From the
PER genes are essential for the biogenesis of
peroxisomes in the yeast Pichia pastoris. Here we describe the
cloning of PER3 and functional characterization of its product
Per3p. The PER3 sequence predicts that Per3p is a 713-amino
acid (81-kDa) hydrophobic protein with at least three potential
membrane-spanning domains. We show that Per3p is a membrane protein of
the peroxisome. Methanol- or oleate-induced cells of per3-1, a
mutant strain generated by chemical mutagenesis, lack normal
peroxisomes but contain numerous abnormal vesicular structures. The
vesicles contain thiolase, a PTS2 protein, but only a small portion of
several other peroxisomal enzymes, including heterologously expressed
luciferase, a PTS1 protein. These results suggest that the vesicles in
per3-1 cells are peroxisomal remnants similar to those
observed in cells of patients with the peroxisomal disorder Zellweger
syndrome, and that the mutant is deficient in PTS1 but not PTS2 import.
In a strain in which most of PER3 was deleted, peroxisomes as
well as peroxisomal remnants appeared to be completely absent, and both
PTS1- and PTS2-containing enzymes were located in the cytosol. We
propose that Per3p is an essential component of the machinery required
for import of all peroxisomal matrix proteins and is composed of
independent domains involved in the import of specific PTS groups.
Peroxisomes (glyoxysomes, glycosomes) are ubiquitous, eukaryotic
organelles that are enclosed by a single membrane and are the site of
hydrogen peroxide-generating oxidases as well as catalase, which
decomposes this toxic by-product into water and oxygen. An unusual
feature of peroxisomes is that the specific pathways that involve the
organelles vary greatly, depending upon the organism
(1, 2) . Furthermore, their size, number, and enzymatic
content within a single organism or tissue can change dramatically in
response to environmental stimuli
(3, 4) . Peroxisomes
have no DNA; therefore, all peroxisomal proteins are likely to be
encoded by nuclear DNA
(5) . Proteins destined for the
peroxisome are synthesized on free polysomes and post-translationally
imported into the organelle in an ATP-dependent manner
(6, 7, 8) . Two peroxisomal targeting signals
(PTSs)
In humans, peroxisomes are
indispensable for survival as demonstrated by a family of lethal
genetic disorders collectively referred to as Zellweger syndrome in
which normal peroxisomes appear to be absent
(26) . In cell
lines derived from these patients, peroxisomal enzymes remain in the
cytosol and are either present and active, or absent due to rapid
degradation
(27, 28) . Cell fusion studies with
Zellweger cell lines indicate that the disease is the consequence of
defects in any one of at least nine different genes
(29, 30) . Recently human cDNA clones were identified
that restored normal organelles to two different Zellweger
complementation groups
(29, 31) . Although normal
peroxisomes are absent from or grossly deficient in Zellweger cells,
abnormal vesicles are observed
(32, 33) . These vesicles
contain peroxisomal membrane proteins, and thus are believed to be
peroxisomal remnants, termed ghosts. Furthermore, the ghost structures
in some Zellweger lines contain the PTS2 enzyme thiolase, suggesting
that these lines have a functional PTS2 import system and must be
defective in one or more other import systems
(34, 35) .
We have selected the methylotrophic yeasts Pichia pastoris and H. polymorpha as model systems to study the molecular
mechanisms involved in the biogenesis of peroxisomes. In P.
pastoris, peroxisome-deficient ( per) mutants have been
isolated that are affected in 10 different genes ( PER genes)
(36, 37) . Each per mutant is defective in the
ability to grow on either methanol or oleic acid, substrates that
require multiple peroxisomal enzymes to be metabolized, but grows at
the wild-type rate on other substrates, such as glucose, ethanol, and
glycerol. Like Zellweger cells, P. pastoris per mutants lack
normal peroxisomes and contain cytosolic peroxisomal matrix enzymes,
e.g. catalase. In addition, alcohol oxidase, an abundant
peroxisomal enzyme required for methanol metabolism, is present at
greatly reduced levels in methanol-induced per mutant cells, a
phenomenon that is also observed with certain peroxisomal enzymes in
Zellweger cell lines. Finally, similar to Zellweger cells, methanol- or
oleate-induced per cells contain abnormal single and multiple
membrane-bound vesicles. We have utilized the per mutants to
clone PER genes by functional complementation. We report that
PER3 encodes a peroxisomal membrane protein that appears to be
involved in peroxisomal protein import. Furthermore, we show that the
abnormal vesicles observed in a per3 mutant contain
peroxisomal matrix enzymes such as thiolase, suggesting that these
structures are peroxisomal in origin.
Polyclonal antibodies against the fusion protein were raised in
rabbits (Josman Laboratories, San Jose, CA). Anti-Per3p antiserum was
affinity-purified by the procedure described by Raymond et al.(41) . Crude rabbit serum was passed two times through a
column containing total soluble E. coli protein from induced
cells of strain TB1 (pMAL-c2). The flow-through from this column was
then passed two times through a column to which total soluble protein
from strain JC121 ( per3
As a first
step in establishing the subcellular location of Per3p, methanol- and
oleate-grown wild-type P. pastoris cells were subjected to
differential centrifugation as described above, and the resulting
pellet and supernatant fractions were examined for Per3p by
immunoblotting. Per3p was present primarily in the organelle pellet
along with a large portion of CAT (Fig. 2 B). A crude
organelle pellet from oleate-grown cells was then layered over a
discontinuous sucrose gradient and centrifuged to separate peroxisomes
from other organelles (mainly mitochondria). In these gradients,
peroxisomes were concentrated at a density of 1.21 g/cm
Phenotypically, the per3
Previously we described the isolation and partial
characterization of a collection of P. pastoris mutants
defective in peroxisome biogenesis ( per mutants)
(36, 37) . In this study, we report the detailed
analysis of mutants in PER3 as well as the isolation and
characterization of PER3 and its product Per3p. The primary
sequence of Per3p predicts a 713-amino acid protein that is hydrophobic
in overall character with three potential
Data base searches revealed only one other
protein with significant sequence similarity to Per3p. Per1p, a protein
required for peroxisome biogenesis in the methylotrophic yeast H.
polymorpha, is 42% identical and 60% similar to Per3p, and may be
its functional homologue (Fig. 8)
(20) . Despite their
similarity, differences between the proteins are evident. First, Per1p
is significantly smaller than Per3p (
Our data suggest that Per3p plays
an essential role in matrix protein import. This conclusion is based on
our observation that the peroxisomal remnant structures in induced
per3-1 cells efficiently import thiolase but not AOX, CAT,
dihydroxyacetone synthase, or luciferase. Thiolase is imported via a
PTS2 pathway in mammals and yeast
(15, 16) , whereas
luciferase is imported via a PTS1 pathway
(10) . Although PTSs
for AOX, CAT, and dihydroxyacetone synthase have not been defined in
P. pastoris, their counterparts in H. polymorpha have
been shown to be PTS1 enzymes
(68, 69) and, therefore,
are likely to be imported via a PTS1 pathway in P. pastoris as
well. Thus, per3-1 cells are primarily affected in PTS1 but
not PTS2 import. The per3-1 strain is not totally defective in
PTS1 import, since small amounts of CAT and AOX are found in the
peroxisomal remnant structures. Interestingly, upon differential
centrifugation of methanol-induced per3-1 preparations,
virtually all of the active AOX enzyme is found in the crude organelle
pellets, indicating that only AOX which gains entry into a peroxisome
assembles into active octameric enzyme, while the majority of AOX
remains in the cytosol as inactive aggregates. That only imported AOX
becomes active suggests that AOX assembly may require the presence of
one or more peroxisomal factors such as the AOX co-factor FAD or a
peroxisomal assembly factor
(70) .
An additional clue to
Per3p function came from studies comparing the per3-1 strain
to a strain deleted for most of PER3 ( per3
To
explain the per3 mutant phenotypes, we propose that Per3p is
an essential component of the cell's machinery for import of both
PTS1 and PTS2 (and perhaps all) matrix proteins. To account for the
continued ability of per3-1 cells to import thiolase, we
suggest that Per3p is composed of several functional domains and that
in the per3-1 allele, a mutant product is synthesized that is
defective in a domain primarily involved in the import of PTS1 proteins
but not PTS2 proteins. Consistent with partially functional Per3p, we
observe that the mutant protein is present in induced per3-1 cells and that it is the same size as in wild-type cells
(Fig. 2 B). To explain the phenotype of the per3
The identification of two PTSs for peroxisomal
protein import has raised the question of whether cells also have two
independent peroxisomal translocation pathways or only have separate
PTS receptors but otherwise share most components of a common
translocation machinery
(53, 71) . Despite the large
numbers of peroxisome-deficient yeast mutants that have been
characterized, only two appear to be selectively defective in a
specific import pathway. P. pastoris pas8 ( S. cerevisiae
pas10; H. polymorpha per3) mutants are specifically
defective in PTS1 protein import and the Pas8p has been shown to
specifically bind a peptide ending in the PTS1 sequence,
Ser-Lys-Leu-COOH
(53, 72) .
We are grateful to Ineke Keizer-Gunnink and Klaas
Sjollema for technical assistance with the electron microscopy
experiments. We thank Dr. W.-K. Kunau for antibodies against S.
cerevisiae and C. tropicalis thiolases, and Dr. S.
Subramani for the AOX1p-LUC plasmid pJAH23. We acknowledge Dr.
H. Waterham for helpful discussions and critical reading of this
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
have been characterized in detail
(9) . The first, PTS1, is a tripeptide motif, Ser-Lys-Leu (and
conservative variants), that is located at the carboxyl terminus of
many peroxisomal matrix proteins
(10, 11) . Proteins
that end in PTS1 are properly imported into peroxisomes of animals and
fungi, glyoxysomes of plants, and glycosomes of trypanosomes
(12, 13) . Thus, the PTS1 system has been conserved
throughout evolution. The second, PTS2, is characterized by the
consensus sequence (RL/IX
Q/HL) located near the amino
terminus of some peroxisomal proteins including: 3-ketoacyl-CoA
thiolases from rats, humans, and several yeast species; watermelon
malate dehydrogenase; and amine oxidase and the peroxisome biogenesis
protein Per1p from the yeast Hansenula polymorpha(14, 15, 16, 17, 18, 19, 20) .
Evidence suggests that additional PTSs exist internally within some
peroxisomal proteins including: Candida tropicalis acyl-CoA
oxidase
(21, 22) , Saccharomyces cerevisiae catalase
(23) , Candida boidinii 47-kDa
peroxisomal membrane protein PMP47
(24) , and H. polymorpha malate synthase
(25) .
Strains, Media, and Microbial Techniques
P.
pastoris strains used in this study are listed in .
P. pastoris cultures were grown at 30 °C in a complex
medium (YPD) composed of 1% yeast extract, 2% peptone, and 2% dextrose,
or in one of the following defined media: YNB medium (0.17% yeast
nitrogen base, 0.5% ammonium sulfate) supplemented with either 0.4%
dextrose, 0.5% methanol, or 0.2% oleate, 0.02% Tween 40, 0.05% yeast
extract. Nutritional supplements for growth of auxotrophic strains were
added to 50 µg/ml as required. Sporulation (mating) medium and
procedures for genetic manipulation of P. pastoris have been
described elsewhere
(36) . P. pastoris transformations
were performed as described in Cregg et al.(38) .
Vector pJAH23, a gift from Dr. S. Subramani (University of California
at San Diego) is composed of the firefly luciferase gene under the
transcriptional control of the P. pastoris AOX1 promoter and
terminator, the S. cerevisiae ARG4 gene, the P. pastoris autonomous replication sequence PARS2, and bacterial plasmid
pBR322.
P. pastoris DNA Library Construction
The P.
pastoris genomic DNA library used to clone PER3 was
constructed in P. pastoris-Escherichia coli shuttle vector
pYM8
(38) . This vector is composed of a P. pastoris autonomous replication sequence (PARS1), the S. cerevisiae histidinol dehydrogenase gene ( HIS4), which is a
selectable marker for transformation of his4 strains of P.
pastoris, and sequences from E. coli plasmid pBR322. To
construct the library, pYM8 was digested with BamHI and
treated with calf intestine alkaline phosphatase. Total DNA was
extracted from wild-type P. pastoris and partially digested
with Sau3AI to generate DNA fragments of 5-10 kilobase
pairs (kb). Fragments in this size range were further size-selected by
sucrose gradient centrifugation. Approximately equal molar amounts of
Sau3AI-digested yeast DNA and BamHI-calf intestine
alkaline phosphatase-treated pYM8 were ligated using T4 DNA ligase and
transformed into E. coli strain MC1061 by selection for
ampicillin resistance. Plasmid DNA was extracted from approximately
56,000 transformed colonies by the alkaline lysis procedure and further
purified by centrifugation in an ethidium bromide/cesium chloride
density gradient
(39) . The library was stored as plasmid DNA in
10 mM Tris-HCl, pH 7.4, 1 mM EDTA buffer at -20
°C.
Isolation and Characterization of PER3
P.
pastoris strain JC120 ( per3-1 his4) was transformed with
5 µg of the above P. pastoris library by the
spheroplast-generation CaCl-polyethelene glycol fusion
method, and transformants were selected for His
prototrophy on YNB dextrose medium
(38) . The
approximately 50,000 transformants that resulted were collected from
the plates as a single pool and inoculated into liquid YNB methanol
medium at an initial OD
of 0.1. After 3 days, the culture
grown on methanol was harvested. Total DNA was extracted from these
cells and transformed into E. coli strain MC1061. Plasmid DNA
was extracted from several resulting transformants and examined by
cleavage with selected restriction enzymes. All recovered vectors
appeared to be identical with inserts of approximately 8.1 kb. One of
the vectors, pYT4, retransformed into JC120 and observed to cotransform
cells to both Mut
and His
with
approximately equal efficiency, was selected for further study. To
define the location of PER3, selected subfragments from pYT4
were subcloned and tested for their ability to complement JC120 for
growth on methanol. In addition, selected segments of pYT4 were
removed, and the truncated vectors were religated and tested for
complementation. Results indicated that the complementing activity was
located within a 5-kb region between ScaI and the
BamHI/ Sau3AI junction. Northern blot studies using
subfragments within this region indicated that a 3.3-kb
EcoRV- ScaI fragment transcribed a methanol-induced
2.1-kb mRNA. Two subfragments from this region, a 2.6-kb
ScaI- HindIII fragment and a 0.7-kb
XbaI- EcoRV fragment, were inserted into appropriately
digested pBluescript II SK+ (Stratagene, La Jolla, CA), and a
series of nested deletions was generated from each of the resulting
plasmids by limited digestion with exonuclease III as described
previously
(39) . The DNA sequence of both DNA strands was then
determined by the dideoxy method
(40) using Sequenase 2.0 (U.
S. Biochemical Corp., Cleveland, OH). DNA and predicted protein
sequences were analyzed using MacVector software (IBI, New Haven, CT).
Sequencing results revealed a long open reading frame whose 5` terminus
extended beyond the EcoRV site. To complete the DNA sequence,
a series of extending oligonucleotide primers was synthesized and
utilized in sequencing reactions along with pYT4 DNA as template. The
predicted amino acid sequence of the open reading frame was compared
with those in the GenBank
data base.
Construction of the PER3 Disruption Strain
A
vector designed to delete most of PER3 was constructed in two
steps starting with plasmid pYM25. This plasmid is composed of a 3.1-kb
HindIII fragment encoding the S. cerevisiae ARG4 gene
inserted into the HindIII site of pBR322. In the first step, a
2.0-kb EcoRI fragment composed of sequences flanking the 5`
terminus of PER3 was inserted into the EcoRI site of
pYM25 to create plasmid pYH2. Second, a 1.5-kb PvuII fragment
composed of 3` PER3 sequences was inserted into the
NruI site of pYH2 to create the PER3-deletion vector
pYH3. The PER3 locus in the vector contains a deletion of 1638
bp of PER3 coding sequence (amino acids 1-546) with the
S. cerevisiae ARG4 gene fragment inserted at the deletion
site. pYH3 was linearized by partial digestion with PvuII and
transformed into GS190 ( arg4). Arg transformants were selected and screened for ones that were
Mut
. Several independent Arg
Mut
transformants were collected and examined
by the Southern blot method to confirm proper insertion of the linear
fragment from pYH3 into the PER3 locus.
Preparation of Anti-Per3p Antibodies
Per3p was
expressed in E. coli as a fusion with the maltose-binding
protein (MAL) using a kit supplied by New England Biolabs. To construct
the strain, the MAL expression vector, pMAL-c2, was first modified to
accept a PER3-containing DNA fragment in the proper reading
frame by digesting the vector with EcoRI and inserting an
adaptor oligonucleotide (AATTGAATGCATTTC). The adaptor insertion
resulted in the introduction of an NsiI site that was in frame
with an NsiI site in PER3. A 1.0-kb
NsiI- BglII fragment from pYH1 encoding amino acids
479-713 of PER3 was then inserted into NsiI-
and BamHI-digested pMAL- NsiI vector to create the
malE-PER3 expression vector, pEH1. MAL-Per3p fusion protein
synthesis in E. coli strain TB1 transformed with pEH1 and
purification were performed as recommended by the supplier.
::SARG4 arg4) had been bound. The
flow-through from this column was collected and used at a 1:1,000
dilution.
Miscellaneous Methods
The E. coli strain
used for recombinant DNA manipulations was MC1061
(39) , except
for expression of malE-PER3 where strain TB1 was employed (New
England Biolabs). E. coli cultivation and recombinant DNA
techniques were performed as described
(39) . Total protein was
determined by Bradford
(42) assay using bovine serum albumin as
standard. Catalase
(43) , acyl-CoA oxidase
(44) , alcohol
oxidase
(45) , luciferase
(46) , cytochrome c oxidase
(47) , and fumarase
(48) activities were
measured according to published procedures. SDS-polyacrylamide gel
electrophoresis and immunoblotting procedures were performed as
described previously
(49) . For immunoblotting experiments,
rabbit polyclonal antibodies against S. cerevisiae thiolase (a
gift of W.-H. Kunau, Ruhr-Universitat, Bochum, Germany), H.
polymorpha catalase, and P. pastoris alcohol oxidase (a
gift of M. Gleeson, SIBIA, San Diego) were used. Antigen antibody
complexes were visualized using the ECL kit following the instructions
of the supplier (Amersham Corp.). Electron microscopy and
immunocytochemistry with rabbit antibodies raised against C.
tropicalis thiolase (a gift of W.-H. Kunau, Ruhr-Universitat,
Bochum, Germany) and H. polymorpha alcohol oxidase were
performed as previously described
(50) . Differential and
discontinuous sucrose gradient centrifugations were performed as
described elsewhere
(5, 36) . Protein was extracted from
peroxisomal membranes using either triethanolamine or carbonate as
described previously
(51, 52) .
per3 Cells Are Defective in Peroxisome
Biogenesis
P. pastoris strain JC111 ( per3-1)
was isolated from a mutagenized culture by screening for strains that
were defective in the utilization of both methanol
(Mut) and oleic acid (Out
) as sole
carbon and energy sources
(36) . These substrates were selected
because each requires several peroxisomal enzymes and functional
peroxisomes to be metabolized
(4) . In methanol- or oleate-grown
wild-type cells, numerous large peroxisomes were readily observed
(Fig. 1, A and B). In contrast, normal
peroxisomes were absent from methanol- or oleate-induced per3-1 cells (Fig. 1 C). Instead, clusters of vesicular
structures were frequently observed.
Figure 1:
Electron micrographs showing
subcellular morphology and location of selected peroxisomal enzymes in
induced per3 cells. Peroxisomes in wild-type cells grown on
methanol ( A) and oleate ( B). In methanol-induced
per3-1 cells, peroxisomal remnants are evident ( C,
arrow); these cells also contain electron-dense aggregates
( D, arrows). In methanol-induced cells of the
per3 strain, peroxisomal remnants are absent
( E). Immunocytochemically, AOX protein is observed in
peroxisomal remnants and cytosolic aggregates in methanol-induced
per3-1 cells ( F, arrow). CAT is also found
in these vesicles ( G, arrow) and in the cytosol and
nucleus ( G). ( A-E, MnO
-fixed cells;
F-G, aldehyde/Unicryl
/uranyl acetate.
Abbreviations: L, lipid droplet; M, mitochondrion;
N, nucleus; P, peroxisomes; V, vacuole.
Bar, 0.5 µm.
Evidence for a defect in
peroxisome biogenesis in per3-1 was obtained from biochemical
and cytochemical experiments designed to determine the location of
peroxisomal enzymes. Cell-free extracts of induced per3-1 cells were examined for activities of selected peroxisomal
enzymes. In per3-1 cells induced with either methanol or
oleate, catalase (CAT) activity was present at approximately the same
level as wild-type cells (Fig. 2 A). In contrast,
activity for alcohol oxidase (AOX), the first enzyme in methanol
catabolism and a major constituent of the peroxisomal matrix of
methanol-grown cells, was consistently present at levels that were less
than 3% of those in wild-type P. pastoris. Initially, AOX
protein levels also appeared to be low, as judged by immunoblots using
anti-AOX antibodies. It was possible that AOX was synthesized in
per3-1 cells but, due to inefficient import, remained mostly
in the cytosol as insoluble aggregates which may have sedimented during
preparation of cell-free extracts. To examine this possibility, total
cell extracts were prepared without centrifugation and assayed for AOX
activity and protein. Results revealed that methanol-induced per3-1 cells actually contained approximately the same amount of AOX
protein as wild-type cells (Fig. 2 A). However, AOX
activity levels remained low, indicating that most of the AOX protein
was inactive. Further evidence of AOX aggregate formation in
methanol-induced per3-1 was provided by electron microscopy
studies, which showed that cytosolic aggregates were common in these
cells (Fig. 1 D) and immunocytochemical experiments which
showed that AOX was concentrated in these aggregates
(Fig. 1 F).
Figure 2:
Activity and location of selected
peroxisomal enzymes in per3 strains. A, AOX and CAT
activity (and AOX protein) levels in methanol-induced strains, and
luciferase ( LUC) and CAT activity (and thiolase protein)
levels in oleate-induced strains reported as a percentage of that in
wild-type cells. Wt, wild-type P. pastoris;
per3, per3-1; per3 ( PER3),
per3-1 strain JC120 ( per3-1 his4) transformed with
PER3-containing plasmid pYT4. B, Specific activity
(or relative concentration of protein) in post-20,000 g organelle supernatant ( S) and pellet ( P) after
differential centrifugation of homogenized protoplasts. ND,
could not be determined due to absence of AOX activity. In immunoblots,
each lane contains 10 µg of protein. Activity units are as follows:
CAT,
E
/min/mg of protein; AOX, micromoles of
product/min/mg; cytochrome c oxidase, micromoles of
product/min/mg
10
; LUC, arbitrary light
units/mg of protein.
To determine the location of selected
peroxisomal enzymes in per3-1 cells, differential
centrifugation experiments were performed on homogenized spheroplasts
prepared from induced cells. With either methanol- or oleate-grown
wild-type cells, a large portion of CAT activity was present in the
20,000 g pellets (Fig. 2 B). In
contrast, in per3-1 cells induced with either substrate, CAT
was located primarily in the supernatant, indicating that the enzyme
was cytosolic. As a control, cytochrome c oxidase, a
mitochondrial marker enzyme, remained mostly in the pellet in
per3-1 preparations. Interestingly, the small amount of AOX
activity remaining in methanol-induced per3-1 cells was
present in the pellet fractions (Fig. 2 B). This
suggested that our per3-1 allele may not be totally defective
in AOX import, and as a consequence, peroxisomes may import a small
fraction of the AOX protein which then assembles into active enzyme.
Evidence that a portion of AOX actually was imported was obtained by
immunocytochemistry which showed that, in addition to the aggregates
described above, AOX was also present within the small vesicular
structures in per3-1 cells (Fig. 1 F). Identical
results were also obtained for CAT (Fig. 1 G) and
dihydroxyacetone synthase, the third peroxisomal enzyme in the methanol
catabolic pathway (data not shown).
per3-1 Cells Are Defective in Import of
Luciferase
Although it is assumed that CAT, dihydroxyacetone
synthase, and AOX are all PTS1 enzymes, this has not been directly
demonstrated in P. pastoris. As direct evidence that
per3-1 was defective in import of PTS1 proteins, we examined
the behavior of heterologously expressed luciferase, the prototypical
PTS1 enzyme
(10) . Wild-type and per3-1 strains were
constructed that contained luciferase under control of the AOX1 promoter of P. pastoris. Both strains synthesized high
levels of luciferase activity in methanol medium, moderate levels in
oleate medium, and no luciferase in glucose medium
(Fig. 2 A)
(53) . After differential
centrifugation of preparations from oleate-induced cells of these
strains, luciferase activity was highly enriched in the pellet fraction
from wild-type cells while activity in per3-1 cells was
present mostly in the supernatant fraction (Fig. 2 B).
From this, we concluded that the per3-1 strain was defective
in the import of luciferase and probably other PTS1-containing proteins
as well.
per3-1 Cells Import Thiolase into Peroxisome
Remnants
The fate of thiolase, a peroxisomal -oxidation
enzyme imported via the PTS2 pathway, was also examined in per3-1 cells
(15, 16, 53) . Crude organelle pellet
and supernatant fractions from oleate-induced cells were immunoblotted
and reacted with antibodies against S. cerevisiae thiolase
(Fig. 2 B). In contrast to CAT, AOX, dihydroxyacetone
synthase, and luciferase, thiolase was concentrated in the pellet,
suggesting that this PTS2 enzyme had either aggregated or was located
in a subcellular compartment. To distinguish between these
possibilities, the location of thiolase was examined
immunocytochemically. In sections from oleate-grown wild-type cells,
peroxisomes were specifically labeled by antibodies directed against
C. tropicalis thiolase (Fig. 3 A). In
oleate-induced per3-1 cells, the gold particles were primarily
observed on the vesicular structures (Fig. 3 B),
indicating that thiolase was located in these structures. Taken
together, our results suggested that per3-1 was specifically
impaired in the ability to import CAT, AOX, dihydroxyacetone synthase,
and luciferase (PTS1 enzymes) but not thiolase (a PTS2 enzyme), which
appeared to be efficiently imported. Furthermore, the presence of
thiolase and other peroxisomal enzymes in the vesicular structures
indicates that these structures represent peroxisomal remnants.
Figure 3:
Immunocytochemical labeling of
oleate-induced cells with anti-thiolase antibodies. Thiolase labeling
is concentrated in peroxisomes in wild-type cells ( A) and in
vesicles in per3-1 cells ( B, arrow), but is
located in cytosol and nucleus in per3 cells
( C). Methanol-grown cells of the per3-1 strain
complemented with PER3-containing plasmid pYT4 contain normal
peroxisomes with AOX protein in the organelles ( D).
Bar, 0.5 µm.
Isolation and Characterization of PER3
The
PER3 gene was isolated from a P. pastoris genomic DNA
library by functional complementation of per3-1 strain JC120
( per3-1 his4). Transformants were initially selected for
histidine prototrophy (His) on glucose medium and
subsequently selected for growth on methanol (Mut
).
Total DNA was extracted from a pool of His
Mut
transformants, and plasmids were recovered
by transforming the DNA into E. coli. A plasmid, designated
pYT4 (Fig. 4 A), was isolated that co-transformed the
per3-1 his4 strain to both His
and
Mut
simultaneously. per3-1 cells transformed
with pYT4 contained normal-appearing peroxisomes
(Fig. 3 D). In addition, AOX activity in methanol-grown
transformants was restored to normal levels and both AOX and CAT were
again present in peroxisomes (Figs. 2 and 3 D). Restriction
mapping of pYT4 revealed a P. pastoris DNA insert of
approximately 8.1 kb. By subcloning selected subfragments from pYT4,
the complementing activity was located within a region of 5.0 kb.
Northern blots using selected fragments from this region revealed a
transcript of approximately 2.1 kb that was present at low levels in
glucose-grown wild-type P. pastoris. Relative to total RNA
levels, this transcript appeared to be induced approximately 5-fold in
methanol- or oleate-grown cells (Fig. 5). Since peroxisomes are
also induced by these substrates, the higher level of this transcript
was consistent with it being the product of a gene coding for a
peroxisomal protein. The DNA sequence of the region revealed an open
reading frame (ORF) of 2,139 bp with the potential of encoding a
polypeptide of 713 amino acids (
81 kDa) (Fig. 6 A).
Further studies indicated that this ORF was PER3. As described
below, a P. pastoris strain in which most of the ORF was
deleted was peroxisome-deficient and was determined genetically to be
an allele of per3-1. In addition, the product of the ORF was
found to be a peroxisomal protein.
Figure 4:
Diagrams of plasmid pYT4 and the
PER3-deletion allele per3. A, restriction map of
the PER3 locus cloned in pYT4. The asterisk marks a
HindIII site in the PER3 gene that was used for
subcloning but is not unique to the vector. B, the structure
of the per3
allele. To construct this allele, PER3 DNA sequences encoding Per3p amino acids 1 through 546 were
replaced with a DNA fragment containing the S. cerevisiae ARG4 gene and inserted into the P. pastoris genome by
homologous recombination. C, correct targeting of the
per3
fragment was demonstrated by Southern blotting of
genomic DNAs cut with ClaI and hybridizing with a labeled
fragment ( P) indicated in B. Lane 1, 2 µg of
wild-type genomic DNA; lane 2, 2 µg of DNA from the
per3
strain JC121.
Figure 5:
Northern blot of PER3 message.
All lanes contain 10 µg of total RNA. Odd-numbered lanes contain RNAs from glucose ( G)-grown cells.
Even-numbered lanes contain RNAs from methanol
( M)-induced cells. Lanes 1 and 2, wild-type
P. pastoris; lanes 3 and 4,
per3; lanes 5 and 6, per3-1.
As a control, the filter was also hybridized with a labeled DNA
fragment encoding the S. cerevisiae actin gene ( ACT).
ACT message levels are significantly lower in methanol-grown
wild-type P. pastoris cells than in glucose-grown cells.
Levels are even lower in methanol-induced cells of the per3 mutants.
Figure 6:
PER3 sequence. A, nucleotide and
predicted amino acid sequences of PER3. Potential
-helical membrane-spanning domains are underlined. B,
hydrophilicity plot of Per3p-predicted primary structure shows that the
peptide is hydrophobic in overall character. These sequence data are
available from EMBL/GenBank
/DDJB under accession no.
L40485.
Hydropathy analysis indicated
that the predicted PER3 product (Per3p) was hydrophobic in overall
character with at least three potential -helical transmembrane
domains (Fig. 6, residues 338-358, 373-392, and
637-660)
(54) . Two additional regions with lower but
significant transmembrane potential were also identified (Fig. 6,
residues 484-510 and 522-540). The primary sequence of
Per3p showed significant similarity to that of Per1p, a protein
required for peroxisome biogenesis in H. polymorpha(20) (see ``Discussion''). Data base searches revealed
no other proteins with overall sequence similarity to Per3p. The only
identifiable sequence feature of Per3p was the three carboxyl-terminal
amino acids (Ala-Lys-Leu-COOH), a known PTS1 sequence variant.
Per3p Is a Peroxisomal Membrane Protein
Per3p was
characterized through rabbit polyclonal antibodies raised against Per3p
expressed in E. coli as a fusion with the maltose-binding
protein. In immunoblots prepared from methanol- or oleate-grown
wild-type extracts, affinity-purified anti-Per3p antibody preparations
recognized an approximately 76-kDa polypeptide that was not present in
extracts from methanol- or oleate-induced cells of a strain in which
most of PER3 had been deleted ( per3)
(Fig. 2 B). The apparent molecular mass of Per3p was
somewhat less than the 81-kDa mass calculated from the predicted
primary sequence. This discrepancy could be due to the high content of
nonpolar amino acids in Per3p (42%) which can cause proteins to migrate
faster in SDS-polyacrylamide gel electrophoresis
(55) . Per3p
was present at a significantly higher level in methanol- and
oleate-grown cells than in glucose-grown cells (not shown), a result
that was consistent with our observations of PER3 mRNA and the
induction of peroxisomes under these growth conditions.
as
judged by the location of CAT and acyl-CoA oxidase activities, and
thiolase protein (Fig. 7 A, fraction 6).
Mitochondria were located at a density of 1.16 g/cm
as
judged by fumarase activity (Fig. 7 A, fraction
12). Per3p was found exclusively in the peroxisomal fraction,
indicating that the protein is peroxisomal. Samples of purified
peroxisomes from these fractions were extracted with either
triethanolamine or sodium carbonate. Following triethanolamine
treatment and centrifugation at 30,000
g, CAT and
thiolase were located mostly in the supernatant, whereas Per3p was in
the pellet, suggesting that Per3p was associated with the peroxisomal
membrane (Fig. 7 B, lanes 2 and 3).
After carbonate extraction and centrifugation at 200,000
g, most Per3p remained in the membrane pellet, suggesting that
Per3p may be an integral peroxisomal membrane protein
(Fig. 7 B, lanes 4 and 5). However, we
repeated the carbonate treatment using crude organelle pellet fractions
from methanol- and oleate-induced cells and found that, in contrast to
the results with purified peroxisomes, only about 25% of Per3p was
resistant to extraction (data not shown). Similar results have been
obtained with the S. cerevisiae 24-kDa peroxisomal membrane
protein (Pmp24p) where the inextractibility of the protein from
purified peroxisomes was believed to be an artifact of the extensive
manipulations involved in the isolation of peroxisomes.
(
)
We conclude that Per3p is tightly associated with the
peroxisomal membrane but is probably not an integral membrane protein.
Figure 7:
Per3p
is a peroxisomal membrane protein. A, sucrose density gradient
profile of crude organelle pellet fraction derived from oleate-induced
wild-type P. pastoris cells. Activity in each fraction is
presented as a percentage of total activity in gradient. Fraction 1 ( left) is gradient bottom and fraction 25 ( right) is gradient top. ACO, acyl-CoA oxidase
activity. Immunoblotting was performed with equal volumes (200 µl)
of selected gradient fractions. Total protein was visualized by
staining with Coomassie Blue dye. B, in lanes 1 through 5, 25 µg of purified peroxisomes isolated
from oleate-grown wild-type cells ( lane 1) were extracted with
20 mM triethanolamine, pH 7.8 ( lanes 2 and
3) or 0.1 M sodium carbonate, pH 11 ( lanes 4 and 5). After extraction, samples were centrifuged, and
supernatant ( even-numbered lanes) and pellet ( odd-numbered
lanes) fractions were immunoblotted with anti-Per3p
antibodies.
A PER3 Deletion Strain Lacks Peroxisomes and Contains
Cytosolic Thiolase
A P. pastoris strain in which most
of PER3 was deleted was created by the gene replacement method
(56) . For the replacement, a plasmid was constructed in which
1638 bp of PER3 coding sequence (nucleotides 1 through 1638
encoding amino acids 1 through 546 in Fig. 6 A) were
removed and replaced with a fragment containing the S. cerevisiae
ARG4 gene. This plasmid was then digested with a restriction
enzyme to release the PER3 deletion allele
( per3) on the linear DNA fragment, shown in
Fig. 4B, and introduced into P. pastoris strain
GS190 ( arg4). Transformants in which the fragment had deleted
the PER3 locus were isolated by selecting for Arg
colonies and then screening for ones which were also
Mut
. Proper targeting of the fragment was confirmed
by Southern blot analysis (Fig. 4 C). In addition,
Northern blots showed that methanol-induced cells of the per3
strains no longer produced the 2.1-kb PER3 message
(Fig. 5 B, lanes 3 and 4). One
per3
-derived strain, JC121 ( per3
::SARG4
arg4), was crossed with JC120 ( per3-1 his4) by selection
for growth on minimal glucose plates. The resulting diploids were
tested for methanol growth and were Mut
. Several
thousand spore products from these diploids were then examined by
replica plating for Mut phenotype and all were Mut
as
well, demonstrating that the per3-1 and per3
alleles were tightly linked and likely to be mutant alleles of the
same gene.
strain was clearly
a per mutant. In addition to being Mut
and
Out
, electron microscopy examination of
methanol-induced per3
cells showed that they lacked
intact peroxisomes, and biochemical studies showed that CAT was induced
to normal levels in both methanol- and oleate-induced cells but was
located in the cytosol (Fig. 2). However, the per3-1 and
per3
strains were different in several respects. First,
methanol-induced cells of per3-1 frequently contained clusters
of peroxisomal remnants, which were absent in methanol- or
oleate-induced per3
cells (Fig. 1 E). Thus,
the phenotype of a per3 null mutant strain appears to be the
complete absence of peroxisomes. Second, methanol-induced per3
cells contained no AOX activity, whereas per3-1 cells
contained low but significant amounts of sedimentable AOX activity
(Fig. 2, A and B). These results support our
suggestion that the per3-1 allele in the mutant strain is
slightly leaky with respect to AOX import and that active AOX enzyme is
solely present in peroxisomal remnants. Third, thiolase was found
biochemically (Fig. 2 B) and immunocytochemically
(Fig. 3 C) to be located in the cytosol. These results
were in contrast to induced per3-1 cells where thiolase was
located primarily within the peroxisomal remnants (Figs. 2 B and 3 B). Thus, although both per3-1 and
per3
were deficient in ability to import CAT, AOX, and
luciferase, per3-1 cells selectively imported thiolase while
per3
cells did not.
-helical transmembrane
domains. Consistent with these predictions, biochemical studies showed
that Per3p has an apparent molecular mass of approximately 76 kDa, is
tightly associated with the peroxisomal membrane, and may even be an
integral membrane protein of the organelle. Per3p terminates in the
tripeptide sequence Ala-Lys-Leu-COOH (AKL), a variant within the PTS1
group that is sufficient to target reporter proteins to peroxisomes
(11, 20) . The presence of a PTS1 on Per3p is surprising
since this targeting signal is thought to be responsible for import of
matrix proteins and not membrane proteins
(14) . Other
peroxisomal proteins that specifically end in AKL include: mammalian
sterol carrier 2
(57) , glycosomal glyceraldehyde phosphate
dehydrogenase of Trypanosoma brucei(58) ,
acetoacetyl-CoA thiolase A of C. tropicalis(59) ,
PMP20 of C. boidinii(60) , and Per1p of H.
polymorpha(20) . However, only a few peroxisomal membrane
proteins have been described and it is not yet clear whether PTS1
participates in the targeting of some membrane proteins. It will be
interesting to determine whether the motif on Per3p is a functional
targeting signal and whether it is responsible for targeting Per3p to
the peroxisomal membrane.
71 versus 81 kDa)
and contains a functional PTS2 sequence not present in Per3p. Second,
P. pastoris and H. polymorpha are closely related
species
(61) , and other proteins show a much greater degree of
similarity between these two yeast species. For example, the primary
sequences for AOX and dihydroxyacetone synthase are each greater than
80% identical
(62, 63, 64) .
(
)
Thus, it might be expected that the H. polymorpha homologue of Per3p would show greater similarity than Per1p.
Second, Per3p is a membrane protein while Per1p is located in the
peroxisomal matrix
(20) . Third, the H. polymorpha PER1 gene does not complement per3 mutants of P.
pastoris, and PER3 does not complement H. polymorpha
per1.
(
)
In contrast, the H. polymorpha genes for AOX and formaldehyde dehydrogenase (another
methanol pathway-specific enzyme) readily complemented P. pastoris mutants in these genes, and Northern blots indicated that
transcription of the H. polymorpha genes initiates correctly
and is properly regulated.
Thus, it is possible that Per3p
and Per1p are not homologues but are different members of a family of
related proteins required for peroxisome biogenesis.
Figure 8:
Comparison of the predicted amino acid
sequences of P. pastoris Per3p and H. polymorpha Per1p. Sequences were aligned using PC gene software. The
character ``'' between sequences indicates residues
that are identical. The character ``.'' indicates similar
residues. Similar residues are defined as: A, S,
T; D, E; N, Q; R,
K; I, L, M, V; F,
Y, W.
Our studies of
per3-1, a nitrosoguanidine-generated mutant, show that the
numerous vesicular structures frequently observed in induced cells of
this strain are peroxisomal in origin. In oleate-induced per3-1 cells, these vesicular structures contain thiolase but only minor
amounts of PTS1 proteins, while the bulk of these enzymes is present in
the cytosol. This is the first direct visual evidence that these
structures, which are observed in most P. pastoris per mutants, are remnants of peroxisomes in P. pastoris. In
Zellweger cell lines, similar peroxisomal remnants, called peroxisomal
ghosts, are also observed
(32) . The additional layer(s) of
membrane that often surround peroxisomal remnants may be the result of
their inclusion within autophagic vacuoles
(65) . In
methylotrophic yeasts, cells shifted from methanol to other carbon
sources are known to rapidly sequester peroxisomes within autophagic
structures where they are degraded by vacuolar enzymes
(66) .
Furthermore, in Zellweger cells, a significant portion of the
peroxisomal ghosts are enclosed within compartments that contain
lysosomal proteins, suggesting that they are subject to rapid turnover
by autophagy as well
(67) .
).
Although neither per3 mutant can grow on methanol or oleate
and both are peroxisome deficient, they display at least two
differences. First, in induced per3
cells, we cannot find
the peroxisomal remnant structures that are so prevalent in per3-1. Second, thiolase is in the cytosol in oleate-induced per3
cells, whereas it is in the peroxisomal remnants in per3-1 cells. Thus, the per3 null phenotype appears to be an
inability to import either PTS1- or PTS2-containing proteins.
strain, we suggest that other domains in Per3p are essential for
PTS2 protein import (or both PTS1 and PTS2 import); therefore, deletion
of PER3 results in a strain that is defective in all matrix
protein import.
(
)
Thus, Pas8p appears to be the PTS1 protein receptor.
Conversely, S. cerevisiae pas7 mutants appear to be
selectively defective in PTS2 import
(73) . (Similar S.
cerevisiae mutants have been reported as peb5 and
peb1 and may be alleles of pas10 and pas7,
respectively
(71) .) The small number of pathway-specific
mutants is most easily explained as a consequence of a shared import
pathway. Our model of Per3p function in which the protein is composed
of domains preferentially involved in import of different PTS protein
classes also appears to be most consistent with a shared import pathway
model.
,
methanol-utilizing; Mut
,
methanol-utilization-defective; ORF, open reading frame;
Out
, oleate-utilization defective; bp, base pair; kb,
kilobase pair.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.