(Received for publication, February 27, 1995)
From the
PER genes are essential for the assembly of peroxisomes
in Hansenula polymorpha. Here we describe the PER3 gene which was cloned by functional complementation of a H.
polymorpha per3 mutant. The complementing PER3 gene
encodes a protein of 569 amino acids (Per3p) with a calculated mass of
63.9 kDa; Per3p belongs to the tetratricopeptide repeat protein family
and is located in both the cytosol and the peroxisomal matrix.
Remarkably, Per3p does not contain a known targeting signal (PTS1 or
PTS2). The PER3 gene product shows similarity to the Saccharomyces cerevisiae Pas10p (40% identity) and the Pichia pastoris Pas8p (55% identity). However, their function
apparently cannot be interchanged since the P. pastoris PAS8 gene failed to functionally complement a H. polymorpha per3 disruption mutant.
The per3 disruption mutant
contained normal but small peroxisomes in which PTS2 proteins (both
homologous and heterologous) were imported. Other matrix proteins (in
particular PTS1 proteins) resided in the cytosol where they were
normally assembled and active.
We argue that Per3p is a component of
the peroxisomal import machinery and most probably shuttles matrix
proteins from the cytosol to the organellar matrix.
Peroxisomes are important cell organelles, which carry out
various metabolic functions in eukaryotic cells (for a review, see (1) ). The organelles do not contain DNA and lack an
independent protein synthesizing machinery; hence all matrix proteins
are synthesized in the cytosol and subsequently sorted to the organelle
(for a review, see (2) ). Two types of peroxisomal targeting
signals (PTS)
Components
essential for peroxisome biogenesis have been identified from an
analysis of various yeast peroxisome assembly mutants (for a review,
see (2) ). The biochemical phenotype of some of these mutants
led to the assumption that in yeasts separate import pathways exist for
PTS1 and PTS2 proteins(7, 8, 9) .
In our
laboratory, we use the methylotrophic yeast Hansenula polymorpha as a model organism for studies on peroxisome biogenesis and
function(10) . In this organism several PTS2 proteins have been
identified (e.g. amine oxidase(11) , thiolase and
Per1p(12) ). Recently, we have demonstrated that the PTS2
import mechanism is conserved because heterologous PTS2 proteins like
watermelon gMDH (6) and Saccharomyces cerevisiae thiolase (13) are normally sorted to H. polymorpha peroxisomes.
In the present study, we provide further evidence
that a separate PTS2 import machinery exists in H. polymorpha.
This was evident from studies on one of the H. polymorpha import (Pim
Escherichia coli strains MC1061, C600, and DH5
For overexpression PER3 was cloned
into pHIPX4 behind the P
The BamHI-NheI fragment of pGF159(6) , containing
the gene encoding the precursor of watermelon (Citrullus
vulgaris) gMDH downstream of the P
Standard recombinant DNA techniques, E.
coli transformation, and plasmid isolation were performed as
described(17) . H. polymorpha was transformed by
electroporation(22) . Mating and random spore analysis were
carried out as described(15) .
Figure 1:
Nucleotide and amino acid sequence of
the H. polymorpha PER3 gene. The amino acids are shown in one-letter code below the second nucleotide of each codon. The
nucleotides are numbered on the left, the amino acid residues
on the right. The region of the TPR motif is underlined.
Upon mating of
The
Figure 2:
Panel A shows a detail of a cell
of
In
Figure 3:
Subcellular location of peroxisomal
proteins in glucose-choline chemostat cells (A) or
ethanol-ethylamine grown batch cells (B) of
Subsequent (immuno)cytochemical
experiments confirmed that amine oxidase protein was solely located
inside the small peroxisomes of
Figure 4:
Cytochemically, amine oxidase is solely
present in the small peroxisomes (arrow) of glucose-choline
grown cells (A). Typically, the membranous protrusions do not or only
very slightly stain (inset). Immunocytochemically, using
specific antibodies against amine oxidase, the protein is also found
confined to the peroxisomal structures (B). A similar location
is observed for watermelon gMDH in methanol-induced cells (C)
and homologous thiolase (inset, D) in oleic-acid
induced
Figure 5:
Subcellular localization of Per3p using
Western blots decorated with affinity purified Per3p antibodies. In
crude extracts of cells which overexpressed PER3, a distinct
protein band with the apparent molecular mass of 70 kDa was detected (lane 1). This band was absent in crude extracts of
methanol-induced
Immunocytochemical experiments, using
Figure 6:
A
and C show the characteristic labeling patterns when specific
antibodies against Per3p are used in the immunocytochemical
experiments. Labeling is found both in peroxisomes and in the cytosol.
Typically, peroxisomal labeling is found in the zone between the
alcohol oxidase crystalloid and the peroxisomal membrane of partly
crystalline organelles in batch cultured cells. In fully crystalline
organelles from methanol-limited cells, the labeling is also found
randomly over the matrix (A). In per1 disruption
cells, which completely lack peroxisomal structures, the labeling is
predominantly associated with the alcohol oxidase crystalloids (B). A similar labeling pattern (peroxisomal and cytosolic) is
seen in ethanol-ethylamine grown cells, which only contain few small
peroxisomes (D). After overexpression of Per3p
(P
Figure 7:
Immunofluorescence. A and C, control labeling of amine oxidase in ethanol-ethylamine
grown cells, showing the expected punctuate structures of the few
peroxisomes present in these cells. A, phase contrast; C, immunofluorescence. B and D, phase
contrast (B) and immunofluorescence (D) image of
methanol-grown WT cells, containing cubic peroxisomes, using affinity
purified anti-Per3p (compare C). E, labeling of
peroxisomes in ethanol-ethylamine grown cells using affinity purified
anti-Per3p; the cytosol shows slight
fluorescence.
Immunocytochemical experiments revealed that also in
these cells Per3p was both in peroxisomes and in the cytosol (Fig. 6E). Compared to WT control cells, the labeling
intensity of the peroxisomal matrix was not significantly enhanced. The
increased levels of Per3p also had no clearcut effect on the overall
peroxisomal morphology and on the localization of typical PTS1
proteins; similar to WT cells, alcohol oxidase (Fig. 6F) and dihydroxyacetone synthase protein (not
shown) were exclusively present in the matrix of the organelles present
in these cells.
In
this paper, we show that in cells of a H. polymorpha per3 disruption mutant (
The cytosolic PTS1 proteins in H. polymorpha
At present, the subcellular location of the ``SKL
receptor'' is still a matter of debate and may even be
species-dependent. In P. pastoris Pas8p was mainly associated
with peroxisomes (8) , whereas in S. cerevisiae Pas10p
was predominantly found in the cytosol.
An analogous situation may exist for PTS2 proteins. As
indicated above, Kunau and co-workers (7) showed that in
bakers' yeast Pas7p is specifically involved in the import of the
PTS2 protein thiolase. Although ultrastructural methods failed to
demonstrate myc-tagged Pas7p in the peroxisomal matrix, biochemical
data pointed to a minor portion of myc-Pas7p being bound to
peroxisomes. This result was strengthened by the finding that in a
thiolase-deficient strain all Pas7p was invariably on the top of the
gradient and thus fully soluble. Therefore, the association of Pas7p
with peroxisomes is possibly dependent on its protein substrate,
thiolase; probably, the EM technique was insufficiently sensitive to
detect these low amounts of myc-tagged Pas7p in the organellar matrix.
If this assumption is correct, this implies that both the PTS1 and
PTS2 import machineries may display the same basic mechanisms in that a
cytosolic receptor (Per3p and eventually also P. pastoris Pas8p and S. cerevisiae Pas10p for PTS1 proteins and S. cerevisiae Pas7p for PTS2 proteins) is essential to
recognize the protein to be imported and to direct it to the matrix of
the target organelle.
How are these proteins imported? Taken
together, the available data on in vivo and (semi) in
vitro import studies are far from conclusive. In vivo the
capacity of individual peroxisomes to incorporate newly synthesized
proteins is only temporal (43) , a property which is not
evident from import studies using semipermeabilized mammalian cells or
protein microinjection(37) . Moreover, recently several
examples have been described in which peroxisomal protein import is not
restricted to proteins having an unfolded
conformation(44, 45) . In addition, complex proteins,
as for instance octameric alcohol oxidase or human serum albumin, to
which SKL-containing peptides were chemically
cross-linked(37) , were succesfully directed to peroxisomes
after microinjection in mammalian cells. One could argue that import of
the above proteins is preceded by their disassembly and unfolding at
some point prior to the import; however, in particular in the case of
octameric alcohol oxidase this is very unlikely(46) .
Therefore, import of preassembled proteins into peroxisomes may indeed
occur. From these results the picture emerges that peroxisomal import
may be highly different from the basic principles underlying import in
other organelles like mitochondria and chloroplasts. McNew and Goodman (45) discussed several interesting alternatives ranging from
static to dynamic pores and membrane internalization processes. To
elucidate the exact mechanisms of peroxisomal protein import is one of
the main future challenges. There is little doubt that several proteins
are involved in this process. For H. polymorpha Per3p genetic
studies predict interactions with the gene products of PER4, PER5 (both members of the AAA family of ATPases and
homologues of PAS1 and PAS8 of S.
cerevisiae), and PER9 (the S. cerevisiae PAS3 homologue)(15).
Based on the amino
acid sequence similarity, we expect H. polymorpha Per3p to
represent the functional homologue of P. pastoris Pas8p and S. cerevisiae Pas10p. However, the P. pastoris PAS8 gene could not functionally complement the H.polymorpha
Both P. pastoris Pas8p and H. polymorpha Per3p do not contain a conserved PTS1 or PTS2. Therefore, it is
unclear how these proteins enter the organelle; one possibility
includes that they are not imported in their free form, but that import
is dependent on binding to their protein substrates. In line with this,
the failure of the P. pastoris PAS8 gene to complement the H. polymorpha
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U26678[GenBank® Link].
We gratefully acknowledge Dr. S. Subramani (La Jolla)
for providing the P. pastoris PAS8 gene and
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)have been identified, namely
PTS1 (located at the extreme C terminus) and PTS2, present at the N
terminus and characterized by the consensus sequence
RL-X
-H/QL(3, 4) . Both PTS1 and PTS2 are
conserved among higher and lower eukaryotes (5, 6) ,
and either one is necessary and sufficient for targeting of homologous
or heterologous proteins to the peroxisomal matrix.
) mutants (14) belonging to
the per3 complementation group. In a per3 deletion
mutant, small peroxisomes were present which were capable to import
PTS2 proteins, while other matrix proteins (e.g. PTS1
proteins) remained in the cytosol. From these data we conclude that
Per3p is a component of a peroxisomal protein import machinery which is
redundant for PTS2 proteins.
Organisms and Growth Conditions
The H.
polymorpha strains used are wild type (WT) CBS4732, the NCYC495
derived leu1.1, leu1.1 ura3 and per3-188
leu1.1, per3-191 leu1.1, per3-229 leu1.1 and per3-237 leu1.1(14, 15) . H.
polymorpha was grown at 37 °C on rich complex medium (YPD)
containing 1% yeast extract, 2% peptone, and 1% glucose, on minimal
media as described (16) or on YNB containing 0.67% yeast
nitrogen base (Difco). The carbon sources used are 0.5% glucose, 0.5%
ethanol, 0.5% methanol, or 0.1% (v/v) oleic acid + 0.02% (v/v)
Tween-80; the nitrogen sources used are ammonium sulfate or
(m)ethylamine at 0.2%. Carbon-limited continuous culturing was carried
out as described previously (16) at a dilution rate of 0.1
h using 0.5% glucose and 0.2% choline. Amino acids
and uracil were added to a final concentration of 30 µg/ml.
were
grown at 37 °C in LB medium or in minimal M9 medium (17) supplemented with ampicillin (50 µg
ml
) or kanamycin (60 µg ml
),
when appropriate.
Cloning and Characterization of the PER3
Gene
per3-237 was transformed with a genomic DNA
library of H. polymorpha(12) . Leucine prototrophs
were replica-plated onto YNB plates containing 0.5% methanol and
screened for the ability to grow on methanol. E. coli MC1061
was transformed by DNA isolated from complemented cells, and a plasmid
containing a genomic insert of approximately 20 kb was recovered.
Retransformation of per3-237 with this plasmid resulted
again in restoration of growth on methanol. The 20-kb insert was
partially digested with Sau3A, and resulting fragments were
cloned in the unique phosphatase-treated BamHI site of the
pHRP2 vector(18) . pHRP2 plasmids containing the complementing
insert were selected by another round of complementation and rescue in E. coli. All complementing plasmids contained a genomic insert
of 4.7 kb, which was subcloned in two orientations in pOK12 (19) . Nested sets of deletions were generated using
exonuclease III (17) . Subclones were sequenced using the
reverse primer (Stratagene). Double-stranded sequencing was performed
in two directions by the dideoxy method(20) . For analysis of
the DNA and amino acid sequence the PC-GENE program
release 6.70 (IntelliGenetics Inc.) was used. The amino acid sequence
was compared with the GenBank(R) Release 84.0.
Plasmid Constructions and Miscellaneous Genetic
Methods
A BglII-HindII 3.4-kb fragment
containing the PER3 gene and approximately 1.4 kb promoter
region was cloned into the BamHI and NheI (blunt)
sites of pHARS1(21) . The same sites of pHARS1 were used for
the cloning of the NotI (blunt)-BglII fragment
containing the alcohol oxidase promoter (P) and the Pichia pastoris PAS8 gene. The latter fragment was constructed
by cloning the BglII (blunt)-XmaI PAS8 fragment from pSP72-PAS8 (a gift from Dr. S. Subramani)
into the HindIII (blunt) and XmaI sites of
pHIPX4(22) .
. HindIII and NdeI sites were introduced upstream the PER3 start
codon using polymerase chain reaction. A 2.1-kb NdeI
(blunt)-SacI fragment was cloned into the HindIII
(blunt)-SacI sites of pHIPX4.
was also cloned
into plasmid pHARS1.
PER3 Disruption
A gene disruption construct was
made by blunt-ended cloning of the 2.2-kb EcoRI-BamHI
fragment of pMK155, containing the LEU2 gene of Candida
albicans (obtained from Dr. E. Berardi, Ancona, Italy) in the ApaI-MscI sites of the complementing fragment. The LEU2-containing insert was subsequently released by digestion
with HindIII and MluI and linearly transformed to H. polymorpha NCYC 495 ura3 leu1.1. Leucine
prototrophic transformants which were methanol-utilization-deficient
were checked for the proper insertion into the genome by Southern
analysis (data not shown). The per3::LEU2 could be
complemented with pHARS containing the 3.4-kb PER3 fragment.
Biochemical Methods
Crude extracts were prepared
as described(14) . Cell fractionation was performed as
described(23) , except that 1 mM phenylmethylsulfonyl
fluoride and 2.5 µg/ml of leupeptine were added to all solutions.
Peroxisomal peak fractions were subjected to carbonate
extraction(24) . Protein concentrations were determined as
described (25) using bovine serum albumin as standard.
SDS-polyacrylamide gel electrophoresis was performed as
described(26) . Gels were used for Western
blotting(27) , and the blots were decorated using the Protoblot
Immunoblotting system (Promega Biotec) and specific polyclonal
antibodies against H. polymorpha peroxysomal proteins.
Generation of Per3p Antibodies
The Protein Fusion
System (New England Biolabs) was used for overexpression of a
maltose-binding protein-Per3 fusion protein in E. coli. A BstXI (blunt)-EcoRI fragment, encoding Per3p except
for the first 15 amino acids, was cloned in frame behind the malE gene in the pMAL-P2 vector. The maltose-binding protein-PER3
protein was recovered from inclusion bodies, subjected to
SDS-polyacrylamide gel electrophoresis, and blotted onto
nitrocellulose. The maltose-binding protein-Per3p band was cut out,
dissolved in MeSO, and upon dilution with
phosphate-buffered saline used to immunize a rabbit. The antiserum was
affinity purified by incubation with crude extracts from
per3 coupled to Sepharose-6B prior to use(28) .
Electron Microscopy
Whole cells and spheroplasts
were fixed and embedded in Epon 812 or Unicryl(12) .
Cytochemical staining of alcohol oxidase and amine oxidase activities
were performed by the methods described previously(29) .
Ultrathin Unicryl sections were labeled using polyclonal antibodies
raised in rabbit and goat-anti-rabbit antibodies conjugated to gold
according to the instructions of the manufacturer (Amersham). Freeze
etching was performed as described previously(12) .
Immunofluorescence
Intact cells were prefixed for
2 h at room temperature in 3% (v/v) formaldehyde in 40 mM potassium phosphate buffer, pH 6.5, and subsequently converted
into protoplasts(30) . Immunofluoresence was performed using
anti-amine oxidase, affinity purified anti-Per3p antibodies and
anti-rabbit fluorescein isothiocyanate(30) .
PER3 Encodes a Protein Belonging to the TPR Protein
Family
The PER3 gene was cloned by functional
complementation of a per3 mutant (per3-237),
using a H. polymorpha genomic library and restoration of
growth on methanol as a selection criterion. Sequence analysis of the
complementing fragment revealed a 1.7-kb open reading frame, encoding a
protein of 569 amino acids with a calculated mass of 63.9 kDa (Fig. 1). In the PER3 gene product (Per3p) no
membrane-associated helices are predicted. The protein shows similarity
to the P. pastoris Pas8p (55% identity; 8) and the S.
cerevisiae Pas10p (40% identity; 9), which both are essential for
import of many matrix proteins (in particular PTS1 proteins) into
peroxisomes. The highest similarity exists in the C-terminal part of
the protein, which contains a tetratrico peptide repeat (TPR) motif,
characterized by a highly degenerate 34 amino acid repeat(31) .
In Per3p seven of these repeats were found.
A mutant in which most
of the PER3 gene is deleted was constructed by replacing the
region surrounding the translation initiation site. The resulting
mutant (per3) was unable to grow on methanol and
contained large cytosolic alcohol oxidase crystalloids, which are
characteristic for constitutive H. polymorpha per mutants(32) . Growth on methanol and normal peroxisome
development was restored upon transformation of
per3 by a
3.4-kb fragment, containing the open reading frame of PER3 or
by a plasmid containing PER3 under the control of the
P
(see below).
per3 (ura3 leu1 per3::LEU2) with an auxotrophic WT strain (leu1 PER3) and subsequent random spore analysis, monogenic
segregation was found for both Leu
and Mut
phenotypes, whereas the LEU2 gene invariably
co-segregated with the Mut
phenotype. Diploids
obtained after crossing
per3 with original per3 mutants carrying four different alleles (per3-188, per3-191, per3-229, and per3-237) all displayed the Mut
phenotype. Linkage analysis of two of the hybrids revealed no
meiotic segregants with a recombinant Mut
phenotype
(384 segregants of each hybrid were tested). Taken together, these data
indicate that the authentic PER3 gene was cloned.
per3 could not be functionally complemented by the P. pastoris PAS8 gene, although few small peroxisomes
were formed in the transformants. Immunocytochemical studies revealed
that the Pas8p was present in the cytosol (data not shown), which is
different from the location of Per3p (see below). Whether the S.
cerevisiae PAS10 gene is able to complement
per3 has
no been tested so far.
Peroxisome Biogenesis Is Not Impaired in the PER3
Disruption Mutant
In order to gain more insight in the function
of the Per3p, we analyzed the phenotype of per3 in
detail. As observed for all other H. polymorpha per mutants,
per3 displayed a Mut
phenotype;
however, the cells grew well on complex media like YPD and on mineral
media containing various carbon (glucose, glycerol, and ethanol) or
nitrogen sources (ammonium sulfate and primary amines). Under these
conditions generally one or a few small peroxisomes were observed per
cell (Fig. 2A). This indicates that Per3p is not
essential for peroxisome biogenesis.
per3 grown in batch culture on ethanol-ethylamine
which characteristically contains one peroxisome. Numerous small
peroxisomes are present when
per3 cells are grown in a
carbon-limited chemostat on glucose-choline (panel B). In
addition membrane protrusions are evident; a magnification of these is
shown in panel C. These membranes (panel D, arrow) are also evident in freeze-fractured cells and thus do
not represent artifacts due to the fixation procedure. In
per3 cells incubated in oleic acid-containing media, numerous
membranous layers also typically develop, associated with one or few
peroxisomes (panel E). Panel F shows the presence of
many normal peroxisomes in cells of the complemented
per3 strain. The abbreviations used are: N, nucleus; P, peroxisome; V, vacuole. Cytosolic alcohol oxidase
crystalloids are indicated by an asterisk (*). The marker
represents 0.5 µm.
The per3 cells
were subsequently grown in glucose-limited chemostat cultures in the
presence of choline as the sole nitrogen source. In WT cells these
growth conditions cause massive proliferation of peroxisomes and a high
level induction of various peroxisomal matrix enzymes(32) . In H. polymorpha choline is first converted to trimethylamine
which is subsequently metabolized via dimethylamine into methylamine
and two formaldehyde molecules(33) . Methylamine in fact serves
as the actual nitrogen source under these growth conditions and is
metabolized by peroxisomal amine oxidase into formaldehyde, hydrogen
peroxide, and ammonium ions. Because glucose is provided at a
growth-limiting rate in these chemostat cultures, peroxisomal enzymes
involved in methanol metabolism are highly expressed due to these
derepressing conditions together with the strong induction by
formaldehyde, released from choline utilization. These peroxisomal
enzymes include alcohol oxidase, catalase, and dihydroxyacetone
synthase(32) , and each reach levels equal to those obtained in
methanol-limited WT cultures.
per3 cells grown in
glucose-choline chemostat cultures, many small peroxisomes developed.
These organelles were frequently observed in conjunction with
membranous protrusions. These membranous structures were also observed
in freeze etch replica's of spray-frozen cells, which implies
that they are not an artifact of the chemical fixation procedure. The
architecture of these membranes resembles those of peroxisomes in WT
cells in that they show smooth fracture faces which are largely devoid
of proteinaceous particles (Fig. 2, B-D). This
morphological phenotype suggests that the biosynthesis of the
peroxisomal membrane is not disturbed or reduced in
per3 cells. This was also indicated from the morphology of
per3 cells incubated in oleic acid-containing medium.
Unlike S. cerevisiae and P. pastoris, H.
polymorpha cannot grow on this compound. However, upon incubation
of WT cells in oleic acid-containing medium, many peroxisomal enzymes
are synthesized, including enzymes of the
-oxidation pathway and
methanol metabolism; these enzymes are present in a peroxisomal
compartment which is associated with excessive amounts of membranous
layers(34) . Upon incubation of
per3 in oleic
acid-containing medium, similar structures developed, indicating again
that peroxisome biosynthesis was not disturbed in the mutant cells (Fig. 2E). However, in contrast to WT cells, these
organelles lacked the typical alcohol oxidase crystalloids which were
instead located in the cytosol (data not shown). Therefore, these data
suggest that Per3p may be involved in matrix protein translocation
across these membranes.
Most Peroxisomal Matrix Proteins Are Located in the
Cytosol of
We tested the location of various
peroxisomal enzymes in cells of per3
per3 grown in chemostat
cultures on glucose-choline. After fractionation of homogenates
prepared from these cells, the PTS1 proteins alcohol oxidase and
catalase were almost exclusively found in the soluble fraction,
suggesting a cytosolic location. Also malate synthase, which contains
neither a PTS1 nor a PTS2(35) , was mainly found in the soluble
fraction. In contrast, amine oxidase, a PTS2 protein(11) , was
clearly pelletable (Fig. 3A).
per3. Cell homogenates were fractionated by differential
centrifugation resulting in an organellar pellet and supernatant
fraction. The organellar pellet was subjected to sucrose density
centrifugation. Equal amounts of protein from the organellar pellet (1), the high speed supernatant (2), and the
peroxisomal peak fractions (3) of the sucrose gradient were
loaded per lane and subjected to SDS-polyacrylamide gel electrophoresis
and Western blotting using antibodies raised against different
peroxisomal matrix proteins as indicated. AMO, amine oxidase; AO, alcohol oxidase; Cat, catalase; MS,
malate synthase.
As mentioned before,
cells of per3 are still able to grow on ethanol in the
presence of ethylamine as the sole nitrogen source. On these compounds
the peroxisomal enzymes amine oxidase, catalase, and malate synthase
are induced, whereas synthesis of the enzymes of the methanol
metabolism is fully repressed. After fractionation of these cells,
catalase and malate synthase were mainly present in the soluble
fraction, whereas the bulk of the amine oxidase protein was
sedimentable (Fig. 3B). Also Per1p, which contains both
a PTS1 and PTS2, was predominantly present in the organellar pellet
(data not shown; 12). After sucrose gradient density centrifugation of
the organellar pellet, amine oxidase sedimented at a density of 52%
(w/w) sucrose, which is typical for peroxisomes of WT
cells(23) . A minor protein band recognized by catalase
antibodies was observed in the peroxisomal peak fraction as well;
however, malate synthase protein was not detectable in this fraction (Fig. 3B).
per3 cells (Fig. 4, A and B). Similar results were
obtained for thiolase (Fig. 4D, inset); a
heterologous PTS2 protein, namely watermelon gMDH, which is known to be
imported in WT H. polymorpha peroxisomes, is also imported in
the small peroxisomes of
per3 cells. In contrast, PTS1
proteins (alcohol oxidase, catalase (not shown), and dihydroxyacetone
synthase) were solely found in the cytosol together with malate
synthase (Fig. 4, D-F). Taken together, we conclude
that PTS1 proteins are not imported in
per3 cells. The
small amount of sedimentable catalase in sucrose gradients most
probably results from aggregation or association of the protein with
membrane vesicles.
per3. Using specific antibodies against
dihydroxyacetone synthase labeling was found in the cytosol (D). Also alcohol oxidase activity (E) and protein (F) were not found in the peroxisomal structures, but instead
were located in the cytosol and nucleus. Due to the high expression
rates in glucose/choline cells, the alcohol oxidase protein is present
in large crystalloids (E and F) in which
dihydroxyacetone synthase is also present (D). N, nucleus.
Per3p Is Located in the Cytosol and the Peroxisomal
Matrix
Antibodies were raised against a Per3p-maltose-binding
protein which was synthesized in E. coli. Using affinity
purified antiserum (-Per3p), a single dominant protein band of
approximately 70 kDa was found in crude extracts of H. polymorpha cells, overexpressing Per3p (P
PER3; see
below). In crude extracts of WT cells, grown on various carbon and
nitrogen sources, Per3p was generally not detectable by Western
blotting, indicating that the levels of Per3p are generally very low.
However, in subcellular fractions from methanol grown cells (organellar
pellet and peroxisomal peak fraction obtained after sucrose density
centrifugation), a single protein band of approximately 70 kDa was
clearly visualized on Western blots decorated with
-Per3p (Fig. 5). A minor band of Per3p was found in blots prepared from
the cytosolic fraction of these cells obtained after differential
centrifugation of cell homogenates.
per3 cells (lane 2). In crude
extracts of methanol-grown WT cells (lane 3), this band was
generally not detectable, although occasionally degradation products
may be present (lane 3). Upon cell fractionation of
methanol-grown WT cells, a dominant Per3p band was present in the
organellar pellet (lane 4) and, less dominant, in the
cytosolic fraction (lane 5). Per3p was clearly detectable in
the peroxisomal peak fraction (lane 6), obtained after sucrose
gradient centrifugation of the organellar pellet. In the mitochondrial
peak fraction (lane 7), Per3p was absent. Upon carbonate
treatment of peroxisomal peak fractions, the membrane pellet (lane
8) and soluble fraction (lane 9) were subjected to
Western blotting; Per3p was only present in the supernatant (lane
9).
After carbonate treatment of
peroxisomal peak fractions, Per3p was clearly present in the soluble
fractions, suggesting that Per3p is not an integral membrane protein.
-Per3p on ultrathin
sections of WT H. polymorpha, revealed a dual location of
Per3p in both the cytosol and the peroxisomal matrix. The peroxisomal
labeling was found both on the alcohol oxidase crystalloids but also
frequently at the periphery of the organelle (Fig. 6, A and C). The latter distribution is similar to the
labeling pattern obtained for catalase, which is shown to be mainly
located in the small zone between the alcohol oxidase crystalloid and
the peroxisomal membrane(36) . In peroxisome-deficient mutants
(
per1; 12) labeling was solely found in the cytosol, in
part associated with the cytosolic alcohol oxidase crystalloid (Fig. 6B). This suggests that Per3p is not
membrane-bound because it resembles the behavior of other soluble
matrix proteins such as amine oxidase and dihydroxyacetone synthase,
which are predominantly found in association with alcohol oxidase
crystalloids in peroxisome-deficient mutant cells(32) .
Immunofluorescence experiments fully confirmed the above location of
Per3p (Fig. 7).
-PER3), the labeling intensity of peroxisomes
is not significantly enhanced, but more label is seen in the cytosol (E). In such cells alcohol oxidase protein is still confined
to the peroxisomal matrix (F, anti-alcohol oxidase). N, nucleus.
Overexpression of PER3 Does Not Affect the Import of
Peroxisomal Matrix Proteins
Transformants, which expressed PER3 under control of the alcohol oxidase promoter
(P), normally grew on methanol. In crude extracts,
prepared from these cells, Per3p was readily detectable by Western
blotting (Fig. 5), indicating that PER3 overexpression
had occurred.
Per3p Is Dispensable for Sorting of PTS2
Proteins
The existence of different PTS-dependent pathways for
peroxisomal protein import in yeasts was already predicted from the
analysis of various import deficient mutants in which the import of
specific subsets of peroxisomal proteins was fully
prevented(7, 8, 9) . As such, these mutants
resembled the phenotypes of six out of nine complementation groups of
human Zellweger syndrome fibroblasts(37, 38) .
per3) PTS2 proteins are normally
sorted to peroxisomes. These results indicate that peroxisomes of
per3 cells contain a fully functional protein
translocation machinery, thus independent of Per3p. PTS1 proteins
however remained in the cytosol. Furthermore, since also heterologous
PTS2 proteins are correctly imported, this PTS2 import pathway is, as
the PTS1 import pathway, conserved to a certain extent.
Per3p Shows Similarity to P. pastoris Pas8p and S.
cerevisiae Pas10p (the PTS1 or SKL receptor)
H. polymorpha Per3p shows significant similarity to Pas8p of P. pastoris and Pas10p of S. cerevisiae. Like the H. polymorpha
per3 mutants, the P. pastoris pas8 and S. cerevisiae
pas10 mutants are specifically defective in import of PTS1
proteins. The regions of strongest identity are found in the C-terminal
parts, in which the TPR motif is located(31) . This motif is
thought to play a role in protein-protein interactions; nevertheless, a
precise biochemical function has not yet been assigned to
it(39) . Most members of the TPR protein family play a role in
mitosis or in regulation of the cell cycle; on the other hand,
MOM72/MAS70, a mitochondrial protein receptor, also belongs to this
family(40) . The receptor function of S. cerevisiae Pas10p is in fact confirmed in recent studies by Brocard et
al.(41) , who showed that Pas10p interacts in vivo with the PTS1 and that the TPR motif is essential for this
interaction.
per3 cells are correctly assembled and enzymatically active;
this suggests that the putative peroxisomal factors(42) ,
involved in the assembly processes, are also not imported and
functional in the cytosol. Remarkably, malate synthase which lacks a
conserved PTS1 or PTS2 signal, also remained in the cytosol of
per3 cells. A likely explanation for this result is that
the yet unknown targeting signal of malate synthase is dependent on a
PTS1 protein to facilitate import. A comparable explanation was
presented by Kunau and co-workers (7) to account for the
import of acyl-CoA oxidase and catalase A, which both have internal
targeting signals, in S. cerevisiae
pas7 cells.
(
)Our
data, however, clearly demonstrated that in H. polymorpha the PER3 gene protein is located in each of these compartments and
is not an integral component of the peroxisomal membrane.
What Is the Function of Per3p?
The easiest
explanation, building on earlier results obtained with S.
cerevisiae Pas10p and P. pastoris Pas8p, is that Per3p
acts as a receptor, which specifically binds the PTS1 of peroxisomal
precursors and shuttles these polypeptides from the cytosol to the
organellar matrix. The fact that a major portion of Per3p is detected
in peroxisomes may indicate that the release of Per3p from the
organelles probably is rather slow compared to the other steps of the
cycle.
(
)
per3 strain although Pas8p was
normally synthesized; immunocytochemistry revealed that the PAS8 gene product was largely cytosolic in H. polymorpha (data
not shown).
per3 mutant may be due to the fact that
minor differences exist between the import machineries of both
methylotrophic yeasts preventing the import, and thus the shuttling
function, of Pas8p in H. polymorpha.
Concluding Remark
With the cloning and
characterization of the H. polymorpha PER3 gene, three genes
involved in peroxisome protein import have been isolated from three
different yeast species. Although likely, it is not yet fully clear
whether the protein products of these genes are functional homologues
of each other and fulfill the same biochemical function. However, the
availability of the deduced amino acid sequences of these proteins
allows initiation of mutational studies in regions of interest to
elucidate their function in detail.
-Pas8p
antibodies and Dr. W. H. Kunau (Bochum, Germany) for
-thiolase
antibodies. We thank Klaas-Nico Faber and Jan Kiel for expert advice in
the molecular genetic part of the work. The skillfull assistance of
Meis van der Heyden, Ineke Keizer-Gunnink, Klaas Sjollema, and Jan
Zagers is gratefully acknowledged.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.