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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. 158 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 ). 160 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. 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Transport of microinjected proteins into peroxisomes of mammalian cells: inability of Zeil weger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal. Mo/ec. Cell Rio/. 12, 531-541, 1992. Note added in pro of: Since submission of this paper part of the results described have been published: Motley, A.M., E.H. Hettema, B. Distel and H.F. Tabak. Differential protein import deficiencies in human peroxisome assembly disorders. J. Cello Bio/. 125, 755-767, 1994. H.F. Tabak et al. 165