XhoI, and the 0.94-kb fragment was inserted into
BamHI-SalI-digested pHIPX4 (Table I) under
control of the alcohol oxidase promoter, resulting in plasmid
pHIPX4-PEX10.MYC. The plasmid pHIPX4-PEX10.MYC
was introduced into a H. polymorpha pex10-1 mutant strain by
electroporation (17) to check the functionality of the hybrid protein.
Also it was linearized with StuI and integrated at the
PAOX locus of H. polymorpha pex5 strain (17).
Transformants were selected on YND plates and site-directed integration
of the plasmid at the PAOX locus was confirmed by Southern blot
analysis. pex5 strains containing two copies of the pHIPX4
derivatives were selected for further studies.
Biochemical Methods--
Crude extracts of H. polymorpha cells were prepared as detailed by Baerends et
al. (18). Cell fractionation procedures (19) and cytochrome
c oxidase activities measurements (20) were performed as
described. Protein concentrations were determined using the Bio-Rad
Protein Assay system (Bio-Rad) using bovine serum albumin as a
standard. SDS-polyacrylamide gel electrophoresis (21) was carried out
as described, and gels were subjected to Western blotting (22).
Nitrocellulose blots were decorated using specific polyclonal antibodies against various H. polymorpha proteins.
Electron Microscopy--
Cells were fixed and prepared for
electron microscopy as described previously (23). Immunolabeling was
performed on ultrathin sections of unicryl-embedded cells, using
specific antibodies against various proteins and gold-conjugated goat
anti-rabbit or goat anti-mouse antibodies (23).
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RESULTS |
Characterization of Peroxisomal Structures in H. polymorpha pex5
Cells--
The presence of putative peroxisomal membrane structures in
H. polymorpha pex5 cells was first determined in cells,
grown at moderate peroxisome-induction conditions on ethanol/ammonium sulfate. Under these conditions, WT cells contain few organelles, characterized by the presence of isocitrate lyase and malate synthase, key enzymes of the glyoxylate cycle. Electron microscopic analysis showed that in such pex5 cells peroxisomal structures were
not detectable despite extensive research also including analysis of
serial sections (Fig. 1A).
This was remarkable since the levels of the membrane protein Pex14p was
equal to WT control cells and over 5-fold enhanced, compared with cells
grown on glucose (Fig. 2). Pex3p was also
detectable but not enhanced compared with glucose-grown WT cells,
independent whether ammonium sulfate or ethylamine was used as the
nitrogen source (Fig. 2). Ethylamine metabolism is mediated by AMO, a
PTS2 protein of H. polymorpha. At the morphological level,
however, replacement of ammonium sulfate by ethylamine led to the rapid
development of peroxisomes in ethanol-grown pex5 cells.
Typically, one or very few peroxisomes developed within 1 h of
cultivation in the new ethylamine environment (Fig. 1B). As
expected, these organelles were characterized by the presence of AMO
protein. This raises the question of the origin of these new
organelles. A reason for the virtual absence of peroxisomal remnants in
ethanol/ammonium sulfate-grown cells could be that these structures
were below the limit of detection of electron microscopy. To enhance
the level of peroxisomal proteins, pex5 cells were
subsequently grown on glycerol/methanol/ammonium sulfate. Since
H. polymorpha pex strains cannot grow on methanol alone (24), glycerol was applied as the growth substrate using methanol as
additional energy source and peroxisome inducer. In this way growth
conditions are created that are similar for both pex5 and WT
control cells, at least until the mid-exponential growth phase. WT
cells grown under these conditions contain several large peroxisomes (25). Western blot analysis revealed that the levels of Pex3p and
Pex14p in glycerol/methanol-grown pex5 and WT cells were
comparable but strongly enhanced compared with the levels in ethanol-
or glucose-grown cells (Fig. 2). This indicated that deletion of the
PEX5 gene did not affect the amounts of peroxisomal membrane proteins in the cells.

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Fig. 1.
Demonstration of the presence of peroxisomal
remnants and peroxisomes containing AMO in H. polymorpha
pex5 cells. A, overall morphology of
ethanol/ammonium sulfate-grown H. polymorpha pex5
cells. In these cells no peroxisomal structures are visible. After 60 min of cultivation on ethanol/ethylamine, a small peroxisome was
observed (B). C, overall morphology of
methanol/ammonium sulfate-induced pex5 cells that contain
few small peroxisomal remnants (arrow = peroxisomal
remnants), characterized by the presence of Pex14p, as was evident upon
labeling using -Pex14p antibodies (C, inset). On
glycerol/methanol/methylamine the cells contained few peroxisomes
(arrow) that were formed within 2 h (D). By
using specific -AMO antibodies, these structures were labeled
(D, inset). A-D, KMnO4 fixation;
insets, glutaraldehyde fixation. Electron micrographs
were taken of glutaraldehyde-fixed cells, poststained with uranyl
acetate, unless otherwise indicated. The abbreviations used are:
M, mitochondrion; N, nucleus; P,
peroxisome; V, vacuole. The bar represents 0.5 µm. Bar insets, 0.25 µm.
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Fig. 2.
Western blot analysis of crude extracts
prepared from H. polymorpha WT and pex5
cells cultivated at various conditions. Cells were grown on
glucose/ammonium sulfate (GA), ethanol/ammonium sulfate
(EA), ethanol/ethylamine (EE),
glycerol/methanol/ammonium sulfate (GMA), and
glycerol/methanol/methylamine (GMM). The levels of the
peroxisomal membrane proteins Pex3p and Pex14p were determined by
decorating the Western blots with specific antibodies against these
proteins. Glucose-grown cells contained minor amounts of these
proteins. Upon growth on ethanol-containing medium, the level of Pex3p
was comparable to glucose-grown cells, but Pex14p levels were enhanced.
Both proteins were strongly induced in cells grown on
glycerol/methanol. The protein band indicated by the
asterisk on the Western blots decorated with Pex3p
antibodies represents an aspecific mitochondrial protein (30).
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Electron microscopic analysis of glycerol/methanol/ammonium sulfate
grown pex5 cells revealed the presence of a cluster of membranous structures (Fig. 1C). These structures apparently
represented peroxisomal membrane remnants, because they could be
specifically labeled in immunocytochemical experiments using antibodies
against the peroxisomal membrane protein Pex14p (Fig. 1C,
inset). However, they did not contain alcohol oxidase,
dihydroxyacetone synthase, and catalase, all PTS1 proteins and key
enzymes of methanol metabolism. In medium with methylamine as sole
nitrogen source instead of ammonium sulfate, the pex5 cells
contained distinct peroxisomes, characterized by the presence of the
PTS2 protein AMO protein (Fig. 1D). This clear-cut
morphological phenotype allowed us to analyze the fate of the membrane
remnants during the adaptation of cells, pre-grown on
glycerol/methanol/ammonium sulfate, to glycerol/methanol/methylamine-containing media to resolve whether these
structures take part in the formation of new AMO-containing peroxisomes
or whether alternative mechanisms exist.
Labeling of the Peroxisomal Membrane Structures in pex5 Cells with
Pex10p.myc--
H. polymorpha has the advantage
over other yeast that PTS2 proteins are repressed or below the level of
detection in cells grown on glucose, glycerol, or methanol in the
presence of ammonium sulfate. Previous data showed that the PTS2 import
machinery is induced by primary amines, used as sole nitrogen source
(26). We have taken advantage of these properties in our studies to determine whether the peroxisomal membrane remnants can develop into
peroxisomes upon induction of AMO protein by (m)ethylamine.
The second crucial condition to conduct the above experiments is the
possibility to tag specifically these membrane structures. To achieve
this, a hybrid gene was constructed that encoded a C-terminal
Myc-tagged version of the peroxisomal membrane protein Pex10p
(Pex10p.myc) and was placed under control of the alcohol oxidase
promoter (PAOXPEX10.MYC). This gene was
selected since it is known that overproduction of Pex10p does not
affect peroxisome integrity and protein import competence (27). Also,
Pex10p.myc has been shown to normally function in man (28).
To test the function of Pex10p.myc in H. polymorpha, we
introduced the PEX10.MYC expression cassette in the original
H. polymorpha pex10-1 mutant (27). The resulting
transformant grew on methanol at WT rates (data not shown). This
suggested that Pex10p.myc functionally complemented the peroxisomal
defect in pex10-1 cells, since any defect in PTS1 (and thus
alcohol oxidase) import would prevent growth of cells on methanol (24).
This was confirmed by electron microscopic analysis, which revealed
that methanol-grown cells of this transformant contained normal
peroxisomes. Moreover, immunocytochemistry showed that the location of
Myc protein was confined to peroxisomal membranes, confirming that
indeed Pex10p.myc was normally sorted to the peroxisomal membrane (not shown).
Next, we introduced the PAOXPEX10.MYC cassette in
the H. polymorpha pex5 strain. A transformant with two
copies of PAOXPEX10.MYC integrated in the genome was
selected and cultivated in glycerol/methanol/ammonium sulfate-containing media to induce Pex10p.myc and peroxisomal protein
synthesis. Electron microscopy demonstrated that these cells contained
clusters of membranous structures (Fig.
3A) that behaved like normal
cell constituents in that they were donated to newly developing buds
(not shown; compare Fig. 9A). These structures were the sole
sites of gold labeling in immunocytochemical experiments, using
-Myc
antibodies (Fig. 3B). Similar results were obtained when
-Pex3p,
-Pex10p, and
-Pex14p antibodies were used (shown for
Pex10p; Fig. 3C), indicating that these structures represent peroxisomal membrane structures. Analysis of series of consecutive sections revealed that these clusters were invariably associated with
the nucleus and generally include over 20 small vesicles (Fig.
4). Taken together, these data imply that
the Pex10p.myc marker protein had specifically accumulated at the
peroxisomal membrane remnants in glycerol/methanol/ammonium
sulfate-grown pex5 cells (Fig. 1C) and, thus, is
a suitable component to tag these structures.

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Fig. 3.
Immunocytochemical analysis of
glycerol/methanol/ammonium sulfate-grown H. polymorpha pex5
cells containing the PAOXPEX10.MYC
expression cassette. The cells contained few small membrane
structures (A, indicated by the arrow) containing
Pex10p.myc (B, -Myc antibodies). Pex10p is also present
in these structures, as was evident upon labeling experiments using
-Pex10p antibodies (C). A, KMnO4
fixation.Electron micrographs were taken of glutaraldehyde-fixed
cells, poststained with uranyl acetate, unless otherwise indicated. The
abbreviations used are: M, mitochondrion; N,
nucleus; V, vacuole. The bar represents 0.5 µm
but the bar in B and C represents 0.25 µm.
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Fig. 4.
Six consecutive sections out of a series of a
glycerol/methanol/ammonium sulfate-grown
pex5::PAOXPEX10.MYC
cell (A F, KMnO4 fixation).
These sections clearly demonstrate the vesicular nature of peroxisomal
membrane remnants in this strain. C, three structures are
selected (white box) that actually represent vesicles, since
in the previous (B) and the following section (D)
of the cell only small fragments are present. These selected
peroxisomal remnants are not detected in other sections of the cell. In
addition, in A and B relatively small membrane
structures, compared with other peroxisomal vesicles, are detected
(indicated by an arrow). Because of their small size, they
may represent newly synthesized peroxisomal membrane remnants.
Remarkably, the location of these small structures seems to be
restricted to a small area (frequently in close association with a
mitochondrion (A)).Electron micrographs were taken of
glutaraldehyde-fixed cells, poststained with uranyl acetate, unless
otherwise indicated. The abbreviations used are: M,
mitochondrion; N, nucleus; V, vacuole. The
bar represents 0.5 µm.
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The co-localization of Pex10p.myc with the peroxisomal membrane
remnants in pex5::PAOXPEX10.MYC
cells was confirmed by cell fractionation experiments. A post-nuclear
supernatant (PNS), prepared from homogenized protoplasts of
glycerol/methanol/ammonium sulfate-grown cells of this strain, was
subjected to sucrose density centrifugation (29).
Analysis of the various fractions obtained indicated that the bulk of
the cellular protein was located at the top of the gradient, representing soluble, cytosolic proteins. Western blot analysis of
these fractions revealed that Pex10p.myc migrated to a density corresponding to ~35% (w/w) sucrose (Fig.
5B, fractions 16-18). A
similar distribution pattern was observed for Pex14p. At 53% (w/w)
sucrose, where intact peroxisomes of H. polymorpha wild type
cells normally sediment (8, 29), Pex10p.myc and Pex14p were not
detectable. The protein peak at ~42% (w/w) sucrose contains mitochondria judged from the distribution of the mitochondrial marker
enzyme cytochrome c oxidase. The minor cytochrome
c oxidase peak, present at 30% (w/w) sucrose, most probably
reflects fragmented and subsequently sealed protoplasts that were still
present in the PNS. From this we conclude that the peroxisomal membrane
structures present in glycerol/methanol/ammonium sulfate-grown H. polymorpha pex5::PAOXPEX10.MYC cells
sediment to a density of 35% (w/w) sucrose. These findings are in line
with earlier data that revealed that peroxisomal remnants from other
H. polymorpha pex mutants defective in matrix protein import
also sedimented to relatively low densities in sucrose gradients
(e.g. pex4 (8), pex8 (30), and
pex14 (29)).

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Fig. 5.
Sucrose gradient, prepared from a
post-nuclear supernatant obtained from homogenized,
glycerol/methanol/ammonium sulfate-grown pex5.
PAOXPEX10-MYC cells (A) showing the
distribution of protein ( ) in mg·ml 1,
sucrose (+) expressed as percentages (w/w), and the activity of the
mitochondrial enzyme cytochrome c oxidase ( ) expressed as
percentages of the activity in the peak fraction, which was arbitrarily
set at 100. Western blots of the fractions (B) to
demonstrate the distribution of the Myc epitope and Pex14p in the
gradient. Equal volumes of each fraction were loaded per lane.
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A Minor Number of the Peroxisomal Membrane Structures Accumulate
Newly Synthesized AMO to Develop into Normal Peroxisomes--
The
approach to analyze whether the peroxisomal remnants in
pex5::PAOXPEX10.MYC cells can
develop into normal peroxisomes includes the initial induction of these
structures by methanol, followed by the synthesis of the peroxisomal
PTS2 protein AMO under conditions that the synthesis of Pex10p.myc is
fully repressed (by glucose).
Pex5::PAOXPEX10-MYC cells were
pre-cultivated on glycerol/methanol/ammonium sulfate to induce the
synthesis of Pex10p.myc containing membrane remnants. These cells were
harvested by centrifugation and incubated for 30 min at 37 °C in
mineral medium, lacking the C- and N-source to remove any Pex10p.myc
mRNAs (31). Subsequently, methylamine (as N-source) was added to
this medium to induce AMO synthesis together with glucose (as C-source)
to concurrently block the synthesis of Pex10p.myc. At various time
points after addition of these substrates, cells were harvested and
analyzed. The protein patterns of AMO, Pex10p.myc, and Pex3p were
determined by Western blotting (Fig. 6).
As evident in Fig. 6A, AMO was rapidly induced within 2 h after the shift to glucose/methylamine medium. Identical blots,
decorated with antibodies against the Myc epitope, revealed a drastic
decrease in Pex10p.myc levels in time. Within 2 h after the shift
the level of Pex10p-Myc had decreased to ~20% of the level in the
inoculum cells. A similar reduction was observed for Pex3p (Fig.
6A).

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Fig. 6.
Western blots prepared from crude extracts of
methanol-grown pex5.
PAOXPEX10-MYC cells shifted to glucose/methylamine-
(A) and glucose/ammonium sulfate-containing media
(B). Pex10p.myc was determined using -Myc antibodies.
Upon the shift of cells to glucose (A and B) as
sole carbon source, a rapid decrease of Pex10p.myc was observed. A
similar decrease was observed for Pex3p, using -Pex3p antibodies. A
and B indicate that the peroxisomal membrane remnants were
degraded. The synthesis of AMO protein is induced after replacing
ammonium by methylamine as sole nitrogen source (A). The
asterisk marks an a-specific protein band, using
-Pex3p antibodies (30). Equal volumes were loaded per lane.
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To analyze the initial events of peroxisome formation, a post-nuclear
supernatant (PNS) of glycerol/methanol/ammonium sulfate-grown pex5::PAOXPEX10.MYC cells,
transferred for 2 h to glucose/methylamine media, was subjected to
sucrose density centrifugation. Analysis of the various fractions
obtained did not allow the unequivocal demonstration of the Myc
epitope, most likely due to the diminished levels of Pex10p.myc
protein. For this reason we tried to enrich the peroxisomal fraction by
conventional differential fractionation of the PNS. Western analysis
revealed that Pex10p.myc pelleted in the 30,000 × g
(P3) organellar fraction (Fig. 7,
inset). Next, this Pex10p.myc-enriched organellar fraction
was subjected to sucrose density centrifugation (Fig. 7). The major
protein peak present in this gradient (Fig. 7A, fractions 12 and 13) represents mitochondria, judged from the
distribution of cytochrome c oxidase activity. Western blot
analysis of the various fractions collected from the gradient revealed
that bulk of the Pex10p.myc had migrated to a density corresponding to
~48% (w/w) sucrose (Fig. 7B, fractions 9 and
10). The major portion of the peroxisomal membrane protein Pex14p was found in the same fractions. Hence, these peroxisomal proteins migrated to higher densities than the peroxisomal remnants of
methanol-induced
pex5::PAOXPEX10.MYC cells
(~35% (w/w) sucrose, Fig. 5) but not yet to the density of mature
peroxisomes of WT methanol-grown H. polymorpha cells
(~53% (w/w) sucrose (8, 29)). This location most probably reflects
the relatively smaller size of the organelles, compared with WT
peroxisomes. The bulk of the AMO protein co-fractionated with
Pex10p.myc and Pex14p in fractions 9-10, suggesting that AMO protein
may be present in the same compartment as Pex10p.myc and Pex14p.
Interestingly, a portion of Pex10p.myc was also found in fraction 8, which contained only a small amount of Pex14p. Hence these fractions
might contain a slightly different type of structures. In addition,
almost no Pex10.Myc was detected at lower densities (fractions 11-12),
which did, however, contain Pex14p and AMO. Possibly these fractions represent very small, newly formed peroxisomes.

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Fig. 7.
Sucrose gradient prepared from an organellar
pellet (P3) obtained from homogenized,
glycerol/methanol/ammonium sulfate-induced pex5.
PAOXPEX10.MYC cells shifted to
glucose/methylamine-containing medium (A), showing the
distribution of the protein ( ) in
mg·ml 1, sucrose (+) expressed as
percentages (w/w), and the activity of the mitochondrial enzyme
cytochrome c oxidase ( ) expressed as percentages of the
activity in the peak fraction, which was arbitrarily set at 100. After
differential centrifugation of the PNS, Pex10p.myc is present in the
30,000 × g pellet (inset). P3 and S3
designate the 30,000 × g pellet and supernatant,
respectively, while P4 and S4 represents the 100,000 × g pellet and supernatant respectively. B, Western
blot analysis of the sucrose gradient demonstrate the distribution of
the Myc epitope, Pex14p, and AMO protein. Pex10p.myc was detected in
fractions 8-10 corresponding to a density of ~48% (w/w)
sucrose. The peak of Pex14p colocalized with Pex10p.myc. Bulk of the
AMO protein was detected in fractions 9-10. Equal volumes
were loaded per lane.
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The morphological adaptations of
pex5::PAOXPEX10.MYC cells
shifted from glycerol/methanol/ammonium sulfate into fresh
glucose/methylamine media were analyzed by electron microscopy (Fig.
8). After 2 h, the fraction of
peroxisomal membrane remnants had strongly reduced. Peroxisomal
membrane structures could still be detected (Fig. 8, B and
C) but were frequently seen surrounded by additional layers
of membranes, which is a typical feature for vacuolar uptake of
peroxisomes to be degraded (Fig. 8B). Immunocytochemistry
demonstrated that Pex10p.myc was still detectable on the remaining
membranous structures (Fig. 8C, inset). After
4 h of incubation on glucose/methylamine, few small peroxisomes
could be observed (Fig. 8D), which contained the PTS2
protein AMO, as was evident from labeling experiments using specific
-AMO antibodies (Fig. 8D, inset). Apart from these peroxisomes, membrane remnant structures were no longer detectable. Remarkably, the number of growing organelles was low compared with the
number of vesicles originally present in the cells. Also, after a shift
of glycerol/methanol/ammonium sulfate to glycerol/methanol/methylamine (thus only changing the nitrogen source), only few (up to maximally 5-6) organelles developed (data not shown).

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Fig. 8.
Morphological analysis of
glycerol/methanol/ammonium sulfate-grown pex5.
PAOXPEX10.MYC cells after the shift to
glucose/methylamine-containing medium. Before the shift, these cells
contained few small peroxisomal membranes, indicated by the
arrow (A). After incubation for 2 h on
glucose/methylamine, these membrane structures could still be detected
(C), although a small portion was surrounded by additional
membrane layers and degraded by the vacuole (B, indicated by
the arrow). Labeling of ultrathin sections of these cells
using specific antibodies against the Myc-epitope showed that the
peroxisomal membrane structures contained Pex10p-Myc (C,
inset). After a longer incubation period (4 h), a few small
peroxisomes could be observed (D), which contained the PTS2
protein AMO, as is evident from the immunolabeling using specific
antibodies against this protein (D, inset). The
abbreviations used are: M, mitochondrion; N,
nucleus; V, vacuole. The electron micrographs
(A-D) are from KMnO4-fixed cells and the
insets are from glutaraldehyde-fixed cells. The
bar represents 0.5 µm, and the bars in the
insets of C and D represent 0.25 µm.
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Double labeling of ultrathin sections of the cells using
-Myc
and
-AMO antibodies revealed that Pex10p.myc and AMO protein co-localized on the peroxisomal structures (Fig.
9). This strongly suggests that the
peroxisomal membrane remnants in glycerol/methanol/ammonium sulfate
grown pex5::PAOXPEX10.MYC cells
had accumulated newly synthesized AMO.

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Fig. 9.
Immunocytochemical demonstration that
the pre-existing peroxisomal remnants containing Pex10p-Myc are the
target for newly synthesized AMO. Glycerol/methanol/ammonium
sulfate-grown
pex5::PAOXPEX10-MYC cells were
transferred to glucose/methylamine medium and cultivated for 2 h.
A double labeling experiment using antibodies against Myc (10 nm
gold-conjugated goat anti-mouse) and AMO (5 nm gold-conjugated goat
anti-rabbit) showed that Pex10p-Myc and AMO co-localized in the
peroxisomal remnants (arrow = peroxisomal remnants). A
small portion of these peroxisomal remnants was transferred to the bud
of this cell. A more detailed image of the co-localization of
Pex10p-Myc and AMO is provided by the inset (the
arrow indicates the 5 nm gold-conjugated goat anti-rabbit).
Electron micrographs were taken of glutaraldehyde-fixed cells,
poststained with uranyl acetate, unless otherwise indicated. The
abbreviations used are: M, mitochondrion; N,
nucleus; V, vacuole. The bar represents 0.5 µm,
and the bar in the inset represents 0.25 µm.
|
|
The biochemical and morphological observations indicate that our shift
experiment probably had initiated two opposite processes that occurred
simultaneously, glucose-induced degradation of peroxisomal membrane
remnants (32) and peroxisome development due to synthesis and import of
newly induced AMO protein (33). To analyze the glucose-induced
degradation process separately from the import process of AMO,
glycerol/methanol-induced
pex5::PAOXPEX10.MYC cells were
shifted to glucose/ammonium sulfate-containing media. Crude extracts
were prepared at various time points after the shift and were analyzed.
The protein patterns of Pex10p.myc and Pex3p were determined by Western
blotting experiments (Fig. 6B). These data indicated that
the levels of both proteins rapidly decreased in time (Fig.
6B), indicating that the Pex10p.myc-tagged peroxisomal
membrane remnants are subject to degradation under these conditions.
This degradation process resembles the selective autophagy that has
been described for peroxisomal remnants in other H. polymorpha
pex mutants (34).
Notably, in glucose/methylamine media the Pex10p.myc and Pex3p
reduction is slightly less than in glucose/ammonium sulfate cultures
lending support to the view that indeed fraction of these membranes
takes part in new peroxisome development. Another indication for this
is the morphological observation that in other H. polymorpha pex deletion strains re-introduction of the corresponding gene invariably results in the development of one (or infrequently few) new
small peroxisome in the vicinity of the cell wall.
 |
DISCUSSION |
In this paper we provide evidence that peroxisomal remnants
present in methanol-induced H. polymorpha pex5 cells may
develop into normal peroxisomes upon the synthesis of the key enzyme in amine metabolism, the PTS2 protein AMO. The generally accepted view is
that peroxisomes develop from already existing organelles by growth and
fission (35). Our data indicate that this rule can now be extended in
that peroxisomes may also develop from remaining residual structures in
pex5 mutant cells. Whether this is also true for the other
pex mutants, e.g. after re-introduction of the
defective gene, is not yet clear. We have demonstrated that the
peroxisomal remnants ("ghosts") in H. polymorpha pex4 and pex14 cells can develop into normal peroxisomes upon
overexpression of the PEX5 gene (8, 29). Peroxisome
re-assembly also occurs in mutant strains, which are known to lack any
peroxisomal membrane remnants, upon re-introduction of the defective
gene. Examples of this include Pichia pastoris pex3 (36) and
Saccharomyces cerevisiae pex19 (37) null mutants, H. polymorpha pex3 (38) and ts6 (31) mutants, as well as human
pex16 mutants (39). This indicates that alternative modes of
peroxisome assembly may exist. It has been suggested that new
peroxisomes may arise from the endomembrane system, e.g.
from specialized regions of the ER. South and Gould (39) presented an
interesting model for peroxisome re-assembly in human pex16
mutant cells and suggested that they may arise by conversion from an
unknown pre-peroxisomal structure, mediated by Pex16p. It remains
unclear whether this model reflects the WT situation or that it serves
as rescue machinery to peroxisome formation. However, as in S. cerevisiae a PEX16 homologue has not been identified
yet, and the human model may not be generally valid. Additional
research is therefore required for elucidating this problem in both
human and yeast.
Characteristic for various pex5 mutants (e.g. of
P. pastoris (9), S. cerevisiae (40),
Yarrowia lipolytica (41-43)) is the cytosolic location of
PTS1 proteins together with the presence of peroxisomal membrane
remnants or, in case PTS2 proteins are synthesized (e.g.
during growth of yeast on oleate), small peroxisomes. A similar
phenotype is described for H. polymorpha pex5 cells, grown
under conditions that both PTS1 and PTS2 proteins are induced (11).
Conversely, in H. polymorpha pex5 cells, grown on glucose or
ethanol in the presence of ammonium sulfate as sole nitrogen source
(conditions that fully repress synthesis of the PTS2 protein AMO),
peroxisomal membrane remnants were below the limit of detection. On the
other hand, peroxisomal remnants were detectable in H. polymorpha
pex5, when grown at high peroxisome induction conditions on
methanol. Therefore, the proliferation of these structures is dependent
on the growth conditions and is most likely related to the ultimate
level of essential peroxisomal membrane proteins.
The membrane remnants in H. polymorpha pex5 cells
display several characteristics, typical for normal WT peroxisomes.
They are the target for artificially produced Pex10p.myc, proliferate under specific growth conditions (this study), and are susceptible to
selective degradation (32, 34).
Our data demonstrated that newly produced AMO protein accumulated at
the Pex10p.myc-containing, pre-existing membrane structures, originally
present in the methanol/ammonium sulfate-grown cells. Since the new
peroxisomes developed under conditions in which the synthesis of
Pex10p.myc was fully repressed, the co-localization of Pex10p and AMO
suggests that these organelles originate from Pex10p.myc-containing
structures. Whether the development of the peroxisomal membrane
remnants involved fusion with small pre-peroxisomal AMO-containing
vesicles, as for instance demonstrated for matrix protein import in
Y. lipolytica, is yet unknown (44).
The possibility that the failure of a portion of the peroxisomal
structures to import AMO is due to the presence of Pex10p.myc is not
very likely. We showed that Pex10p.myc is functional and can complement
the pex10 mutant phenotype.
In conclusion, our data suggest that peroxisomal membrane remnant
structures present in glycerol/methanol/ammonium sulfate-grown H. polymorpha pex5 cells can develop into peroxisomes upon subsequent synthesis of the PTS2 protein AMO. Notably, this also happens when the
cells are placed in conditions (shift from glycerol/methanol/ammonium sulfate to glucose/methylamine) that induce two oppositely directed processes, namely degradation of the Pex10p.myc-containing peroxisomal structures versus the need for a protein import-competent
structure. Essentially similar results have been obtained on H. polymorpha WT cells, shifted from methanol/methylamine to
glucose/methylamine (45). Given the rate of Pex10p.myc degradation, our
results lend support to the view that few import-competent structures escape degradation and remain available for peroxisome formation. Apparently, these structures maintained the functions essential for
PTS2 protein import of matrix proteins and thus could develop into
AMO-containing peroxisomes. These results are in line with recent data
on a PEX5-deficient Chinese hamster ovary cell line by
Yamasaki et al. (46). These authors showed that peroxisomes reappeared after microinjection of Pex5p together with green
fluorescent protein fused to a PTS1 signal. They observed green
fluorescent protein fluorescence in particulate structures in which
PMP70 co-localized, suggesting that all peroxisomal remnants were able to gradually take up PTS1 proteins in time. The reasons for the apparent discrepancy with the structures in H. polymorpha
pex5 cells are unknown. A possible explanation may be related to
the fact that microinjection in fact confronts the cell with an excess of matrix proteins. In view of the finding that under WT conditions the
maximal import capacity of peroxisomes apparently is not fully used
(47), it can be envisaged that the sudden excessive amounts of matrix
proteins disturbs the normal balance between matrix protein synthesis
and import capacity thus leading to additional protein import in
existing organelles. However, more research is required to solve this
specific question.