Download Comparative Studies on Peroxisome Biogenesis in S. Cerevisiae

H.F. Tabak, A. Motley, M. Franse, Y. Elgersma, M. van den Berg,
T. Voorn-Brouwer, W. Hemrika and B. Distel
Comparative Studies on Peroxisome Biogenesis in S. Cerevisiae
and Human Fibroblasts
Introduction
When you inspect a biochemistry textbook for a description of peroxisomes, you
can consider yourself lucky if you find a single page describing a meagre set of
properties relating to their contribution to cellular metabolism. Even in an
idealised drawing of a typical eukaryotic cell to be found in: 'The Living Cell'
written by their discoverer Christian de Duve, they feature as inconspicuous
small circ1es, while the remaining organelles are illustrated with artistic cartoons
[2]. This situation is rapidly changing, however, and peroxisomes are now
attracting the attention they deserve. Two aspects contribute to this development.
- some years ago it was found th at a number of serious human diseases are
caused by malfunction of peroxisomes (reviewed in 11). Cell fusion studies
between fibroblasts of such patients followed by scoring for the rescue of
peroxisomal function has identified at least nine complementation groups for the
more serious disorders (reviewed in 18). The first gene has been identified, the
defective allele of which is responsible for the disease of one of these complementation groups [19] , while a second gene was accidentally discovered in a human
DNA sequence project [15].
- genetic approaches to analyse peroxisomal function and maintenance in
various yeasts have turned out to be very rewarding, and a number of genes
have already been cloned whose products contribute to the biogenesis of peroxisomes (reviewed in 12).
The discovery th at peroxisome growth and proliferation can be induced in
Saccharomyces cerevisiae by growth on the fatty acid oleate [22] was especially
important in this respect, since it disc10sed an organism for peroxisomal research
that is gene rally considered a simp Ie model eukaryote. Moreover, the genome of
S. cerevisiae may well be completely sequenced within the next few years, thus
providing a wealth of new information. In the Departments of Pediatrics and
Biochemistry of the Academic Medical Center in Amsterdam we are trying to
follow and integrate these leads into a collaborative effort to understand better
H.F. Tabak et al.
155
the biogenesis of peroxisomes and their essential contribution to optimal cellular
performance.
S. cerevisiae as a model eukaryote for studying peroxisomes
Genetic approaches
The first S. cerevisiae mutants deficient in peroxisomal functions were isolated
using a time-consuming replica plating technique to identify cells unable to grow
on culture media containing oleate as sole carbon source [5]. Biochemical fractionation of cell homogenates was used to study the sedimentability of peroxisomal marker enzymes, which allows one to distinguish between the mutants
with a general impairment in biogenesis and the ones with single enzyme
deficiencies. We have tried to set up positive selection screens in order to analyse
larger mutant populations with the hope of finding new genes in addition to the
ones already cloned.
Catabolism of fatty acids in the p-oxidation pathway results in the production
of H 2 0 2 , which is subsequently decomposed by catalase. In the presence of
3-amino-triazole catalase is inhibited, and in a wild type cell growing on a fatty
acid as the exclusive carbon source, H 2 0 2 will accumulate (Fig. IA). Mutants
disturbed in fatty acid oxidation will not produce H 2 O 2 and therefore can survive under the selection conditions. Such mutants may comprise not only cells
deficient in an enzyme of the p-oxidation pathway, but also and more interestingly, cells with gross alterations in peroxisomal biogenesis or protein import.
Application of this screen has led to the isolation of various mutants, which were
unable to grow on oleate. These mutants were further characterised by biochemical subfractionation and electronmicroscopy to single out the mutants
with generalised impairment in peroxisome assembly from the ones with single
enzyme deficiencies [20]. After a complementation analysis including the
mutants isolated by the group of Kunau, a number of complementation groups
were found (possibly) representing different affected genes (Tabie 1). Further
research on this set of mutants has given the impression th at import mutants
sensu stricto are underrepresented, and this has stimulated us to set up a new
positive selection screen th at more specifically was aimed at finding rep resentatives of this latter class of mutants. lts principle is based upon protein
import selection screens worked out by other groups such as Beckwith and
coworkers [16], Schekman and coworkers [3] and Meyer and coworkers
[ 13]. Our variation on this idea is the use of the phleomycin-binding protein,
which can protect a cell against the toxic action of the antibiotic phleomycin. By
coupling this protein to luciferase through gene fusion of the corresponding
reading frames, a hybrid protein is produced that is imported into peroxisomes
in wildtype cells (Fig. lB). In a peroxisomal protein mutant, the hybrid protein
will remain in the cytoplasm, however. In reconstitution experiments, we have
shown that this results in a differential resistance to phleomycin: a cell contain-
156
Comparative studies on peroxisome biogenesis in S. cerevisiae and human fibroblasts
ing the phleomycin-binding protein within peroxisomes is much more sensitive
to phleomycin than in cell containing the protein in the cytoplasm. Thus a
protein import mutant can be selected on the basis of its increased resistance to
phleomycin. This genetic screen has turned out to be very successful: all the
mutants selected are of the pas phenotype characterised by generalised impairments of peroxisomal biogenesis [4]. Apart from the already established complementation groups, also a number of new complementation groups have been
found (Tabie 1). When we compare the two methods, it is clear from Table 1 that
the phleomycin selection is more specific. for in stance, pas7 (a PTSII-import
deficient mutant) was not found, which was expected because import of SKLcontaining proteins (PTSI route) is still functional. AIso, pas 14 ( = SNFI) and
pas 19 (= AD RI) were not isolated. Both mutants have deficiencies that
indirectly impair peroxisome biogenesis: PAS14 functions in a signal transduction pathway and is an essential component in the chain of events required for
induction of genes when cells are relieved from glucose repression [20]. PAS19
is a transcription factor th at is more directly involved in expres sion of gen es coding for peroxisomal proteins [20]. Our choice to use the catalase promoter
(itself inducible by oleate) to con trol the expres sion of the phleomycin-Iuciferase
hybrid pro te in may be responsible for the lack of appearance of these transcription factors or signal transducing proteins in the phleomycin selection screen.
Table I. Peroxisome assembly complementation groups in S. Cerevisiae*
Compilation of peroxisomal assembly mutants of S. cerevisiae. A recent update of the mutants of
the Kunau collection is found in references 10.
Mutant
Protein characteristics
past
pas2
pas3
pas4
pas5
pas6
pas7
pas8
pas9
pas 10
pasll
pasl2
pasl4
pasl9
pas20
pas2l
pas22
AAA type protein
UBC protein
Peroxisomal membrane protein
Protein with Zn finger motif
Protein with Zn finger motif
SKL containing protein
Involved in PTS2 import
AAA type protein, transmembrane domain
Transmembrane domain
Involved in PTSl import, TPR family
No. of alleles isolated
H202
Phleomycin
Protein with farnesylation consensus sequence
= SNFl , SerjThr protein kinase
=ADRl, transcription factor
(being sequenced)
(being sequenced)
Putative NLS, Calcium binding site (?)
I
2
1
4
2
5
3
3
I
2
2
4
3
1
5
2
6
1
2
1
1
*Mutants isolated by Kunau and coworkers are included in this tabIe.
H.F. Tabak et al.
157
Principle of the Phleomycin Selection Procedure
g;A1
ISleomycin I
Luciferase
Wild-type cell
Import mutant
~
_
Phleomycin
Bleomycin-Iuciferase
Phleomycin sensitive
Phleomycin resistant
1
1
Cell dies
Principle of the
Wild-type cell
H:202 accumulates
SKL I
Cell survives
~02
selection procedure
Peroxisomal mutant
No
~2
produced
Cell dies
Cell survives
Fig. 1. Positive selection screens [or the isolation of yeast mutants disturbed in peroxisomal functions and biogenesis.
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Comparative studies on peroxisome biogenesis in S. cerevisiae and human fibroblasts
Cloning of PAS genes by functional complementation of mutants
We have recently and sequenced the wild type genes corresponding to two complementation groups. In the PAS8 mutant, proteins of the PTSI as weIl as of the
PTSII class remain in the supernatant upon biochemical subfractionation, and
electronmicroscopy does not show even a trace of peroxisomes or residual membranes. As such it represents a typical pas phenotype, and it is remarkable therefore that this mutant in its laboratory life is considered to be a wild type stain
(YP102) and th at a similar phenotype in human ceIls results in severe disease
(Zenweger syndromes). The P AS8 gene encodes a protein of 1030 amino acids
with a number of interesting features [23]. It contains a conserved domain of
at least 185 amino acids with sequence motifs characteristic for ATP binding
and/or hydrolysis: the Walker motifs A and B. This 185 amino acid long region
is present once or twice in a group of other proteins, such as NSFp and SEC18p
(involved in the secretory process), CDC48p (involved in cen cycle progression),
BSPlp (a protein involved in the formation of the bel complex of the
respiratory chain), SUGlp (functioning in gene expression) and PASlp, which,
like PAS8p, is involved in peroxisome biogenesis (reviewed in 10).
PAS 1P has two co pies of the 185 amino acid domain, of which one is rather
degenerate, while PAS8p has only one copy. Moreover, the similarities are
limited to the 185 amino acid domains since outside these regions no significant
similarities remain. Although both the proteins possess the accessory ATP binding domain, their primary functions may thus be quite ditTerent. A hydrophilicity
plot of P AS8p indicates the possible existence of two hydrophobic regions
(between amino acids 245-269 and 540-555), but whether the protein is in deed
associated with membranes remains to be demonstrated.
The PASI0 mutant and its corresponding 'knock-out' shows a ditTerential
pro te in import deficiency [21]. A number of peroxisomal enzymes that come
down in the penet after fractionation of a wild type homogenate remain in the
supernatant after fractionation of aPASlO homogenate. These include multifunctional enzyme, catalase A and heterologously expressed luciferase, the
import of which is dependent on a functional PTSI import route. Thiolase,
which enters the peroxisome via the PTSII import pathway, is associated with
leaflet-like membrane structures present in PASlO ceIls and is still recovered in
the penet fraction after fractionation of a PAS 10 homogenate. Since
immunogold-electronmicroscopy can not distinguish thiolase adhering to the
cytoplasmic surface of these membranes and translocation across these membranes, we have also studied and compared the protease sensitivity of thiolase
in homogenates of PASlO, PAS7 and wildtype cens. Thiolase is degraded via a
somewhat stabIe intermediate in PAS7 extracts, which is in line with the characterisation of PAS7 as a PTSII import incompetent mutant. Under identical conditions, thiol ase in PASlO and wildtype homogenates is completely resistant to
protease indicating true import of thiolase in the residual membrane structures
of PASI0. The question can be raised as to wh ether these membranes represent
residual peroxisomal structures or whether their accumulation is merely a non-
H.F. Tabak et al.
159
specific secondary effect of the mutation. The membranes are decorated with
gold particles using not only a thiolase antibody but also using an antibody
directed against p-galactosidase [9] in cells expressing a hybrid protein consisting of the N-terminal part of the peroxisomal membrane protein PAS3p coupled
in frame to p-galactosidase. Moreover, in a PAS7jPAS10 double mutant, neither
the morphologically normal-Iooking peroxisomes of PAS7, nor the membrane
leaflets found in PAS 10 cells can be observed, indicating a synergistic effect
of both mutations. It suggests that the PASlO and PAS7 gene products function
in the same biological pathway: peroxisome biogenesis. We believe therefore
that the membranes accumulating in PASlO are of bona fide peroxisomal
origin.
We have cloned the wild type PASlO gene by functional complementation of
the PASlO mutant and sequenced it [21]. Comparison of its 612 amino acid
sequenced with other proteins in data bases indicates that it is a member of the
TetratricoPeptideRepeat (TPR) protein family. As the name implies, such
proteins contain a 34 amino acid repeat unit which is present in multiple direct
repeats in various proteins [7]. These proteins are involved in diverse biological
processes, which does not help very much to pinpoint a particular function of
the TPR motifs. Based on structural analysis and computer graphics, it is argued
that TPR motifs can interact with each other in a 'knobhole' like interaction
which can be either intra- or intermolecular [8]. Of special interest to us are
TPR proteins involved in mitochondrial protein import such as MAS70j
MOM72 (containing multiple TPR repeats) and MOM19 (containing a single
TPR unit). Both are considered to be receptors with overlapping specificities for
import of proteins into mitochondria [17].
To investigate whether PASlOp could also function as a receptor mediating
import of PTSI-containing proteins into peroxisomes, we are studying its subcellular location. Initial attempts to tag PAS 1Op with the myc epitope yielded
ambiguous results. Although the tagged derivative complemented the PAS10
mutant, erratic results were possibly caused by overexpression of the protein,
which is required to detect the myc epitope. More recently we have used a
polyclonal antibody, which was raised against a (his) 6 tagged PASlOp
derivative overproduced in E. coli and purified on a Ni-chelating column. After
fractionation of wild type homogenates, most of the PAS10p fractionates in the
pellet when analysed by Western blotting using this polyclonal antibody. When
the pellet fraction is further analysed by sucrose density gradient centrifugation,
most of the PAS 1Op is found at the characteristic density of peroxisomes
together with catalase. These results indicate th at PASI0p is associated with
peroxisomes. We have not yet been able to determine the precise location of
PASlOp within the peroxisome, however. PASlOp can interact with the SKL
carboxy-terminal aminoacid sequence of luciferase because PASlO and the terminal part of luciferace can reestablish the transactivation function of GAL4 in
the yeast 'two hybrid' system developed by Fields [6]. This interaction is
abolished when an SEL mutant derivative of luciferase is used, which underlines
the specificity of this interaction (M. Franse, unpublished observations ).
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Comparative studies on peroxisome biogenesis in S. cerevisiae and human fibroblasts
A PAS 10 homologue (P AS8p) has been found in Pichia pastoris [14]. Subfractionation of peroxisomes in P. pastoris suggests that part of PAS8p is
strongly bound to the peroxisomal membrane, while the remainder is loosely
bound or present in the cytosolic fraction. Moreover, binding of an SKL-containing peptide to PAS8p has been demonstrated, suggesting a link between
PTSI containing proteins and their import into peroxisomes. Interestingly, the
polyclonal antibody raised against S. cerevisiae PAS 10p cross-reacts with a
pro te in is induced by treatment of rats with clofibrate and is present in a sucrose
gradient at a position at which the peroxisomal marker enzymes are located. We
are currently investigating whether this protein is a true homologue of S.
cerevisiae PASI0p in higher eukaryotes.
Human fibroblasts to study peroxisomes in relation to disease
The S. cerevisiae mutants PAS7 and PASlO illustrate the existence of the differential PTSI and PTSII protein import pathways. PAS7 contains morphologically normal looking peroxisomes and thusfar only thiolase has been shown to
be mislocalised, indicating that the PTSII route represents only a minor import
pathway. The PASI0 mutant shows a drastically altered morphological
phenotype and is more reminiscent of the generalised peroxisomal impairments
encountered among cells derived from Zellweger patients. To investigate whether
differential PTSI and PTSII protein import deficiencies occur among fibroblast
celllines derived from patients with peroxisomal disorders, we have extended
our search to cell lines derived from rhizomelic chondrodysplasia punctata
(RCDP) patients in addition to the generalised impairments found in the
Zellweger fibroblasts. The capacity to import PTSI and PTSII proteins was
assessed by micro-injecting into the nucleus reporter plasmids encoding a hybrid
protein consisting of the N-terminal prepiece of thiolase followed by the chloramphenicol acetyl transferase (CAT) protein (PTSII reporter) or luciferase
(PTSI reporter) (Fig. 2). Injection of a few plasmid molecules per nucleus
resulted in enough production of protein to allow detection by immunofluorescence using antibodies directed against CAT or luciferase after approximately
18 h of further incubation. This procedure allows the cells to recover from the
trauma of injection and prevents overloading of the cells with the injected
material. RCDP cells injected with the plasmid encoding luciferase developed
punctate fluorescence suggesting that the PTSI protein import pathway was still
operational. This result is expected and confirmed earl ier results obtained by
others using different methodologies. Production of prethiolase-CA T did not
lead to punctate fluorescence. This is more surprising because it has been rep orted that precursor thiolase (44 kDa) equilibrates in light density fractions of a
sucrose gradient and is protease-resistant, implicating that thiolase could be present in a vesicle-enclosed compartment [1]. We therefore also measured the
protection of endogenous thiolase to protease in digitonin permeabilised RCDP
cells and observed that thiol ase was completely sensitive to protease. If there is
H.F. Tabak et al.
161
CMV - - - I
RSV
luciferase
prethiolase/CAT
Fig. 2. DNA constructs encoding reporter proteins for testing the functionality of two
peroxisomal import pathways. CMV, cytomegalo virus promoter. RSV, Rous sarcoma virus
promoter. Luciferase is used as PTSI and prethiolasejCAT as PTS2 reporter.
a compartment in RCDP cells containing precursor thiolase, the lipid composition of this compartment is very distinctive to th at of peroxisomes, which are
difficult to permeabilise using digitonin due to the low cholesterol content of
their membranes. Our interpretation of these results is that the RCDP fibroblasts are remarkably similar in their phenotype to the PAS7 mutant of S. cerevisiae. Both have morphologically normal peroxisomes and are deficient in the
import of proteins containing the PTSII signal.
In the set of fibroblasts with generalised impairments of peroxisomal function
included in this survey, production of luciferase did not lead to punctate fluorescence, as has been reported earlier [24]. In fibroblasts of complementation
group IV (neonatal adrenoleukodystrophy: NALD) however, production of
prethiolase-CAT resulted in punctate fluorescence. The number of fluorescent
spots was much lower compared to wild type fibroblasts and they appeared
larger in size. In experiments using double immunofluorescence the endogenous
thiolase of these cells colocalised with antibody directed against the 69 kDa
peroxisomal membrane protein, indicating the association of thiolase with the
residual peroxisomal structures. After permeabilisation of the plasma membrane
with digitonin, punctate fluorescence was obtained with the anti69 kDa antibody, but not with antibody directed against CAT, nor with the monoclonal
directed against endogenous thiolase. Positive immunofluorescence with the latter two antibodies was detected only after treatment with digitonin in the
presence of Triton XIOO, wh en both plasma and the peroxisomal membrane are
permeabilised. Protease treatment of homogenates followed by Western blot
analysis confirmed that endogenous thiolase was protected from degradation by
protease. As a fin al control to exclude the possibility that protease resistance
could be due to the formation of aspecific protein/phospholipid clumps, we have
performed immunogold electronmicroscopy. These experiments show colocalisation of the 69 kDa peroxisomal membrane protein with thiolase (Fig. 3). The
thiolase labelling was surrounded by membranes (in some cases even by multiple
layers), thus indicating th at the most plausible reason for the observed protease
resistance of thiol ase is its presence within a membrane. Based on this set of
properties, the NALD complementation group IV cells are the opposite of the
RCDP cells, proficient in the PTSII import pathway but lacking in import of
PTSI-containing proteins. Thus human group IV resembles the S. cerevisiae
162
Comparative studies on peroxisome biogenesis in S. cerevisiae and human fibroblasts
.'
Fig. 3. Electronmicrograph of residual peroxisomal structures in a Zellweger fibroblast of complementation group 4. Large gold partic1es indicate the presence of thiolase, small gold partic1es
indicate the presence of the peroxisomal membrane protein PMP70.
PASlO mutant in phenotype (see Note added in proof). Final proof for this
proposal would be to test if introduction of a mammalian PAS 1Op homologue
can restore the function of the PTSI pro te in import capacity in group IV
fibroblasts.
Concluding remarks
It is rather pleasing to notice on the basis of this comparative study th at the
yeast mutants have their counterparts in human diseases, supporting the view
that yeast can be used as a valid model eukaryote to study complex biological
processes such as peroxisome biogenesis. On the other hand, a note of caution
is also in order. Yeast homologues of proteins identified in higher eukaryotes
with an important role in peroxisome function or biogenesis such as: PMP70
( = 69 kDa), PAFI and X-ALD have not yet been discovered in the genetic
H.F. Tabak et al.
163
screens applied thusfar. Thus, there is every reason to further sharpen our tools
to unravel the intricacies of peroxisomal biogenesis.
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