(Received for publication, November 22, 1995; and in revised form, January 18, 1996)
From the
We have cloned and characterized the Hansenula polymorpha
PER9 gene by functional complementation of the per9-1 mutant of H. polymorpha, which is defective in peroxisome
biogenesis. The predicted product, Per9p, is a polypeptide of 52 kDa
with sequence similarity to Pas3p, a protein involved in peroxisome
biogenesis in Saccharomyces cerevisiae. In a per9 disruption strain (per9), peroxisomal matrix and
membrane proteins are present at wild-type levels. The matrix proteins
accumulated in the cytoplasm. However, the location of the membrane
proteins remained obscure; fully induced
per9 cells
lacked residual peroxisomal vesicles (``ghosts''). Analysis
of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced
during growth of cells on peroxisome-inducing substrates. The highest
expression levels were observed in cells grown on methanol.
Localization studies revealed that Per9p is an integral membrane
protein of the peroxisome. Targeting studies suggested that Per9p may
be sorted to the peroxisome via the endoplasmic reticulum.
Overexpression of PER9 induced a significant increase in the
number of peroxisomes per cell, a result that suggests that Per9p may
be involved in peroxisome proliferation and/or membrane biosynthesis.
When PER9 expression was placed under the control of a
strongly regulatable promoter and switched off, peroxisomes were
observed to disintegrate over time in a manner that suggested that
Per9p may be required for maintenance of the peroxisomal membrane.
Peroxisomes are cell organelles that are present in virtually all eukaryotic cells. They perform specific metabolic functions that are often related to the developmental stage and/or the organism in which they occur(1) . The metabolic importance of peroxisomes in humans is demonstrated by the fact that the absence of the organelles leads to severe abnormalities, followed by an early death (e.g. Zellweger syndrome(2) ). Consequently, many studies are now devoted to unravel the molecular mechanisms of peroxisome biogenesis and function. Yeasts are excellent model systems for such studies having the advantages that (i) the induction and protein composition of peroxisomes can readily be manipulated by varying growth conditions and (ii) in the absence of peroxisomes, yeasts are viable(3, 4) . Hence, peroxisome-deficient mutants have been isolated from different yeast species(4) , and the corresponding genes are being cloned and characterized.
In
yeast, peroxisomes normally develop by growth and fission from
pre-existing ones. Peroxisomal matrix proteins are nuclear-encoded,
synthesized in the cytoplasm, and directed to the organelle by
topogenic signals (PTSs). ()Two PTSs have been identified
and are located either at the extreme C terminus (PTS1) or the N
terminus of the protein (PTS2)(4) . Our knowledge on the
sorting of peroxisomal membrane proteins is still limited, and
consensus topogenic sequences have yet to be identified(5) .
In our laboratory, we use the methylotrophic yeast Hansenula polymorpha as the model organism for studies on peroxisome biogenesis and function. We have isolated a collection of peroxisome-deficient (per) mutants of this organism that comprises 28 different complementation groups and that includes constitutive and conditional (temperature-sensitive) mutants. In previous reports, we described the cloning and characterization of three H. polymorpha PER genes, namely PER1, PER3, and PER8(6, 7, 8) , the protein products of which are part of the protein import (Per1p and Per3p) (6, 7) or peroxisome proliferation machinery (Per8p)(8) . Here, we describe the molecular cloning and characterization of the PER9 gene and its protein product (Per9p). Per9p shares sequence similarity with Saccharomyces cerevisiae Pas3p (9) and plays a key role in peroxisome biogenesis and maintenance.
To clone the PER9 gene, mutant per9-1 was transformed with an H.
polymorpha genomic library constructed in vector pYT3(8) .
Leucine prototrophic transformants were screened on YNM plates for the
ability to grow on methanol. The complementing plasmids of positive
clones were rescued and transformed to E. coli DH5(15) . To facilitate sequencing, restriction
analysis, and construction of subclones, a 2.7-kb complementing DNA
fragment was subcloned as a SalI fragment into SalI-digested phagemid pBluescript II KS
(Stratagene, La Jolla, CA) in both orientations. Sequencing of
both strands was carried out on an Applied Biosystems 373A automatic
sequencer using the Taq dye deoxy terminator cycle sequencing
kit supplied by the manufacturer. For DNA and amino acid sequence
analysis, the PC-GENE
program (Release 6.70,
IntelliGenetics, Mountain View, CA) was used. The TBLASTN algorithm (17) was used to search the GenBank
Data Bank
(Release 91.0, October 15, 1995) for DNA and protein sequences showing
similarity to the PER9 gene and its protein product.
Figure 2:
Schematic representations of the 2.7-kb per9-1 complementing fragment containing open reading frame 2 (ORF2) and the PER9 gene (A) and the
disruption of PER9 using C. albicans LEU2 or H.
polymorpha URA3 (B). The SalI site to the left
originates from vector pYT3. The nucleotide sequence has been deposited
in the GenBank Data Bank under accession number
U37763.
Figure 1:
Ultrathin sections of
methanol-incubated cells of the original per9-1 mutant and the
wild-type strain of H. polymorpha. The per9-1 mutant (A) lacks peroxisomes, which are evident in the wild-type cell (B) (KMnO). Electron micrographs are taken from
aldehyde-fixed cells, unless otherwise indicated. Bar =
0.5 µm.
From 10 colonies, three Mut
transformants were selected,
each of which carried the library vector pYT3 with a 7.5-kb insert. By
subcloning, the complementing activity was found to reside within a
2.7-kb SalI fragment. Sequence analysis of the 2.7-kb
complementing fragment revealed two open reading frames of 1374 and 621
base pairs (Fig. 2A). Further subcloning analysis
demonstrated that the 1374-base pair open reading frame represented the
complementing gene. This 1374-base pair open reading frame, hereafter
referred to as the PER9 gene, encoded a putative protein
(Per9p) of 457 amino acids with a calculated mass of 52 kDa. Hydropathy
analysis (32) predicted one membrane-spanning region (amino
acids 16-36) and one membrane-associated region (amino acids
159-179). A data base search revealed strong similarity to the
integral peroxisomal membrane protein Pas3p of S. cerevisiae (Fig. 3)(9) . H. polymorpha Per9p and S. cerevisiae Pas3p display a similar hydropathy pattern, and
both have one predicted membrane-spanning region and one
membrane-associated region.
Figure 3: Amino acid sequence alignment of H. polymorpha Per9p (HpPer9p) and S. cerevisiae Pas3p (ScPas3p). The putative membrane-spanning region and membrane-associated region in both proteins are underlined. Identical residues are indicated by asterisks, and similar ones by dots. Gaps were introduced to maximize the similarity.
The per9 strain grew at a wild-type
rate on rich medium (e.g. glucose/ammonium sulfate) and also
on selected carbon (e.g. ethanol) and nitrogen (e.g.D-alanine and methyl- and ethylamine) sources known to
require the activity of peroxisomal enzymes(33) . However,
growth on methanol as sole source of carbon and energy was totally
defective (data not shown). Electron microscopic analysis of thin
sections of
per9 cells demonstrated that under all growth
conditions employed, intact peroxisomes were invariably absent (data
not shown). The subcellular morphology of
per9 was
studied in cells grown in a continuous culture on a glucose/methanol
mixture. Enzyme activity measurements (data not shown) and Western blot
analysis (Fig. 4) revealed that each peroxisomal protein
examined (alcohol oxidase (AO), catalase (CAT), dihydroxyacetone
synthase, malate synthase, Per8p(8) , and Per10p) (
)was present at normal wild-type levels. Ultrastructural
analysis showed that peroxisomes were absent. As we have observed in
other H. polymorpha per mutants(34) , the
per9 cells contained a large cytoplasmic crystalloid (Fig. 5A). Immunocytochemistry indicated that the other
matrix proteins were also not in peroxisomes, but were located in the
cytoplasm (shown for AO and CAT in Fig. 5, B and C, respectively; the wild-type CAT control is shown in Fig. 5F). Membranous remnants of peroxisomes (vesicles,
ghosts) were not observed. In addition, overproduction of the H.
polymorpha peroxisomal membrane protein Per8p (8) also
failed to resolve such structures. Instead, Per8p was found in small
aggregates that were often associated with a mitochondrial profile (Fig. 5E). Since peroxisomal vesicles are readily
detected in other per disruption strains overexpressing PER8 (e.g.
per1)(6, 35) , we
concluded that membranous remnants were either absent in
per9 cells or present at a level below the limit of detection of the
ultrastructural methods.
Figure 4:
Protein levels of peroxisomal matrix and
membrane proteins in H. polymorpha per9 and wild-type
cells. Western blots are prepared of crude extracts from methanol-grown
wild-type (WT) or
per9 cells and decorated with
antibodies against H. polymorpha AO, CAT, dihydroxyacetone
synthase (DHAS), malate synthase (MAS), Per8p, and
Per10p. 30 (AO, CAT, dihydroxyacetone synthase, and malate synthase),
40 (Per10p), and 60 (Per8p) µg of protein were loaded on each
lane.
Figure 5:
A,
shown is the overall morphology of the per9::LEU2 disruption
mutant. Cells were incubated for 36 h in batch cultures containing 0.5%
methanol. Peroxisomes were absent; instead, a large cytosolic
crystalloid was present (*). B, these crystalloids were
labeled with anti-AO antibodies, including the crystalloids in the
nucleus. C, CAT was predominantly localized in the cytoplasm
(anti-CAT antibodies). D, shown is the immunocytochemical
localization of Per9p on the peroxisomal membrane (anti-Per9p). E, shown is a characteristic picture of the labeling obtained
after incubation of ultrathin sections of per9 cells,
overexpressing the H. polymorpha PER8 gene, with specific
antibodies against Per8p. Peroxisomal ghosts were never seen in such
cells; instead, low but specific Per8p labeling was observed, generally
associated with a mitochondrial profile. F, shown is the
typical labeling pattern of catalase protein in peroxisomes of H.
polymorpha wild-type cells. m, mitochondrion; n,
nucleus. Bar = 0.5 µm.
Figure 6:
A, shown is the specificity of the
anti-Per9p antiserum. Western blots were prepared from crude extracts
of H. polymorpha wild-type cells (WT; lanes 1 and 4), per9 cells (lane 2), and
cells overexpressing the PER9 gene (lane 3). No
signal was observed when the preimmune serum (lane 4) was
used. Cells were grown/incubated in batch cultures on 0.5% methanol.
Equal amounts of protein were loaded per lane. B, shown is the
localization of Per9p. The 30,000
g supernatant (S3; lane 2) and organellar pellet (P3; lane 1), obtained after differential centrifugation of
homogenates prepared from methanol-grown wild-type cells of H.
polymorpha, as well as the peroxisomal (Per; lane
3) and mitochondrial (Mit; lane 4) fractions,
obtained after subsequent sucrose density centrifugation of the P3
organellar pellet, were subjected to Western blotting. The data show
that Per9p is a peroxisomal protein. As a control, the H.
polymorpha integral peroxisomal membrane protein Per8p was used. C, shown are Western blots demonstrating the distribution of
Per9p over the soluble (S; lanes 1 and 3)
and pelletable (P; lanes 2 and 4) fractions
after high salt treatment (NaCl; lanes 1 and 2) and sodium carbonate extraction (CO
; lanes 3 and 4) of the peroxisome-enriched peak fractions. D, as
controls for the location of Per9p (see C), the distribution
of the matrix protein CAT and Per8p (an integral component of the
peroxisomal membrane of H. polymorpha) was studied. Per8p was
pelletable after both high salt treatment (NaCl; lanes 1 and 2) and sodium carbonate extraction (CO
; lanes 3 and 4) of the peroxisomal peak fractions; instead, catalase was
partly solubilized after NaCl treatment (lanes 1 and 2), but completely soluble after carbonate treatment of these
fractions (lanes 3 and 4). These data confirm that
Per9p is an integral membrane protein of peroxisomes of H.
polymorpha.
To gain information on the
topogenic signals of Per9p, we expressed selected chimeric genes
composed of PER9 sequences encoding different N-terminal parts
of Per9p fused to a CAT gene encoding CAT lacking a functional
PTS1 (CAT(-PTS1)). In previous studies, we showed that this
PTS1-mutated CAT gene product is not sorted to peroxisomes of H. polymorpha, but instead accumulates in the
cytosol(36) . The fusion products were synthesized in an H.
polymorpha CAT disruption strain (cat).
Immunocytochemical experiments performed on a strain producing
the first 16 amino acids (N
) of Per9p fused to
CAT(-PTS1) revealed that the N
Per9p-CAT(-PTS1)
hybrid protein was located at membranous layers, most probably derived
from the ER, since also the nuclear envelope often showed labeling (Fig. 7, A and B). In addition, low labeling
intensities were found on the peroxisomal membrane (Fig. 7A). When larger parts of Per9p (N
or N
) were used for fusion, labeling was
predominantly on the peroxisomal membrane (data not shown), indicating
that additional information is required for Per9p to insert in the
peroxisomal membrane. Infrequently, small vesicles were seen associated
with these membranes, which were also labeled (Fig. 7B). CAT protein was not, or to a very low extent
as judged by the labeling patterns, in the peroxisomal matrix; it must
be emphasized that the immunocytochemical methods allow us to
discriminate between an intraperoxisomal and membrane-bound location in
batch-cultured cells(37) . From these results, we conclude that
the first 16 amino acids of H. polymorpha Per9p contain
topogenic information that is able to direct a reporter protein
(CAT(-PTS1)) to the ER.
Figure 7:
Shown in A is the typical
labeling patterns after incubation of ultrathin sections of H.
polymorpha cat cells, synthesizing
N
Per9p-CAT(-PTS1), with specific anti-CAT
antibodies. Labeling is predominantly located on layers of membranes,
associated with the nucleus, and on the peroxisomal membrane. In the inset (B), labeled small vesicles are seen. m, mitochondrion; n, nucleus; p, peroxisome. Bar = 0.5 µm.
Figure 8:
Activity of the PER9 promoter
determined by P-driven
-lactamase
synthesis under selected growth conditions.
-Lactamase enzyme
activities are expressed as units/milligram of
protein.
We examined the kinetics of the
biogenesis of peroxisomes by electron microscopy in
P-PER9-containing
per9 cells after
a shift from ammonium sulfate- to methylamine-containing medium. These
experiments indicated that new small peroxisomes were first detected
30 min after the shift. Typically, only one small organelle per
cell initially developed, which was characterized by the presence of AO
and Per9p (Fig. 9, A and B). These organelles
subsequently increased in size and multiplied during further
cultivation, as described before for a shift of wild-type cells from
glucose to methanol(38) . The mechanisms of peroxisome
reintroduction in these cells are currently being studied in depth and
will be detailed in a separate paper.
Figure 9:
A
and B show details of cells of per9::URA3 with
genomically integrated P-PER9 4 h after the
shift of cells from glucose/ammonium sulfate- to
methanol/methylamine-containing medium. Small peroxisomes were present
in the cells, characterized by the presence of AO (A; anti-AO)
and Per9p (B; anti-Per9p). After incubation for 12 h in
methanol/methylamine, the cells were transferred back to
methanol/ammonium sulfate, thus repressing Per9p synthesis. 8 h after
the shift, the first manifestation of disintegration of the peroxisomal
membranes (arrows) was observed (C and D),
associated with the appearance of AO protein in the cytoplasm (D; anti-AO antibodies). E and F show the
presence of AO protein in cytosolic aggregates (*), present in
per9 cells synthesizing truncated Per9p from which the
membrane-spanning region is deleted. In these aggregates, besides AO (F; anti-AO antibodies), also truncated Per9p was present (E; anti-Per9p antibodies). G shows the presence of
increased numbers of peroxisomes of various sizes in transformed
methanol-grown H. polymorpha cells expressing a plasmid
containing PER9 under the control of the strong AO promoter. m, mitochondrion; n, nucleus; p, peroxisome; v, vacuole. Bar = 0.5
µm.
Subsequently, cells grown on
methanol/methylamine were shifted to methanol/ammonium
sulfate-containing medium, thus repressing Per9p synthesis, and the
effect on peroxisome morphology was followed by electron microscopy. In
the initial hours after the shift, morphological alterations of
existing organelles were not detectable. After 8 h of incubation,
a partial disintegration of the peroxisomal membranes was observed (Fig. 9C). This result was highly reproducible with
regard to both the time interval (±8 h) and the deterioration
effect on the membrane. At this stage, soluble AO protein was also
first observed in the cytosol (Fig. 9D). At later
stages, AO crystalloids that lacked a surrounding membrane appeared in
the cells; a number of these crystalloids were subsequently degraded in
the vacuole (data not shown). These results indicate that Per9p plays a
role in maintenance of the peroxisomal membrane in vivo.
Per9p may also play a role in matrix protein assembly. This aspect
was further investigated in per9 cells transformed with
an expression plasmid carrying a truncated PER9 lacking the
region coding for the predicted membrane-spanning region (amino acids
16-36). These transformants could not grow on methanol and lacked
peroxisomes, suggesting that the peroxisomal location of Per9p is
essential for its functioning. Surprisingly, AO was not assembled and
active in these cells, as it is in
per9 cells, but
instead, AO was present in cytoplasmic aggregates in which the
truncated Per9p protein was also located (Fig. 9, E and F).
Finally, we studied the effect of PER9 overexpression on peroxisome biogenesis. An H. polymorpha strain was constructed that expresses PER9 under the control of the strong AO promoter. Electron microscopic analysis of such cells grown on methanol revealed that they contained higher than normal numbers of peroxisomes, often accompanied by vesicular structures (Fig. 9G).
Disruption of the PER9 gene had a
drastic effect on the overall morphology of derepressed H.
polymorpha cells in that (i) peroxisomes are absent and (ii)
peroxisomal membrane remnants are also not detected. Peroxisomal matrix
proteins like AO, CAT, dihydroxyacetone synthase, and malate synthase
are normally synthesized and active in the cytosol as in other per disruption strains; peroxisomal membrane proteins (Per8p (8) and Per10p) were also present at approximately
normal levels in
per9 cells. However, vesicular
structures (``ghosts'') (39) were never observed. The
method used (overproduction of the H. polymorpha peroxisomal
membrane protein Per8p as marker protein) enabled such vesicles in
other H. polymorpha
per strains to be readily
discerned(35) . By a comparable method, Purdue and Lazarow (40) demonstrated the presence of peroxisomal membrane vesicles
in S. cerevisiae peb mutants. The apparent absence of
peroxisomal vesicles in
per9 cells makes this mutant
attractive for molecular studies on the reintroduction of peroxisomes
as occurs in transformants that synthesize Per9p under the control of a
substrate-inducible promoter. This reintroduction is in line with
earlier observations that peroxisomes in H. polymorpha do not
necessarily derive from pre-existing peroxisomes(41) . The
mechanisms of the reintroduction of peroxisomes in per disruption strains, including
per9, are currently
being studied in our laboratory.
Moreover, Per9p seems to be involved in peroxisome proliferation. The multiplication of organelles, observed under conditions of PER9 overexpression, could simply reflect a Per9p-mediated enhanced synthesis of peroxisomal membranes. On the other hand, it is tempting to speculate that the mechanisms controlling peroxisome proliferation and membrane biogenesis are functionally related in that the proteins involved in these processes are associated (or only can function) in one complex. Such functional interactions were already predicted from a classical genetic study in which various H. polymorpha genes, including PER9, were shown to be functionally linked(12) .
H. polymorpha Per3p may also play a role in this putative protein complex. Recently, Per3p was identified as the H. polymorpha PTS1 receptor(7) , showing both functional and structural similarities to the Pichia pastoris PTS1 receptor, Pas8p(42, 43) . We postulated a model in which Per3p acts as the cytosolic receptor of newly formed PTS1 proteins that shuttles these polypeptides from the cytoplasm into the organellar matrix. One of the missing links in this model is that it does not explain how the Per3p-PTS1 protein complex reaches the peroxisomal membrane. Per3p lacks any known PTS and does not enter the peroxisome after overexpression of PER3 in H. polymorpha wild-type cells(7) . Hence, we proposed that Per3p undergoes a conformational change due to or after binding to the PTS1 protein. The modified Per3p is subsequently recognized by a second protein that mediates sorting to the peroxisome. We speculate that Per9p could represent this protein; the involvement of Per9p in this process is also suggested by the simultaneous aggregation of AO and truncated Per9p lacking its membrane-spanning region. This hypothesis is in line with genetic studies by Titorenko et al.(12) , who predict a functional interaction between Per9p and Per3p; it also does not conflict with the proposed function of Pas3p of S. cerevisiae(9) , which was suggested to act as a protein receptor.
Unexpectedly, the first 16 amino acids of PER9 were already sufficient to sort CAT, lacking its
functional PTS, to the ER and nuclear membrane. One possible but
unlikely explanation for this result is the cryptic nuclear targeting
signal present in the 16 N-terminal amino acids of Per9p. The low
labeling of CAT on peroxisomes can then be explained as a result of
``piggybacking'' (44, 45) in that the
NPer9p-CAT fusion protein associates with authentic Per9p,
which is also synthesized in these cells. The alternative is that the
ER is directly involved in peroxisome biogenesis and that protein
import and membrane biosynthesis are coupled processes. This can be
envisaged in the view that peroxisomes are compartments, filled with
proteins, that can only incorporate additional protein when the
internal volume is simultaneously increased by recruiting phospholipids
from the ER. The view that the ER may be involved in the biogenesis of
peroxisomes is further supported by the finding that brefeldin A
prevents peroxisome formation, resulting in the accumulation of
peroxisomal matrix proteins at the ER. (
)It is interesting
to note in this context that Bodnar and Rachubinski (46) described a 50-kDa integral membrane protein of mammalian
peroxisomes that was synthesized on membrane-bound polysomes. It is
still unclear, however, whether Per9p has properties similar to this
protein.
The concept of protein import coupled to membrane insertion, e.g. via vesicle formation(45) , of course leaves many questions unsolved, but takes its attractiveness from the fact that it unites a number of yet unexplained observations in various organisms in one model. Among these are the observations of Bellion and Goodman (47) on the import of AO in Candida boidinii. They showed that import and subsequent octamerization of AO was prevented in the presence of ionophores and resulted in the formation of peroxisomal membrane-associated protein complexes consisting of AO, dihydroxyacetone synthase, and several other unknown proteins(47) . It can be envisaged that these proteins are not imported because membrane vesicle formation and/or fusion is prevented due to the lowered ATP levels in the cell. This mode of import could also explain how mature complex proteins or even gold particles enter the peroxisome(44, 45, 48) . Further studies on the putative role of the ER in peroxisome biogenesis are currently in progress.