Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000
Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX-HA). Coexpression of AOX-HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX-HA present in octameric active AOX, suggesting that newly synthesized AOX-HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.
ALCOHOL oxidase (AOX)1 is a homooctameric flavoprotein consisting of eight identical subunits of
~74 kD, each containing a flavin adenine dinucleotide molecule (FAD) as a prosthetic group (van der Klei
et al., 1991 Genes encoding AOX have been cloned from P. pastoris (AOX1 and AOX2; Ellis et al., 1985 Recent observations indicate that the peroxisomal protein import mechanism may differ significantly from those
known for other organelles (McNew and Goodman, 1996 To investigate whether oligomeric import is a general
characteristic of peroxisomal matrix protein import, we
have examined the targeting, import, and assembly of P. pastoris AOX. Our results demonstrate that AOX is targeted by a COOH-terminal PTS1 motif and that assembled and active AOX is only found inside peroxisomes. In
addition, we show that the efficiency of import and assembly of AOX without its PTS1 is not enhanced by coexpression with wild-type AOX. Thus, unlike other peroxisomal
proteins, AOX cannot initiate assembly in the cytoplasm.
Strains, Media, and Microbial Techniques
Yeast strains used in this study are listed in Table I. Shake-flask cultures
were incubated for 10-15 h at 30° (P. pastoris strains) or 37°C (H. polymorpha strains) in selective minimal YND or YNM medium (0.17% [wt/
vol] yeast nitrogen base without amino acids {Difco Laboratories Inc., Detroit, MI} supplemented with 0.5% [wt/vol] glucose [dextrose] or 0.5%
[vol/vol] methanol). For growth of auxotrophic strains, amino acids were
added to a final concentration of 50 µg/ml. Transformations of P. pastoris (Becker and Guarente, 1991 Table I.
Yeast Strains
). The protein catalyzes the oxidation of methanol to formaldehyde and hydrogen peroxide, the first step in the methanol utilization pathway of certain yeasts including Pichia pastoris, Hansenula polymorpha, and Candida boidinii (van der Klei et al., 1991
). AOX is normally
localized in the matrix of single membrane-bound organelles called peroxisomes. During methanol growth, the
peroxisomes also contain large amounts of dihydroxyacetone synthase, the first enzyme in the methanol assimilatory pathway, and catalase (CAT), which converts the hydrogen peroxide generated by oxidases such as AOX into
water and oxygen (Veenhuis and Harder, 1991
). As a result, peroxisomes, which are small and few in number in
glucose-grown cells, are massively induced in methanol-grown cells (Veenhuis and Harder, 1991
). In previous studies, we have shown that functional peroxisomes are essential for growth of P. pastoris and H. polymorpha on
methanol, but not on glucose, and have exploited this observation in the isolation of numerous mutants that are defective in the biogenesis/assembly of peroxisomes (pex
mutants) (Cregg et al., 1990
; Gould et al., 1992
; Liu et al.,
1992
; Waterham et al., 1992
; Tan et al., 1995a
).
; Cregg et al.,
1989
; Koutz et al., 1989
), H. polymorpha (MOX; Ledeboer
et al., 1985
), and C. boidinii (AOD1; Sakai and Tani, 1992
).
The predicted primary sequences of their products are 73-
85% identical. In all of these yeast species, the transcription of genes encoding AOX is highly repressed during
growth on glucose or ethanol, and maximally induced during growth on methanol (Tschopp et al., 1987
). Like other
peroxisomal proteins, AOX is translated on free cytosolic
ribosomes and posttranslationally imported into the peroxisomal matrix (Roa and Blobel, 1983
). The four COOH-terminal amino acids of H. polymorpha AOX are capable of targeting a nonperoxisomal reporter protein to peroxisomes, suggesting that AOX is targeted by alanine-arginine-
phenylalanineCOOH (ARF), an uncommon variant of the
type 1 peroxisomal targeting signal (PTS1) motif (Hansen
et al., 1992
; Subramani, 1993
). The typical PTS1 motif is a
tripeptide of the sequence serine-lysine-leucineCOOH (SKL;
and conserved variants) found at the extreme COOH terminus of many matrix proteins (Gould et al., 1989
, 1990
;
Swinkels et al., 1992
; Subramani, 1993
). The motif is specifically recognized by a PTS1 receptor protein, Pex5p, as
an early step in the peroxisomal protein import process
(McCollum et al., 1993
; Terlecky et al., 1995
). Pex5p is
thought to deliver PTS1-containing polypeptides to the surface of the peroxisome and then cycle back to the cytoplasm for further rounds of PTS1 protein binding and targeting
(Dodt and Gould, 1996
; Waterham and Cregg, 1997
).
).
In particular, newly synthesized peroxisomal proteins
need not be in an extended monomeric conformation to be
imported, but can assemble/oligomerize in the cytoplasm before import. For example, Saccharomyces cerevisiae thiolase, without its NH2-terminal PTS2 motif, is not imported into peroxisomes (Glover et al., 1994
). However, if
coexpressed with wild-type thiolase, it is efficiently imported. The interpretation of this result is that thiolase, a
homodimer, must be capable of oligomerizing before import, and that PTS2-less thiolase monomers are capable of
dimerizing with wild-type monomers in the cytoplasm, and
being coimported (piggybacked) into the organelle. Similar results have been obtained with three PTS1-targeted
proteins: a chloramphenicol acetyl transferase PTS1 fusion
protein (CAT-PTS1; McNew and Goodman, 1994
), human
alanine/glyoxylate amino transferase 1 (AGT; Leiper et al.,
1996
), and S. cerevisiae malate dehydrogenase 3 (MDH3; Elgersma et al., 1996
). Further evidence supporting the
notion that peroxisomes can import preassembled structures has been provided from microinjection studies of human cell lines that show that colloidal gold particles
9
nm and coated with a human serum albumin-PTS1 conjugate can be imported (Walton et al., 1995
).
Materials and Methods
) or H. polymorpha (Faber et al., 1994
) were
performed by electrotransformation. Cultivation of Escherichia coli strain
DH5
and standard recombinant DNA techniques were performed essentially as described (Sambrook et al., 1989
).
Construction of -Lactamase Fusion Vector Strains
Plasmids encoding chimeric proteins composed of selected AOX amino
acid sequences fused to a modified E. coli -lactamase protein were constructed. The modified
-lactamase was composed of amino acid residues
H24-W286 (Sutcliffe, 1978
) preceded by the amino acids methione-serine-glycine (MSG) as previously described (Waterham et al., 1994
, 1997
).
DNA sequences encoding the COOH termini of wild-type AOX (AOX-WT), AOX-RSC, and AOX-HA (see below) were ligated in reading frame to
the 3
end of the bla gene using either the StuI site located at 1,924 bp or
the AgeI site (made blunt ended with the Klenow fragment of DNA polymerase I) located at 1,972-bp downstream of the AOX1 start codon
(Koutz et al., 1989
). The proper DNA sequence at each fusion junction
was verified by DNA sequencing. The primary sequence of each fusion
protein is shown in Table II. All
-lactamase fusion proteins were expressed under transcriptional control of the constitutive GAP promoter
(PGAP) from the P. pastoris GAP gene (Waterham et al., 1997
) in vector
pHWO10K. This vector was created from the HIS4-based vector pHWO10
by replacing the ampicillin-resistance gene with the kanamycine-resistance gene. The vectors were integrated into the genomic HIS4 locus of P. pastoris strains GS115 or pex5
(Table I) after linearization with SalI, a
unique site in the HIS4 gene of each vector.
Table II. Plasmids |
Construction of Mutant AOX Expression Strains
The P. pastoris AOX1 gene was amplified from plasmid pPG5.4 (Cregg
et al., 1989) by the PCR using a forward primer, composed of the first 18 bp
of the AOX1 open reading frame preceded by an EcoRI site, and a reverse primer, composed of 19 bp located 247-266-bp downstream of the
AOX1 stop codon which includes a genomic HindIII site (Koutz et al., 1989
).
After digestion with EcoRI and HindIII, the AOX1 gene was first subcloned in EcoRI-HindIII-digested pBS-SK (Stratagene, La Jolla, CA),
and subsequently as an EcoRI-ClaI fragment under transcriptional control of the P. pastoris AOX1 promoter (PAOX1) into EcoRI- and ClaI-digested
vector pHIL-AI (Invitrogen, San Diego, CA; Waterham et al., 1997
). The
resulting vector was named pHWO40. To modify sequences encoding the
COOH terminus of AOX-WT, vector pHWO40 was digested with AgeI
(located 1,972-bp downstream of the AOX1 start codon) and then the resulting termini were made blunt with Klenow fragment of DNA polymerase I and religated. This resulted in a frame shift changing the COOH-terminal residues of AOX-WT leucine-alanine-arginine-phenyl-alanineCOOH (LARF) to arginine-serine-cysteineCOOH (RSC). The resulting vector that
expressed AOX-RSC was named pHWO41. A second vector encoding a
COOH-terminal modified AOX was created by inserting two complementary oligonucleotides, which changed the four COOH-terminal residues from LARF to that for the human influenza virus epitope (HA) tag at
the AgeI site of pHWO40. These residues are recognized by the mouse
monoclonal antibody 12CA5 (Boehringer Mannheim Biochemicals, Indianapolis, IN). The resulting modified AOX protein is called AOX-HA
(vector pHWO42). Finally, a vector was created that encodes a modified
AOX (AOX-
22) in which the last 22 amino acids of the protein are deleted. This vector (pHWO43) was generated by inserting a blunt AgeI
adaptor encoding an in-frame stop codon between the StuI and AgeI sites
of AOX1 in vector pHWO40. The primary sequences of the COOH termini of the modified AOX proteins are shown in Table II. All constructs
were verified by DNA sequencing. The constructs were integrated into
the genomic HIS4 locus of P. pastoris strains MC100-3, GS115, and pex5
(Table I) by linearization at the unique EcoNI site in the HIS4 gene of
each vector.
Two-hybrid System Experiments
Interactions between the P. pastoris PTS1 receptor protein Pex5p and selected sequences from AOX were studied with the yeast two-hybrid system (Matchmaker; Clontech Laboratories, Inc., Palo Alto, CA). The P. pastoris PEX5 open reading frame was released from pSP72 (a gift from
S. Subramani, University of California at San Diego, San Diego, CA) by
digestion at a SacII site located 42-bp downstream of the PEX5 start
codon, treated with mung bean exonuclease, and then digested at a PstI
site located downstream of the PEX5 stop codon. This fragment was ligated into SmaI-PstI-digested pGAD424 which resulted in the expression
of Pex5p fused in reading frame with the GAL4 activation domain
(pHWO51). The COOH termini of AOX-WT, AOX-RSC, and AOX-
HA were released from vectors pHWO40, pHWO41, and pHWO42, respectively, by digestion with StuI (located 1,924-bp downstream of the
AOX1 start codon) and PstI (located downstream of the AOX1 stop
codon). These fragments were then ligated into pGBT9 that had been cut
with XmaI, filled with Klenow, and then digested with PstI. The resulting
vector expressed the GAL4-DNA binding domain protein fused in-frame
to each of the AOX-derived peptides described above. The COOH-terminal sequences of the resulting constructs named pHWO48 through
pHWO50 are shown in Table II. All constructs were verified by DNA sequencing. These constructs and control vectors were transformed by electrotransformation into the S. cerevisiae reporter strain SFY526 (Becker
and Guarente, 1991).
-Galactosidase activity in each strain was determined qualitatively by the filter assay method and quantitatively by the
cell-free activity assay method described in the Clontech technical manual.
Expression of Modified AOX1 Genes in P. pastoris
AOX-HA was coexpressed with AOX-WT by inserting vector pHWO42
in GS115 (GS-HWO42), and expressed alone by insertion of the vector
into the AOX1 and AOX2 deletion strain MC100-3 (MS-HWO42). Octamerization of AOX proteins was examined using a modified version of
the velocity sedimentation method described by Goodman et al. (1984).
Cells (50 OD600 U) were lysed by vortexing for 15 min at 4°C with 1 vol of
glass beads in 0.3 ml TENT buffer (10 mM Tris-HCl, pH 8, 5 mM EDTA,
50 mM NaCl, 1% Triton X-100) and then centrifuged for 10 min at maximum speed in a microcentrifuge (Eppendorf model 5415C; Brinkman Instruments, Westbury, NY).
Supernatant samples of 0.1 ml were separated through 5-30% (wt/vol) sucrose gradients (6 layers of 1.5 ml sucrose in TENT) by centrifugation for 5 h at 40,000 rpm and 2°C in a rotor (SW41Ti; Beckman Instruments, Palo Alto, CA). Fractions of 0.5 ml were removed from the top and analyzed for sucrose density, AOX, and CAT activities, and for AOX-WT or AOX-HA protein by immunoblotting using polyclonal antibodies against AOX or monoclonal antibodies against the HA epitope.
Heterologous Expression of AOX1 and MOX Genes
The H. polymorpha MOX gene was expressed under transcriptional control of its own promoter by integration of KasI-linearized vector pHWO56
into the HIS4 locus of the P. pastoris AOX1 and AOX2 deletion strain
MC100-3 and by integration of SacI-linearized vector pHWO55 into the
PAOX1 locus of the P. pastoris pex2 aox1
aox2
strain MC200. The
MC200 strain was generated by disruption of the PEX2 gene in MC100-3
using BamHI-digested pUZ12 (Waterham et al., 1996
). The P. pastoris
AOX1 gene was expressed under transcriptional control of its own promoter (pHWO57) and the H. polymorpha PMOX (pHWO58) in a H. polymorpha mox
pex10-1 strain. This strain was constructed by disruption of
the MOX gene in the H. polymorpha pex10-1 mutant strain using AlwNI-
SalI-digested pHWO52.
Indirect Immunofluorescence
P. pastoris cells (25 OD600 U) were washed twice with water and fixed in 1 ml
of 40 mM KPO4 buffer, pH 6.5, with 3.7% (vol/vol) formaldehyde for 1 h
at room temperature. After two washes with 1 ml MOPS+ (5 mM 3-[morpholino]propane-sulfonic acid, 10 mM Na2SO3, and 0.5 M KCl), fixed cells
were converted into spheroplasts in 0.5 ml MOPS+ supplemented to 85 mM
-mercaptoethanol and 0.08 mg/ml Zymolyase-100T for 1 h at 30°C. The
spheroplasts were washed twice with 1 ml MOPS+, incubated for 5 min at
20°C in 100% methanol, washed twice with 1 ml PBS+ (PBS, pH 7.2, 1 M
sorbitol, and 1% bovine serum albumin), and finally suspended in 1 ml
PBS+. Suspended cells (0.2 ml) were incubated for 2 h at room temperature with 0.5 µl of polyclonal antibodies raised against CAT or
-lactamase (5 Prime, 3 Prime, Boulder, CO). After three washes with 1 ml PBS+,
cells were suspended in 0.2 ml PBS+ and incubated for 1 h at room temperature in the dark with 0.5 µl FITC-conjugated goat anti-rabbit antiserum (Boehringer Mannheim, Indianapolis, IN). After three washes with 1 ml
PBS+, cells were suspended in 0.1 ml PBS+. For microscopic observation,
small samples of the cell suspensions were mixed with equal volumes of
0.1 M n-propyl gallate in 90% glycerol. Cells were examined with a microscope equipped for indirect immunofluorescence (Leitz Laborlux S; Wild
Leitz, Rockleigh, NJ) at 1,000× and photographed (T-MAX 400 film;
Eastman Kodak Co., Rochester, NY).
Biochemical Methods
Subcellular fractionation of P. pastoris and H. polymorpha cells was performed as described previously (Waterham et al., 1996) except that H. polymorpha cells were converted to spheroplasts in the presence of 3 M
sorbitol and homogenized in the presence of 2 M sorbitol. Cell-free extracts were made using a glass bead method (Waterham et al., 1997
). Peroxisomal AOX and CAT, mitochondrial cytochrome c oxidase, cytosolic
glyceraldehyde-3-phosphate dehydrogenase, and
-lactamase activities
were assayed as described previously (Waterham et al., 1996
, 1997
). Protein concentrations were determined with the bicinchoninic acid protein
assay kit (Pierce Chemical Co., Rockford, IL) using bovine serum albumin as a standard. The transfer of proteins to nitrocellulose after SDS-PAGE electrophoresis was performed using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) as directed
by the manufacturer. Immunoblotting experiments were performed using
the Western Light Kit (Tropix Inc., Bedford, MA) with specific polyclonal
antibodies against AOX and monoclonal antibody 12CA5 against the hemagglutinin epitope (Boehringer Mannheim). Immunoprecipitations were
performed as described in Rehling et al. (1996)
except that protein A-Sepharose (Pharmacia Biotech. Inc., Piscataway, NJ) was used instead of DynaBeads. Native (nonreducing nondenaturing discontinuous) gels were
electrophoresed at 200 v for 2 h at 4°C through 5% polyacrylamide using a
Mini-Protean II apparatus (BioRad Laboratories) as described in Ausubel
et al. (1996)
.
Nucleotide Sequence Accession Numbers
Sequence data for the P. pastoris AOX1 and AOX2 genes, as published by
Koutz et al. (1989), have been submitted to the databases and are available from GenBank/EMBL/DDBJ under accession numbers U96967
(AOX1) and U96968 (AOX2).
Active AOX Is Present Only in Peroxisomes of P. pastoris
Our previous studies with P. pastoris pex mutants indicated that methanol-induced pex cells contain little or no
AOX activity, although significant amounts of AOX protein are present in the cells (Liu et al., 1992, 1995
; Waterham et al., 1996
). The level of residual AOX activity in an
individual pex mutant allele strongly correlated with the
severity of the peroxisome-deficient phenotype of that strain.
For example, in methanol-induced cells of pex2-1 and pex8-1
(two slightly leaky mutants generated by chemical mutagenesis), numerous small peroxisomal remnants were
observed that retain the ability to import small amounts of
peroxisomal enzymes (Liu et al., 1995
; Waterham et al.,
1996
). In both of these mutants, small but significant
amounts of active AOX were present and these appeared
to be exclusively within the peroxisomal remnants as judged by subcellular fractionation studies (Table III). In
contrast, methanol-induced cells of pex2 and pex8 deletion
strains (pex2
and pex8
), which have a more severe peroxisome-deficient phenotype with few, if any, peroxisomal
remnants and no measurable import ability, contained no
active AOX, although AOX protein was present (Fig. 1).
The inactive AOX protein was concentrated in cytoplasmic protein aggregates which sedimented with organelles
during subcellular fractionation (Fig. 1; Liu et al., 1995
;
Waterham et al., 1996
). These results indicate that active
AOX exists only in the peroxisomal matrix and suggest
that AOX precursors require the presence of functional
peroxisomes to assemble into active octamers.
Table III. Peroxisomal Enzyme Activities in Organelle Pellet and Cytosolic Supernatant Fractions of Methanol-induced P. pastoris pex strains |
AOX from H. polymorpha Is Active in the Cytoplasm of P. pastoris
Unlike P. pastoris, AOX from H. polymorpha (MOX) efficiently assembles into an active octameric enzyme in the
cytoplasm of H. polymorpha pex mutants (van der Klei et al.,
1991). To determine whether this difference is a characteristic of the AOX proteins themselves or the cellular environment provided by their hosts, we constructed pex
strains of each yeast species that expressed AOX/MOX from
the heterologous species. For H. polymorpha, a vector containing the P. pastoris AOX1 gene was inserted into
the genome of an H. polymorpha pex10 mox
strain. AOX
activity could not be detected in the AOX-expressing
pex10 mox
strain CW111 at either 30° or 37°C (Table
IV). Immunoblot analysis performed on crude organelle
pellet and cytosolic supernatant fractions from methanol-induced cells of this strain showed that AOX protein was
synthesized and located primarily in pellet fractions (Fig.
2, group 2 and 3 lanes). The pellet location was most likely
a consequence of AOX protein aggregation as seen in P. pastoris pex mutants (Fig. 1). Identical results were obtained when AOX1 was expressed under control of the H. polymorpha MOX promoter (data not shown). Thus, in
contrast to endogenous H. polymorpha MOX, P. pastoris
AOX is not assembled into active protein in the cytosol of
an H. polymorpha pex mutant.
Table IV. Peroxisomal Enzyme Activities in Organelle Pellet and Cytosolic Supernatant Fractions of Methanol-induced Strains |
The converse result was obtained when H. polymorpha
MOX was expressed in a P. pastoris pex mutant. As expected, MOX was able to complement for methanol growth
in a P. pastoris strain on MC100-3 that is deleted for both
AOX genes (aox1 aox2
). When the PAOX-MOX vector
was then inserted into the genome of a P. pastoris pex2
aox1
aox2
strain (MC200), methanol-induced cells of
the transformed strain contained active MOX (Table IV).
Upon subcellular fractionation, both MOX activity and
protein were found in the cytosolic supernatant (Fig. 2, 1 lanes). The same result was obtained when MOX was expressed in a P. pastoris pex5
aox1
aox2
strain (data
not shown). Since H. polymorpha MOX assembles into an
active enzyme in the cytoplasm of either H. polymorpha or
P. pastoris pex strains, the inability of P. pastoris AOX to
properly assemble in these yeasts must be a characteristic
of the protein itself and not of the environment provided
by the P. pastoris cytoplasm.
A Peroxisomal Targeting Signal Is Located at the COOH Terminus of AOX
To further investigate the targeting and import of AOX, it
was first necessary to define the AOX PTS. The three
COOH-terminal amino acids of AOX, ARF, are similar in
sequence to the PTS1 motif and, therefore, were a good
candidate for the AOX PTS. Preliminary evidence in support of this was provided by Hansen et al. (1992) who
showed that the last four amino acids of H. polymorpha
MOX (LARF) are capable of targeting a nonperoxisomal
protein to peroxisomes. To examine targeting of P. pastoris AOX, we first expressed a protein composed of the
bacterial reporter enzyme
-lactamase fused to the four
COOH-terminal amino acids of P. pastoris AOX (LARF)
and determined whether this fusion protein (
-lac-LARF) was targeted to P. pastoris peroxisomes. Methanol-grown
cells expressing
-lac-LARF were fractionated into an organelle pellet, consisting mainly of peroxisomes and mitochondria, and a cytosolic supernatant (Fig. 3 B). Biochemical analysis of these fractions revealed that a significant
portion of
-lactamase activity colocalized with CAT activity in the organelle pellet fraction, suggesting that
-lac-
LARF was targeted to peroxisomes. (Typically, a significant amount of peroxisomal matrix protein is also found in
the supernatant fraction due to breakage of these fragile
organelles.) Peroxisomal targeting of
-lac-LARF was
confirmed by indirect immunofluorescence using
-lactamase- and CAT-specific antibodies (Fig. 3 B) and by further fractionation of the organelle pellet through sucrose density gradients (Fig. 4). In these gradients,
-lac-LARF
was present at the same density as CAT and not with mitochondrial cytochrome c oxidase. As a control,
-lactamase
without LARF expressed in P. pastoris fractionated to the
cytosolic supernatant along with the cytoplasmic marker
enzyme glyceraldehyde-3-phosphate dehydrogenase and
appeared to be cytoplasmic in immunofluorescence assays
(Fig. 3 A). We concluded a PTS is located within the
LARF sequence.
LARF Is Necessary for Efficient Targeting of AOX
We next investigated the necessity of LARF for targeting
and import of AOX. In one set of experiments, the 22 COOH-terminal amino acids of AOX were fused to the
COOH terminus of -lactamase (
-lac-AOX). As expected, this fusion protein was efficiently targeted to peroxisomes of P. pastoris (Fig. 3 D). We then deleted the
four COOH-terminal residues from this construct and replaced them with either the non-PTS amino acid sequence
RSC (
-lac-AOX-RSC) or the hemagglutinin-epitope-tag sequence (
-lac-AOX-HA). When expressed in methanol-grown P. pastoris cells, both of these chimeric proteins were localized to the cytoplasm as judged by indirect immunofluorescence and subcellular fractionation assays
(Fig. 3, E and F).
The necessity of LARF for AOX targeting was further
investigated by expressing mutant versions of AOX in
which the tetrapeptide sequence had been removed and
replaced with either RSC (AOX-RSC) or the HA tag
(AOX-HA). Each mutant protein was expressed under control of the P. pastoris AOX1 promoter in a P. pastoris
aox1 aox2
strain. Both the AOX-RSC and AOX-HA
constructs partially complemented the strain for growth
on methanol (18 and 22 h/generation, respectively, versus
3.5 h/generation for wild type), and both contained low
but significant levels of AOX activity (16 and 3% of wild
type; Table V). Subcellular fractionation studies of the
AOX-RSC and AOX-HA expression strains showed that
the majority of AOX activity was in the organelle pellet
(Table V), suggesting that the active portions of the mutant AOXs were properly targeted to peroxisomes. Further fractionation of the organelle pellet from the AOX-HA
strain through a sucrose density gradient confirmed that
the AOX activity was peroxisomal (Fig. 5 B). Thus, a portion of both AOX-RSC and AOX-HA synthesized in P. pastoris cells is properly imported into peroxisomes where
it assembles into an active enzyme.
Table V. Peroxisomal Enzyme Activities in Organelle Pellet and Cytosolic Supernatant Fractions of Methanol-induced P. pastoris Strains Expressing Mutant AOX1 Genes |
To estimate the proportion of total AOX-RSC and
AOX-HA protein that assembled into an active enzyme,
total cell extracts from methanol-induced cells of the
AOX-RSC and AOX-HA-expressing strains were prepared by mechanical disruption and subjected to sucrose velocity gradient sedimentation (Goodman et al., 1984).
This glass bead-based procedure was chosen because it
avoids the long incubation period needed to prepare yeast
protoplasts for subcellular fractionation, during which unstable inactive AOX protein is rapidly degraded relative
to stable active AOX octamers. As a marker for the velocity gradients, we assayed activity for CAT, a tetrameric
protein of ~300 kD. In a control gradient with total lysates
prepared from strains expressing AOX-WT (Fig. 6 C),
AOX activity and protein comigrated at one position
through the velocity gradient, the same position as purified octameric AOX with a molecular mass of ~600 kD
(Fig. 6 A). Gradients prepared from cells of strains expressing either AOX-RSC or AOX-HA alone contained
small amounts of AOX activity at the normal octameric
AOX position (Fig. 6, D and E). However, most of the
AOX protein was spread over fractions representing
lower molecular masses. Control gradients containing chemically denatured (mainly monomeric) AOX (Fig. 6
A, AOX*) or extracts prepared from a pex5
mutant
strain expressing AOX-HA (Fig. 6 B) indicated that the
inactive AOX proteins at these lower mass positions were
most likely monomeric and aggregated forms of AOX.
We concluded from these studies that a PTS sufficient for targeting proteins to peroxisomes is present within LARF (most likely ARF), and that this PTS is essential for the efficient targeting and import of AOX. However, since small but significant amounts of AOX are imported in the absence of LARF, a second less efficient PTS exists in another part of the protein. This second PTS may be located within the next 18 COOH-terminal amino acids of AOX, since a mutant protein in which the 22 COOH-terminal amino acid residues were deleted was completely inactive (Table V).
The COOH Terminus of AOX Interacts with the PTS1 Receptor
The similarity of ARF to the consensus PTS1 motif, SKL,
suggests that AOX may be imported via the PTS1 pathway. Furthermore, import of AOX or -lac-AOX is
blocked in a pex5
mutant which is specifically defective
in the PTS1 receptor Pex5p (Fig. 3 C; McCollum et al.,
1993
). Additional evidence that AOX is targeted to peroxisomes by the PTS1 pathway was obtained in yeast two- hybrid system assays in which the 22 COOH-terminal
amino acids of AOX were expressed as a fusion with the
GAL4 DNA-binding domain (GAL4B-AOX) in combination with the P. pastoris PTS1 receptor protein Pex5p
fused to the GAL4 activation domain (GAL4A-Pex5p). This combination produced a strong response in the system (Fig. 7). The interaction was specific since expression
of GAL4B-AOX with unfused GAL4A or GAL4A-Pex5p
with unfused GAL4B produced little response. We then
tested GAL4B-AOX variants in which the COOH-terminal LARF sequence was replaced by either RSC or the HA tag. Neither of these constructs produced a specific response in combination with GAL4A-Pex5p.
The Import Efficiency of AOX-HA Is Not Improved by Coexpression with AOX-WT
We examined whether the import efficiency of AOX without its PTS1 motif was increased when coexpressed with
AOX-WT. To distinguish between AOX without its PTS1
motif and AOX-WT, we used the AOX-HA construct in
which the four COOH-terminal amino acids had been replaced with the HA-epitope tag. Immunoblot analysis of
total extracts prepared from P. pastoris strains expressing
either AOX-WT or AOX-HA alone demonstrated that the
anti-HA monoclonal antibodies exclusively recognized the
AOX-HA protein, whereas polyclonal antibodies against
AOX protein recognized both AOX-WT and AOX-HA (Fig. 5 A). To estimate the proportion of imported versus
cytoplasmic AOX-HA, we again made use of the sucrose
velocity sedimentation method to separate imported, assembled, and active octameric AOX from cytoplasmic inactive monomers and aggregated forms of the protein
(Goodman et al., 1984). Velocity gradients prepared with lysates from a strain expressing only AOX-HA showed a
small amount of AOX activity at the position expected for
octameric AOX (Fig. 6 E). However, immunoblot analysis
revealed that most AOX-HA protein migrated to lower
molecular mass positions in the gradient. This distribution
was not because of the HA tag since a similar result was
obtained with a strain expressing AOX-RSC (Fig. 6 D).
As described above, control gradients performed with purified and then chemically denatured AOX (Fig. 6 A,
AOX*), or with a lysate prepared from a pex5
strain expressing AOX-HA (Fig. 6 B), indicated that the AOX
protein at the lower positions represented monomeric
and/or aggregated forms of AOX.
A P. pastoris strain coexpressing both AOX-HA and AOX-WT was first examined by subcellular fractionation. After the organelle pellet from methanol-grown cells of the strain were centrifuged through a sucrose density gradient, a significant portion of AOX-HA was found to be peroxisome-bound as indicated by the comigration of AOX-HA protein with activities for AOX and CAT (Fig. 5 C). When a lysate prepared from this strain was analyzed by sucrose velocity gradient sedimentation, large amounts of AOX activity migrated to the position of octameric protein (Fig. 6 F). Immunoblot analysis with anti-AOX antibodies that recognize both AOX-WT and AOX-HA confirmed that the major portion of AOX protein comigrated with the AOX activity. However, immunoblot analysis with the anti-HA antibodies did not show a shift of AOX-HA protein to the position of octameric AOX (compare the distribution of AOX-HA in Fig. 6, F and E). Thus, the import efficiency of AOX-HA, as measured by octamerization, was not improved by coexpression with AOX-WT, indicating that AOX-HA could not oligomerize with AOX-WT in the cytoplasm before import into peroxisomes.
AOX-HA Is Not Impaired in Ability to Assemble with AOX-WT
An alternative interpretation of the coexpression results
was that AOX-HA could not oligomerize with AOX-WT
in the cytoplasm because of interference by the HA tag in
AOX-HA/AOX-WT assembly or to the rapid assembly
kinetics of AOX-WT, as suggested for MDH by Elgersma
et al. (1996). If this were true, then the small amount of
AOX-HA that does assemble into active enzyme would
be expected to be present as AOX-HA homooctamers
and not as AOX-HA/AOX-WT heterooctamers. To examine whether or not active AOX-HA oligomerized with
AOX-WT in cells coexpressing the two proteins, two experiments were performed. In the first, sucrose velocity
gradient fractions containing octameric AOX from the coexpression strain (Fig. 6 F, fraction 9) and from the strain
expressing AOX-HA alone (Fig. 6 E, fraction 9) were
immunoprecipitated using anti-HA antibodies. The immunoprecipitates were subsequently examined via immunoblot for the relative amounts of HA and AOX cross-
reacting protein (Fig. 8 A). If active AOX-HA in the
coexpression strain was a homooctamer, the relative amount
of HA- and AOX-reacting protein should be the same as
in the AOX-HA fractions. In contrast, results showed that
the HA antibodies precipitated significantly more AOX
protein from the coexpression strain fractions, indicating that AOX-WT protein was coprecipitated along with
AOX-HA. As a control for the specificity of the HA antibody preparation, no AOX protein was immunoprecipitated from AOX-WT gradient fractions (Fig. 6 C, fraction
9; Fig. 8 A, lane 4). These results suggested that AOX-WT
and AOX-HA subunits exist as heteromers in octameric fractions from the coexpression strain.
In the second experiment, the presence of AOX-HA and AOX-WT protein in the same species of AOX molecule was examined directly by subjecting active octameric AOX (Fig. 6, from the gradients in fraction 9) to native PAGE and immunoblotting (Fig. 8 B). In these native gels, AOX-WT homooctamers migrate slower than AOX-HA homooctamers (despite the fact that AOX-HA is slightly higher in predicted molecular mass than AOX-WT). In fractions from the coexpression strain, AOX-HA migrated at approximately the same rate as homooctameric AOX-WT. Thus, most of the AOX-HA was present as heterooctamers with AOX-WT. Furthermore, the fact that heteromers migrated at about the same rate as AOX-WT homomers indicated that the heteromers were composed mostly of AOX-WT with only one or two AOX-HA subunits, indicating that the small amount of AOX-HA that reaches the peroxisomal matrix had no difficulty oligomerizing with the large amount of AOX-WT present in the organelle.
In this paper, we examined the targeting and assembly of
peroxisomal AOX in the yeast P. pastoris. In particular,
we were interested in determining whether newly synthesized AOX is capable of oligomerizing in the cytoplasm
before import, as recently reported for certain other peroxisomal proteins. Previous results from our laboratory
suggested that P. pastoris AOX could not assemble outside the peroxisome. Methanol-induced pex mutants of this
yeast contain little or no activity for AOX, although substantial amounts of AOX protein are present in the cytoplasm (Liu et al., 1992, 1995
; Waterham et al., 1996
). Here
we show that the amount of residual AOX activity in the
pex mutants closely correlates with the severity of the peroxisomal biogenesis defect with chemically induced (and
slightly leaky) pex mutants typically containing some residual AOX activity, while most pex
strains contain no detectable AOX activity. In addition, we show that residual active AOX in the pex mutants is located inside the
few small peroxisomes or peroxisomal remnants present in
these cells. Although these results do not eliminate the
possibility that small amounts of AOX can octamerize into
an active enzyme in the cytoplasm of pex mutants, they
strongly suggest that the bulk of AOX protein must reach
the peroxisome to efficiently assemble.
The inability of AOX to properly assemble in the cytoplasm of pex mutants contrasts with other peroxisomal enzymes which appear to be fully active in the cytoplasm and,
therefore, have little difficulty assembling there. These enzymes include CAT from P. pastoris, an enzyme that must
properly fold, incorporate a heme cofactor, and tetramerize to become active (Gould et al., 1992; Liu et al., 1992
).
Thus, the inability of AOX to assemble is not the result of
some general inability of peroxisomal enzymes to incorporate cofactors or form homooligomers in the cytoplasm. The assembly problem does not appear to be because of
the inability of AOX to oligomerize outside the peroxisome, since Evers et al. (1995)
were able to efficiently reassemble active octameric AOX in vitro from FAD-containing monomeric subunits obtained from either P. pastoris
or the related yeast H. polymorpha. They further showed
that, in a riboflavin auxotrophic mutant of H. polymorpha, AOX protein is found in misfolded aggregates, indicating
that insertion of FAD into AOX is an essential step in the
assembly of H. polymorpha AOX (Evers et al., 1994
,
1996
). They postulated that the FAD insertion step may
be mediated by an unknown assembly factor since, once
removed, FAD could not be reinserted into AOX in vitro
(Evers et al., 1995
). Thus, in the absence of peroxisomes, the inability of P. pastoris AOX to assemble in the cytoplasm may also be related to a failure of FAD insertion
(e.g., FAD and/or FAD insertion factor concentration in
the cytoplasm may be too low to support efficient FAD
binding).
Interestingly, AOX (MOX) from H. polymorpha efficiently assembles into an active enzyme in the cytoplasm
of H. polymorpha pex mutants (Cregg et al., 1990; van der
Klei et al., 1991
). We found that MOX is active in the cytoplasm of P. pastoris pex mutants while AOX is inactive in
the cytoplasm of pex mutants of H. polymorpha. Thus, this
distinction between P. pastoris and H. polymorpha AOXs must be due to differences between their AOX polypeptides and not to the environment provided by their hosts.
This is a surprising result given the high degree of sequence similarity shared by these polypeptides (~85%
identical). Perhaps, for proper assembly, P. pastoris AOX
requires a higher concentration of FAD or FAD-insertion factor, or is more dependent on the acidic environment of
the peroxisome than H. polymorpha AOX.
Recent studies suggest that at least some peroxisomal
proteins are not only capable of cytoplasmic assembly but
are also imported into peroxisomes in a preassembled oligomeric state. The key experiment in these studies is the
coexpression of the peroxisomal protein without its PTS
along with the wild-type protein. In each case, although
the PTS-less protein expressed alone is not imported, coexpression resulted in the efficient coimport or piggybacking of the PTS-less polypeptides. Coimport has been reported for the human PTS1 enzyme, alanine/glyoxylate
amino transferase (Leiper et al., 1996), a chloramphenicol
acetyl transferase PTS1 fusion protein (McNew and Goodman, 1994
), a yeast PTS1 enzyme, MDH3 (Elgermsa et al.,
1996), and the yeast PTS2 enzyme thiolase (Glover et al., 1994
). The ability of each of these proteins to piggyback
into peroxisomes strongly suggests that these proteins can
oligomerize in the cytoplasm before import.
To perform the coexpression study with P. pastoris
AOX, it was first necessary to identify and characterize its
PTS. We found that critical information for efficient peroxisomal targeting of P. pastoris AOX is located within its
four COOH-terminal amino acids, LARF. Previously, a
similar conclusion was drawn for the same four COOH-terminal amino acids of H. polymorpha MOX (Hansen et al.,
1992). This conclusion was based on immunocytochemical data which indicated that these four amino acids were capable of targeting
-lactamase to peroxisomes in H. polymorpha. We show that these same four COOH-terminal
amino acids are also sufficient to target
-lactamase to P. pastoris peroxisomes. In addition, we show that these four
amino acids are critical for targeting since their removal
and substitution with either RSC or an HA-epitope tag results in proteins that are only inefficiently imported into peroxisomes. Interestingly, a small portion of both AOX-
RSC and AOX-HA is properly targeted to peroxisomes.
Cell fractionation studies confirmed that these activities
represent AOX protein that is correctly imported in peroxisomes, although the efficiency of import is not sufficient to support normal methanol-growth rates. One explanation for the residual import of LARF-less AOX is
that, in addition to a COOH-terminal PTS, AOX contains
a second independent but less efficient PTS. A second PTS
has been reported for other peroxisomal matrix proteins
including S. cerevisiae catalase A (Kragler et al., 1993
) and
H. polymorpha Per1p (Waterham et al., 1994
). This second PTS may be located within the 18 amino acids immediately adjacent to LARF in AOX, since a mutant AOX deleted for the 22 COOH-terminal amino acids is not imported at all. Furthermore, a
-lactamase fusion containing the 22 COOH-terminal amino acids of AOX is targeted more efficiently than one fused to just the LARF
sequence. A second explanation for the residual import of
AOX without LARF is that the protein oligomerizes in the cytoplasm with another methanol pathway protein,
such as dihydroxyacetone synthase, and is coimported.
The preimport coassembly of AOX and dihydroxyacetone
synthase was previously suggested from the results of ionophore experiments with the yeast C. boidinii (Bellion and Goodman, 1987
). We observed that, in glucose-grown cells
that do not synthesize dihydroxyacetone synthase, AOX-
RSC (expressed under control of the constitutive GAP
promoter) is not imported, although AOX-WT is imported (not shown), a result that is consistent with the notion that residual import of AOX without LARF may be dependent on another methanol pathway protein.
AOX is clearly targeted to peroxisomes via the PTS1
import pathway. Previous results demonstrated that AOX
behaves in a manner similar to luciferase, the prototypical
PTS1 protein, in P. pastoris wild-type and pex mutant cells
(McCollum et al., 1993; Spong et al., 1993; Liu et al., 1995
;
Waterham et al., 1996
). Here, we extend these results by
demonstrating that neither AOX nor
-lactamase fused to
LARF is targeted to peroxisomes in a pex5
strain that is
specifically defective in the import of PTS1 proteins
(Spong et al., 1993). In addition, two-hybrid system results
indicate that AOX strongly interacts with the PTS1 receptor Pex5p, and that this interaction is dependent upon the
last four amino acids of AOX. We conclude that the primary targeting signal for AOX is a PTS1 located within
LARF and that the PTS most probably consists of the
COOH-terminal tripeptide sequence ARF. The first two
amino acids of the motif in AOX, alanine and arginine, are
known functional variants of the prototypical PTS1 sequence (SKL; Gould et al., 1989
; Swinkels et al., 1992
).
The substitution of a phenylalanine for leucine at the ultimate position was shown to abolish peroxisomal targeting
in mammalian cells (Swinkels et al., 1992
). However, while
this work was in progress, Elgersma et al. (1996)
reported that PTS1 motifs ending in phenylalanine were imported
in S. cerevisiae.
To investigate the effect of coexpressing AOX-WT and
AOX without its primary PTS, it was not possible to examine samples by differential or sucrose density gradient centrifugations, as done in previous studies of this kind. These
techniques require prolonged incubation of cells to remove cell walls, conditions that result in degradation of
improperly folded AOX (Liu et al., 1992, 1995
; and Waterham et al., 1996
). Furthermore, improperly folded AOX forms into protein aggregates which sediment upon differential centrifugation and, thus, appear in organelle pellets
along with properly imported and assembled AOX. To observe import of AOX, cell extracts were prepared by the
glass bead disruption method at a low temperature and
were subjected to sucrose velocity sedimentation (Goodman et al., 1984
). The proportion of AOX protein present at the position of fully assembled AOX octamers was then
noted in the gradients. Previous studies (Liu et al., 1992
,
1995
) as well as those presented here indicate that active
octameric AOX is only found in the peroxisomal matrix.
Thus, the presence of AOX-HA at the position of octameric AOX in velocity gradients represents AOX-HA
that was properly imported into the organelle.
The coexpression studies showed that the efficiency of
AOX-HA import is not improved by AOX-WT. Thus, it
appears that AOX-HA cannot initiate cytoplasmic assembly with AOX-WT in the cytoplasm and be piggybacked
into the peroxisome. An alternative explanation for the failure of AOX-HA to efficiently oligomerize with AOX-WT in the coexpression experiment was that the HA tag somehow interfered with its assembly. Elgersma et al. (1996)
proposed that the apparent inability of S. cerevisiae PTS1
enzyme MDH to coimport PTS1-less MDH was because
of a reduction in the assembly kinetics of the mutant polypeptide. As a result, the mutant subunits were outcompeted by the wild-type subunits that rapidly homodimerize
in the cytoplasm. Our results indicate that AOX-HA is not
impaired in its ability to assemble with AOX-WT. When
we examined the small portion of AOX-HA in the coexpression strain that does reach the peroxisomal matrix and
assemble into active octameric enzyme, we found that virtually all of the mutant protein was present as heterooligomers with AOX-WT. Thus, it appears that despite the
presence of large amounts of AOX-WT, most AOX-HA
remains in the cytoplasm because AOX cannot initiate oligomerization in this compartment and, without its primary
PTS1, the mutant AOX is inefficiently targeted to and imported into the peroxisome. In summary, although peroxisomes are capable of importing some proteins as preassembled structures, AOX must be targeted to peroxisomes
as monomers for import and assembly to occur properly.
Despite recent progress, basic features of protein import
into peroxisomes remain largely unknown (Waterham and
Cregg, 1997). Although the organelle appears to be morphologically simple with a single membrane and uncomplicated matrix, the import mechanism is unexpectedly complex. Peroxisomes have evolved at least two matrix protein
import pathways and a third independent pathway specific
for peroxisomal integral membrane proteins. As shown by
several recent studies, the matrix protein import machinery is capable of importing large preassembled structures
9 nm in diameter (Walton et al., 1995
), a size much greater
than AOX octamers at ~600 kD. Therefore, the necessity
of importing AOX as unassembled monomeric polypeptides is probably not related to its size but may reflect the
necessity to incorporate FAD before octamerization, a
process proposed to occur in the peroxisomal matrix (Evers
et al., 1996
).
Although this is the first description of a yeast peroxisomal protein that cannot assemble in the absence of functional peroxisomes, this phenomenon may be relatively
common in patients afflicted with the lethal peroxisomal
biogenesis disorder Zellweger syndrome (Lazarow and
Moser, 1994). In cells of these patients, peroxisomes are
absent and peroxisomal matrix enzymes are left in the
cytoplasm. Some of these enzymes are stable and active,
whereas several others (including the plasmalogen biosynthetic enzymes, acyl-CoA:dihydroxyacetone phosphate acyltransferase and alkyl-dihydroxyacetone phosphate synthase, and the peroxisomal
-oxidation pathway enzymes,
acyl-CoA oxidase, bifunctional enzyme, and 3-oxoacyl-CoA thiolase) are deficient (Lazarow and Moser, 1994
).
Pulse chase studies on acyl-CoA oxidase and thiolase demonstrated that the proteins are synthesized normally but
remain largely inactive in the cytosol where they are rapidly degraded (Schram et al., 1986
; Suzuki et al., 1986
). The inability of these and other enzymes to assemble into
stable active enzymes may be the reason that specific peroxisomal metabolic pathways are defective in Zellweger
patients. Further investigations into the fate of AOX in P. pastoris mutants may shed light on the molecular details of
this phenomenon and the molecular etiology of this aspect
of Zellweger syndrome.
Received for publication 9 May 1997 and in revised form 9 October 1997.
This research was supported by a National Institutes of Health grant (DK-43698) and a National Science Foundation grant (MCB-9514289) to J.M. Cregg.We thank S. Subramani (University of California at San Diego) for the gift of vector pSP72 containing the P. pastoris PEX5 gene and the pex5 deletion strain. We thank T. Hadfield (Oregon Graduate Institute, Portland, OR) for assistance in preparing the manuscript.
AOX, alcohol oxidase; AOX-WT, wild-type AOX; ARF, alanine-arginine-phenylalanineCOOH; CAT, catalase; FAD, flavine adenine dinucleotide; LARF, leucine-alanine-arginine-phenylalanineCOOH; MDHC, malate dehydrogenase 3; MOX; AOX from Hansenula polymorpha; MSG; methione-serine-glysine; pex, peroxisome biogenesis defective; PTS, proximal targeting signal; SKL, arginine-serine-cysteineCOOH.
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