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From
Received for publication, August 9, 2001, and in revised form, September 19, 2001
We have analyzed the function of
Hansenula polymorpha Pex14p in selective peroxisome
degradation. Previously, we showed that Pex14p was involved in
peroxisome biogenesis and functions in peroxisome matrix protein
import. Evidence for the additional function of HpPex14p in selective
peroxisome degradation (pexophagy) came from cells defective in
HpPex14p synthesis. The suggestion that the absence of HpPex14p
interfered with pexophagy was further analyzed by mutational analysis.
These studies indicated that deletions at the C terminus of up to 124 amino acids of HpPex14p did not affect peroxisome degradation.
Conversely, short deletions of the N terminus (31 and 64 amino acids,
respectively) of the protein fully impaired pexophagy. Peroxisomes
present in these cells remained intact for at least 6 h of
incubation in the presence of excess glucose, conditions that led to
the rapid turnover of the organelles in wild-type control cells. We
conclude that the N terminus of HpPex14p contains essential information
to control pexophagy in H. polymorpha and thus, that
organelle development and turnover converge at Pex14p.
Hansenula polymorpha is a methylotrophic
yeast that is used as a model organism in contemporary peroxisome
research. Methylotrophic yeast species, also including Candida
boidinii and Pichia pastoris, have the advantage that
the ultrastructural changes accompanying peroxisome development and
degradation are much more pronounced, relative to Saccharomyces
cerevisiae. Also, enhanced numbers of growth
substrate-dependent peroxisome functions can be induced compared with bakers' yeast.
In H. polymorpha highest peroxisome induction rates are
observed during growth of cells on methanol. Under these conditions the
organelles may occupy up to 80% of the cytoplasmic volume and are
essential for growth as they contain the key enzymes of methanol
metabolism, alcohol oxidase
(AO),1 catalase, and
dihydroxyacetone synthase. These enzymes all possess a C-terminal
targeting signal and require the function of the PTS1 import
machinery for sorting to peroxisomes (1, 2). In H. polymorpha, Pex14p is involved in matrix protein import and
functions (probably together with Pex13p and Pex17p) as the putative
docking site for PTS1 receptor-cargo complexes at the peroxisomal
membrane (3). Recent data, however, indicate that the protein is not
essential for import but most likely enhances the efficiency of the
process (4).
Peroxisome degradation may occur aselectively during general autophagy
(5) or in a selective way, when the organelles have become
dysfunctional or, alternatively, redundant for growth. In H. polymorpha the selective degradation process is morphologically characterized by three subsequent steps, namely (i) tagging, followed by sequestration of the organelle to be degraded by multiple membranous layers, (ii) heterotypical fusion of the sequestering membranes with
the vacuolar membrane, and (iii) hydrolysis of the organelle contents
in the vacuole (6). This process, designated macropexophagy, is also
observed in another methylotrophic yeast species, P. pastoris (7, 8). In this yeast, but not in H. polymorpha, a second mode of selective degradation is described,
specifically induced by glucose, which involves uptake of clusters of
peroxisomes by engulfment by the vacuole (micropexophagy). Selective
peroxisome degradation has also been described in the yeasts S. cerevisiae and Yarrowia lipolytica (9); however, the
exact mode of degradation is as yet unknown.
Various mutants affected in pexophagy have been isolated from P. pastoris (gsa and pag mutants) (10-13) and
H. polymorpha (pdd mutants) (14, 15). The
analysis of the corresponding genes has shown that pexophagy has
several components in common with non-selective autophagy, vacuolar
protein sorting, endocytosis, and the cytosol-to-vacuole transport
pathway in bakers' yeast (16, 17).
In H. polymorpha pex mutants, peroxisomal remnants are
normally susceptible to glucose-induced selective degradation, except for those present in Organisms and Growth Conditions--
All H. polymorpha strains used in this study are derivatives of strain
NCYC495 (leu1.1 ura3) (25):
H. polymorpha cells were grown in batch cultures at 37 °C
on (a) selective minimal medium (YND) containing 0.67%
(w/v) yeast nitrogen base without amino acids (DIFCO) supplemented with
1% (w/v) glucose, (b) rich medium (YPD) containing 1%
(w/v) yeast extract, 1% (w/v) peptone, 1% (w/v) glucose, or
(c) mineral medium (26) supplemented with 0.5% (w/v)
glucose, 0.5% (v/v) methanol or a mixture of 0.1% (v/v) glycerol,
0.5% (v/v) methanol as a carbon source, together with 0.25% (w/v)
ammonium sulfate as a nitrogen source. When required leucine (30 µg/ml) was added.
For analysis of peroxisome degradation, cells were extensively
precultivated to the mid-exponential phase on mineral medium with
glucose and then shifted to mineral medium containing glycerol + methanol for a period of 16 h to induce peroxisome biogenesis. Subsequently 0.5% glucose was than added to induce selective
peroxisome degradation. Escherichia coli DH5 Molecular Techniques--
Standard recombinant DNA techniques
were carried out essentially according to Sambrook et al.
(27). Transformation of H. polymorpha was performed by
electroporation as described previously (28). Restriction enzymes and
biochemicals were obtained from Roche Molecular Biochemicals and
used as detailed by the manufacturer. Protein sequences were aligned
with the program CLUSTALX (29).
Construction of Plasmids--
To express WT and truncated forms
of PEX14 in Biochemical Methods--
Crude extracts using
trichloroacetic acid-precipitated H. polymorpha cells
were prepared as described (30). SDS-polyacrylamide gel
electrophoresis and Western blotting were performed by established procedures. Proteins on Western blots were detected using a
chemiluminescent Western blotting kit (Roche Molecular Biochemicals)
after decoration with polyclonal antibodies against various H. polymorpha proteins.
In vivo phosphorylation of HpPex14p was determined as
described (31); cells were pregrown on YPD, depleted for phosphate, harvested, and subsequently suspended in the same volume of
phosphate-depleted glycerol/methanol medium containing
[32P]orthophosphate for 16 h.
32P-labeled H. polymorpha Pex14p was recovered
from crude extracts by immunoprecipitation, separated by
SDS-polyacrylamide gel electrophoresis, and detected by autoradiography.
Electron Microscopy--
Whole cells were fixed and prepared for
electron microscopy and immunocytochemistry as detailed before (32).
Immunolabeling was performed on ultrathin section of
unicryl-embedded cells, using specific antibodies against
various proteins and gold-conjugated goat anti-rabbit antibodies
according to the instructions of the manufacturer (Amersham
Pharmacia Biotech).
In an initial series of experiments we have analyzed the fate of
peroxisomes that had developed in cells of a pex14 deletion strain that overproduced Pex5p
( Ultrastructural and biochemical analyses revealed that, upon exposure
of such cells to glucose-excess conditions, selective peroxisome
degradation (pexophagy) was inhibited. Electron microscopy revealed
that after the addition of glucose the initial event in peroxisome
degradation, namely sequestration of the organelle to be degraded, was
never observed (data not shown). Also, immunocytochemistry failed to
demonstrate any AO protein in the vacuole, a typical morphological
characteristic of pexophagy, in the same time interval (Fig.
1B). In WT control cells both phenomena were frequently observed (data not shown; see Ref. 6). Biochemical experiments showed
that the amount of Pex10p, an integral component of the peroxisomal
membrane, had slightly increased 4 h after addition of glucose to
Peroxisome Biogenesis and Selective Degradation Converge
at Pex14p*
§,
,**,
, and§§
Eukaryotic Microbiology, Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen, P. O. Box 14, 9750 AA Haren, The Netherlands and ¶ Laboratory
of Cellular and Molecular Biology, Department of Veterinary Science,
Graduate School of Agriculture and Biological Sciences, Osaka
Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pex14 cells (18). This raised the
question whether Pex14p, besides being involved in matrix protein
import, could have additional functions in the control of the
susceptibility of individual organelles for selective degradation. This
aspect was analyzed in cells of constructed mutants that contained
peroxisomes that either completely lacked Pex14p or contained truncated
forms of this peroxin. The results of this work are included in this paper.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pex14(leu1.1) (20), pex14::PAOX.PEX5mc
(4), and pex14 strains expressing full-length or
truncated forms of PEX14 under control of the
PEX14 promoter (PPEX14) (this study).
(27)
was used for plasmid construction and was grown on LB medium
supplemented with the appropriate antibiotics.
pex14 cells under control of its
endogenous promoter, we first constructed the expression vector
pHIPX10, a derivative of pHIPX4-B (20). The PEX14 promoter
was isolated by PCR using the PPEX14 primer (5' GGG GAT CCG
GTG AGG AAG AAA AAG AG 3'), the M13/pUC universal primer, and plasmid
pBS3.2P14 (20) as template. The PCR fragment was digested with
EcoRV and BamHI and inserted between the
NotI (blunted) and BamHI sites of plasmid
pHIPX4-B thus replacing the alcohol oxidase promoter. To obtain
pX10-PEX14-WT we inserted the 1.5-kilobase
BamHI-EcoRV fragment of pPAOXPEX14
(20) between the BamHI and SacI sites of pHIPX10.
Plasmids expressing truncated versions of PEX14 were
constructed as follows: pX10-PEX14-C58 was constructed by filling in
the EcoRI site in the PEX14 coding region
followed by self-ligation. Similarly, pX10-PEX14-C124 was obtained
by blunting the PstI site in the PEX14 coding
region followed by self-ligation. Plasmids pX10-PEX14-N31 and
pX10-PEX14-N64 were constructed by PCR using the N31 primer (5' AGA
GGA TCC ATG GCC AAA AAG GTC GAA TTT C 3') or the N64 primer
(5' AA GGA TCC ATG TCA CAG CAG TCC GTT GTA 3') in
combination with the M13/pUC sequencing primer and pBS1.3P14 as
template. Subsequently, for pX10-PEX14-N31, a BamHI + HindIII-digested PCR fragment was cloned between the
BamHI and HindIII sites of pHIPX10. For
pX10-PEX14-N64 a BamHI + EcoRV-digested PCR
fragment was cloned between the BamHI and SmaI
sites of pHIPX10. Plasmids were transformed into H. polymorpha pex14 (leu1.1) (20) and
produced Pex14p levels similar to those observed in WT cells.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pex14::PAOX.PEX5mc),
(4), after a shift of cells from methanol to glucose-excess conditions.
To this end cells were pre-grown on a mixture of glycerol and methanol
until an optical density (OD663) of 1.5 before excess glucose (final concentration 0.5%) was added. As described before, glycerol-/methanol-grown
pex14::PAOX.PEX5mc
cells contained several well developed peroxisomes (Fig.
1A) that lacked Pex14p but
contained the bulk of two key enzymes in methanol utilization,
AO and dihydroxyacetone synthase (data not shown), in
conjunction with minor amounts of these proteins in the cytosol
(4).
View larger version (68K):
[in a new window]
Fig. 1.
In
pex14::PAOX.PEX5mc
selective peroxisome degradation is prevented. Electron
micrographs are taken of cells, fixed with glutaraldehyde, and embedded
in unicryl for immunocytochemistry, unless otherwise indicated.
M, mitochondrion; N, nucleus; P,
peroxisome; V, vacuole. The marker represents 0.5 µm.
A, overall morphology of KMnO4-fixed
mutant cells, grown in glycerol-/methanol-containing media. The cells
contain several medium-sized peroxisomes. B, these
organelles are not degraded upon a shift of cells to glucose-excess
conditions. 4 h after the shift -AO-dependent
labeling was not detectable in the vacuole.
pex14::PAOX.PEX5mc
cells whereas in WT controls this marker protein markedly decreased during this time period (Fig. 2). Taken
together these data suggest that the peroxisomes present in
pex14::PAOX.PEX5mc
cells were not susceptible to glucose-induced pexophagy.
View larger version (46K):
[in a new window]
Fig. 2.
Western blots, prepared from crude extracts
of glycerol-/methanol-grown WT and
pex14::PAOX.PEX5mc
cells upon induction of selective peroxisome degradation by
glucose. Blots were decorated with antibodies against the
peroxisomal membrane protein Pex10p. Equal volumes of samples were
taken 0 and 4 h after the addition of glucose, and trichloroacetic
acid was precipitated. For the WT (A) strain a strong
reduction of the Pex10p level is observed whereas in the mutant
(B) strain the Pex10p level has increased indicative for the
inhibition of peroxisome degradation.
Mutational Analysis of Pex14p--
To delineate the region of
Pex14p controlling peroxisome turnover, we constructed mutant genes
that encoded various truncated Pex14ps and transformed these into a
PEX14 deletion strain (pex14). These proteins
lacked either the initial 31 or 64 N-terminal amino acids (designated
N31 and N64, respectively) or the extreme 58 or 124 C-terminal
amino acids (designated C58 and C124, respectively). A
pex14 strain expressing full-length PEX14 was
taken as control (designated WT). Cells of these strains were
subsequently analyzed for growth on methanol, Pex14p synthesis and
location, peroxisome development, and the susceptibility of these
organelles to selective degradation.
The Mutant Pex14ps Are Normally Synthesized and Sorted to
Peroxisomes--
Cells of the various constructed strains were
analyzed for their capacity to grow on methanol as the sole
source of carbon. As shown in Table
I only the WT control and mutant C58
grew normally on methanol at WT rates, whereas the other strains showed no or severely retarded growth. The reason for this became clear from
electron microscopy, which revealed that WT and C58 cells displayed
normal peroxisomes that were the sole site of AO protein, judged from
immunocytochemistry (Fig. 3, A
and B). The three other strains contained several
peroxisomes of smaller size (Table I). This most likely reflects the
observation that only a portion of AO protein was present in
peroxisomes, whereas the remaining portion resided in the cytosol (Fig.
3, C and D). However, in these three mutants the
amount of AO imported into peroxisomes was substantially higher
compared with the residual import in peroxisomal remnants in
pex14 cells. Because AO is a PTS1 protein, these data
indicate that the long (124 amino acids) C-terminal deletion, as well
as N-terminal deletions of HpPex14p, affect Pex5p-dependent
protein import. We showed before that a minor amount of active
cytosolic AO prevents growth of H. polymorpha cells on
methanol (19). Therefore, the cytosolic portion of AO protein in
C124, N31, and N64 cells most likely explains the failure of
the cells to grow on methanol.
|
, reduced;
|
Subsequently, all constructed strains were analyzed for the presence of
Pex14p by Western blotting, using -Pex14p antibodies and crude
extracts prepared from glycerol-/methanol-grown cells. These
experiments revealed that all truncated Pex14ps were normally synthesized and were of the expected mass, judged from their migration pattern in the gel (Fig. 4A).
In these blots Pex14 protein is observed as a double band at ~47 kDa,
of which the upper band represents the phosphorylated state of the
protein (20). As evident in Fig. 4B, normal phosphorylation
of Pex14p can also be observed in N64 and C58 cells but not in
C124 cells, in which phosphorylation of Pex14p was hardly
detectable. This suggests that the putative phosphorylation site(s) of
the protein are present within the region of amino acids 228 to
293.
|
The subcellular location of these Pex14p variants was subsequently
analyzed biochemically and immunocytochemically. Upon differential centrifugation of homogenized protoplasts, prepared from
methanol-induced cells of the various strains, WT and mutant Pex14ps
were predominantly found in the 30,000 × g organellar
pellets (Fig. 5). This suggests that the
proteins are indeed organelle-bound. Immunocytochemically, using
-Pex14p antibodies, the specific labeling was exclusively localized
on the peroxisomal membrane (Fig. 6).
From this we concluded that all truncated Pex14ps (WT, C58, C124,
N31, and N64) were normally synthesized and sorted to the correct
target membrane (summarized in Table I).
|
|
N-terminal Deletions of Pex14p Affect Selective Peroxisome
Degradation--
Cells of the various strains were grown on
glycerol/methanol mixtures and subsequently exposed to glucose-excess
conditions. Electron microscopical analysis revealed that in the strain
producing full-length Pex14p peroxisomes were normally degraded. A
similar phenomenon was observed for C58 and C124 cells. 30 min
after addition of glucose the first cells were observed that contained AO protein in the vacuole (Fig. 7), a
characteristic feature for selective peroxisome degradation under these
conditions (6). It should be stressed that the cytosolic portion of AO
is not subject to degradation under these conditions (21) and therefore cannot be the source of vacuolar AO protein.
|
The above observations were confirmed by biochemical data. After
Western blotting of crude extracts, prepared from the various strains
at different time points after the addition of glucose, a rapid
decrease in the levels of Pex10p and mutant Pex14ps were observed (Fig.
8). Opposite results were obtained in
identical experiments, using N31 and N64 cells. In these cells,
electron microscopical analysis failed to resolve any sign of
peroxisome turnover in the first 4 h after addition of glucose
(Fig. 7, C and D), a time interval that is
sufficient to remove the bulk of the peroxisomal population in WT
control cells. Also, the levels of Pex10p and mutant Pex14p hardly
diminished in this period (Fig. 8). From this we conclude that the
N-terminal deletions of Pex14p prevent glucose-induced pexophagy in
H. polymorpha.
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DISCUSSION |
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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In this paper we have provided evidence that the H. polymorpha PEX14 gene product (HpPex14p) plays a role in the
selective degradation of peroxisomes (pexophagy). We showed that
considerable portions of the C terminus of the protein could be deleted
without affecting PTS1 protein import (C58 cells) and pexophagy
(C58 and C124 cells). In contrast, deletion of the first 31 N-terminal amino acids affected both peroxisome biogenesis (by reducing
the rate of PTS1 protein import) and selective peroxisome degradation (pexophagy). The general function of Pex14p in PTS1 and PTS2 matrix protein import as a component of the docking site for the cytosolic receptors of the PTS1 and PTS2 targeting signals was demonstrated before (3). Very recently, we showed, however, that in H. polymorpha Pex14p most likely is not essential for PTS1 protein
import but enhances the efficiency of this process (4). Our current
data imply that Pex14p may have multiple roles and functions in both the biogenesis and selective degradation of the organelle. This mode of
controlling peroxisome biogenesis versus degradation
(homeostasis) in one "switch" has obvious physiological advantages
in that it enables the cell to rapidly adapt peroxisome numbers and
function upon changes in the environment. This is particularly
important in methanol-grown H. polymorpha cells, in which
specific organelles have become dysfunctional through, e.g.
chemically induced damage of their membrane (22) resulting in the
leakage of matrix proteins into the cytosol. It has been shown before
that the presence of only minor amounts of enzymatically active AO
protein in the cytosol prevents growth of cells on methanol because of
the major energetic disadvantages related to cytosolic hydrogen
peroxide metabolism (19).
In H. polymorpha pexophagy is a remarkable fast process; individual organelles can be degraded within a time interval of 15 min. Thus, a molecular switch controlling organelle homeostasis (both biogenesis and degradation) allows taking rapid decisions and consequently, may be of great value as a component in the control of cell viability. In surface plasmon resonance analyses Schliebs and colleagues (23) demonstrated that the N terminus of human Pex14p could efficiently bind to the PTS1 receptor, Pex5p . This indicates that the N terminus of the protein may be a prime target for the PTS1 receptor-cargo complex. These data are in line with our observations that N-terminal deletions of both 31 or 64 amino acids resulted in a partial AO protein-import defect. However, the same stretch of N-terminal amino acids also appears to control pexophagy. The mechanisms on how the discrimination is made between these functions of HpPex14p are still largely speculative. One possible option is that this may be related to conformational changes of HpPex14p, for instance by oligomerization from monomers to dimers or even by alteration of the location of the N terminus of Pex14p from inside to outside the organellar matrix. On the topology of Pex14p in yeast nothing is known yet; only for human Pex14p has evidence been presented that the N terminus of the protein is (at least in part) exposed to the lumen of the organelle (24). Our experiments strongly suggest that modification of the protein by phosphorylation is most likely not discriminative in this respect.
Sequence comparison of the various Pex14ps currently available,
reveals that the similarity predominantly resides in the N-terminal part of the protein (Fig. 9). This
similarity becomes even stronger when only the proteins of the
methylotrophic yeast species H. polymorpha and P. pastoris are compared. We have now initiated further studies to
analyze the significance of the conserved residues in peroxisome
homeostasis in depth.
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ACKNOWLEDGEMENTS |
|---|
We thank Ineke Keizer-Gunnink and Klaas Sjollema for expert technical assistance.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an Erasmus grant.
Supported by grants-in-aid for scientific research from the
Ministry of Education, Culture, Sports, Science and Technology of Japan.
** Holder of a PIONIER fellowship from Aard-en-Levenswetenschappen (ALW), which is subsidized by the Dutch Organization for the Advancement of Pure Research.
Supported by a grant from ALW.
§§ To whom correspondence should be addressed. Tel.: 31-50-363-2176; Fax: 31-50-363-8280; E-mail: M.Veenhuis@ biol.rug.nl.
Published, JBC Papers in Press, September 19, 2001, DOI 10.1074/jbc.M107599200
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ABBREVIATIONS |
|---|
The abbreviations used are: AO, alcohol oxidase; WT, wild-type; PCR, polymerase chain reaction; aa, amino acid; PTS, peroxisome-targeting signal.
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REFERENCES |
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES
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