CELL BIOCHEMISTRY AND FUNCTION VOL.
10: 167-174 (1992)
A Genetic Approach to the Biogenesis of Peroxisomes in the Yeast Saccharomyces cerevisiae RALF ERDMANNt AND WOLF-H. KUNAU Instituf .fur Pliysiologische Chemie, Medizinische Fakultat der Ruhr- Universitat Bochum, 0-4630 Bochum, Federal Ropuhlir~of' Germany
INTRODUCTION
structure of protein components involved in peroxisome biogenesis in S. cerevisiae.
The budding yeast Succhurom,vces cerevisiue offers the advantages of combined genetic, molecular genetic and biochemical approaches to study cell PEROXISOMAL MUTANTS biological processes. S. cerevisiae mutants have been powerful tools to dissect the biogenesis of Like other yeasts, S. cerevisiue does not contain a subcellular structures, such as mitochondria,'.2 en- mitochondria1 P-oxidation system.' Consequentdoplasmic r e t i ~ u l u m ,g~~. ~l g ivacuoles,6 ,~ and most ly, for the utilization of fatty acids as carbon source, S. cerevisiue depends on a functional peroxisomal recently p e r o x i ~ o m e s . ~ - ' ~ /I-oxidation system. As a first step in identifying The presence of peroxisomes in S. cerevisiue was peroxisomal mutants, cells that were able to grow first demonstrated by Avers and Federman,' but on non-fermentable carbon sources such as glyin contrast to other yeasts and filamentous cerol and ethanol, but unable to grow on oleic acid fungi,' 2 . ' ' peroxisomes of S. cerevisiue have not as single carbon source were isolated (Figure 1). been characterized in great detail. This lack of This screening step defined oleic acid non-utilizing information is explained by the observation that under the growth conditions originally chosen, strains (onu-mutants) and therefore enriched for peroxisomes in S. cerevisiue are very rare and mutants affected in the degradation of fatty acids7 ~ r n a 1 l . lIn ~ several yeasts, proliferation of this or- The lack of a functional P-oxidation system in the ganelle can be induced by growth on distinct car- onu-mutants could be due to defects in individual bon and nitrogen sources1' but attempts to induce fi-oxidation enzyme activities or to defects in perthe proliferation of peroxisomes in S. cerevisiue oxisome biogenesis which in turn would prevent failed for a long time. It was not until Veenhuis et the proper assembly of a functional P-oxidation d.'" and Kunau et al.' demonstrated the inducibi- complex inside peroxisomes. By ultrastructural lity of peroxisomal P-oxidation as well as perox- analysis some of the onu-mutants were shown to be isome proliferation in yeasts by growth on oleic peroxisome-deficient. In order to identify these acid as the single carbon source that remarkable mutants by biochemical means, fi-oxidation enamounts of peroxisomes were also found in S. zyme activities as well as catalase activity were ccw~ci.siur~.'4.1This finding opened the way for the determined in whole cell extracts and subcellular development of a genetic screen to isolate peroxiso- fractions of onu-mutants. This screening step allowed the detection of mutants with individual ma1 mutants of S. ~erevisiue.~ ,fitty acid oxidation enzyme deficiencies In this report we review the first attempts to (fox-mutants)" as well as mutants with peroxisocharacterize peroxisome biogenesis in S. cerevisiue ma1 matrix enzymes mislocalized to the cytosol by a combined genetic and biochemical approach. (peroxisome assemblyor p ~ s - m u t a n t s )The . ~ misWe describe the different types of peroxisomal mutants and our current knowledge of the primary localization of peroxisomal matrix enzymes to the cytosol indicated a defect in peroxisome biogenesis. Used in this broad sense, peroxisome biogenesis t Present address: Laboratory of Cell Biology, Rockefeller Uni- includes all reactions required for import of peroxisomal proteins from the cytosol, reactions which versity, New York, New York 10021-6399, U.S.A.
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0263 6484/92,'030167 08$09.00 \( 1992 by John Wiley & Sons, Ltd.
168
R. ERDMANN AND W-H. KUNAU
SCREENING PROTOCOL FOR PEROXISOMAL MUTANTS mutagenesis
++I1
COLONY SCREENING
screening for inability to
grow onoleote
-01e a te - 5 on - _uti Ii z i n g
strains
(zstra i nsl BIOCHEMICAL SCREEN I N G
mislocal i za t i o n of perox i s omal enzymes (25,00O*g)
deficiency o f a p-oxidation enzyme i
electron microscopy
+r
e l
MORPHOLOGICAL SCREENING
fox m u t a n t s
t y p e I- 111
Figure 1. Screening protocol for peroxisomal mutants
oxidase was cloned accidentally and identified by its homology to the acyl-CoA oxidase genes of C. tropicalis. The fox2 mutant showed concomitant loss of three P-oxidation enzyme activities, indicating the presence of a trifunctional fl-oxidation enzyme in S. cerevisiue. FOX2 has been cloned by functional complementation and the multidomain structure of the corresponding gene product is reflected in its primary sequence.I8 Thefox3 mutants are affected in 3-oxoacyl-CoA thiolase. As judged by sequence comparison, FOX3, which recently has been cloned by functional complementaFox MUTANTS ARE AFFECTED IN tion, indeed encodes yeast peroxisomal thiolase.” PEROXISOMAL ,!I-OXIDATION The thiolase deficiency and the onu-phenotype of The fox mutants fall into three complementation fox3 mutants indicate that unlike the situation in only one thiolase involved in groups. This result is consistent with biochemical mammalian results, indicating that peroxisomal fl-oxidation in peroxisomal 8-oxidation exists in S. cerevisiue. Other yeast mutants with defects in peroxisomal S. cerevisiue is performed by three gene product^.'^ All three corresponding genes have been cloned. enzymes have been isolated by different screening The FOXI or POXI” gene, encoding the yeast procedures and the corresponding wild-type genes peroxisomal P-oxidation entry enzyme acyl-CoA have been cloned by functional complementation: control peroxisome proliferation and those which provide the lipids for peroxisomal membranes. Further characterization of peroxisomal mutants was achieved by electronmicroscopical and immunocytochemical investigations. Recently, this screening protocol has been improved by introducing a positive selection step.” This resulted in the isolation of a number of additional pas mutants, among which five new complementation groups could be defined.
GENETICS OF PEROXISOMAL BIOGENESIS
CTAI,” encoding catalase A; A L ~ D H ~encoding ,’~ malate dehydrogenase 2 ; and CIT2,23 encoding citrate synthase 2. Fox mutants do contain peroxisomes which, apart from the deficiency of the corresponding b-oxidation enzyme, exhibit wildtype properties. Therefore, yeast mutants deficient in endogenous peroxisomal proteins might represent suitable systems to study homologous and heterologous expression and in uiuo import of peroxisomal proteins. For example, by taking advantage of a thiolase deficient fox3 mutant, very recently the peroxisomal targeting signal of yeast 3oxoacyl-CoA thiolase has been defined.24
PUS-MUTANTS ARE AFFECTED IN PEROXISOME ASSEMBLY Originally, pas mutants were defined by the (i) inability to grow on oleic acid (onu-phenotype), (ii) mislocalization of peroxisomal matrix enzymes to the cytosol and (iii) absence of detectable peroxisomes at the ultrastructural leveL7 However, detailed analysis of newly identified mutants revealed that mislocalization of peroxisomal proteins is not always due to the absence of peroxisomes. All the pas mutants so far characterized show the onuphenotype and the cytosolic mislocalization of one or all of the peroxisomal matrix enzymes tested. Based on these criteria three different subgroups of pas mutants, designated type I to I11 have been distinguished.
Type I pus Mutants are AfSected in Peroxisome Formation Type I pus mutants (pasl, pus2, pus3, p a d ) are characterized by the absence of morphologically detectable peroxisomes. The lack of peroxisomes was demonstrated electronmicroscopically using serial sections and cytochemical staining for catalase activity. Subcellular fractionation studies and immunogold labelling confirmed mislocalization of peroxisomal matrix enzymes to the ~ y t o s o l . ’ , ~ ~ Pus mutants of type I resemble the phenotype of fibroblasts from Zellweger patients, suffering from a human peroxisomal and peroxisome-deficient mutants of CHO-cell line^.^'-^^ This raises the question of whether peroxisomal membrane structures, called ‘ghosts’, that have been detected in both peroxisome-deficient cell types33-35 are also present in type I pus mutants.
169 The availability of antibodies against the peroxisoma1 integral membrane protein P A S ~ Pshould ~ ~ now open the possibility of detecting peroxisomal ghosts in pas mutants by a combined biochemical and immunocytochemical approach. Another intriguing question is whether or not these ghosts actually are precursors (pre-peroxisomes) of functionally and morphologically mature peroxisomes. In s. cerevisiae different lines of results strongly suggest the existence of such prestructures. Accumulated evidence indicates that peroxisomes are not formed de novo (for reviews see references 36 and 37). Complementation analysis demonstrated that crossing of two haploid type I pus mutants resulted in diploid cells with peroxisomes, Moreover, even transformations of type I pus mutants with plasmids carrying in their inserts single genes rescued the pus phenotype. This approach has been successfully used to clone the respective PAS genes. Thus, presuming that peroxisomes cannot be formed de nouo, these results imply the existence of peroxisomal remnants functioning as a starting point for peroxisome biogenesis. Because this argues for peroxisomal prestructures in complementable type I pus mutant, the absence of morphologically detectable peroxisomes may be due to defects directly or indirectly affecting the import of peroxisomal matrix proteins. If prestructures exist and are the prerequisite (conditio sine qua non) for functional complementation one can envisage defects resulting in their absence. Due to the lack of an initiative point for peroxisome formation, none of those pus mutants should be complementable by transformation. Such mutants have not yet been identified. Type II pus Mutants are Affected in Peroxisome Proliferation Type I1 pus mutants (pad, pus6) differ significantly from type I mutants by the presence of morphologically detectable peroxisomes. However, the number and size of peroxisomes of induced type I1 mutant cells are drastically decreased in comparison to wild-type ~ e l l s . ~The * , ~phenotype ~ of oleic acid-grown type I1 mutant cells resembles a glucose-repressed state rather than an induced state of wild-type cells. In type I1 mutants, peroxisomal matrix enzymes are present in wild-type amounts but, consistent with the pas phenotype, they are mainly mislocalized to the cytoplasm. Nevertheless, a small portion of peroxisomal matrix proteins is
170 correctly localized, indicating that peroxisomal import can occur. Therefore, mislocalization of peroxisomal proteins in these mutants is most likely to be due to the deficiency of sufficient host organelles for their uptake. These results and the observation that oleic acid-induced peroxisome proliferation is controlled by the level of PAS4p expression (see below) suggest that type I1 pas mutants are affected in peroxisome proliferation. A number of pas mutants with a similar phenotype have been isolated by Tabak and coworkers and are described in more detail in their review."
Type III pas Mutants are Characterized by the Misloculization of Individual Peroxisomal Proteins to the Cytoplasm Pas7, the only known mutant of type 111, shows mislocalization of 3-oxoacyl-CoA thiolase to the c y t ~ s o l .All ~ ~ other peroxisomal proteins tested were found within the organelle. Genetic and molecular biological results demonstrated that thiolase mislocalization in pas7 is not due to a defect in the thiolase itself. Therefore, an as yet undefined protein, essential for thiolase import into peroxisomes seems to be affected in the pas7 mutant. As FOX2p," carrying the known C-terminal peroxisomal targeting signal SKL*4 1 . 4 2 appears to be imported normally in the pas7 mutant, the occurrence of this mutant gives the first evidence for a SKL-independent peroxisomal import pathway. This conclusion is supported by recent findings, indicating that the targeting signals of peroxisomal thiolase of rat liver43.44 and S. cereai.siac~~~ resides at the amino terminus of the proteins. An amino terminal targeting signal has also been suggested for the peroxisomal malate dehydrogenase of the ~ a t e r m e l o n Also . ~ ~ the peroxisomal integral membrane protein PAS3p of S. cerevisiae appears to have an amino-terminal targeting signal.2s However, it is imported into peroxisomal membranes of p a ~ 7 , ~ ' suggesting the presence of a second SKL-independent peroxisoma1 import pathway in S. cereitsiae. Whether these import pathways converge or whether they are entirely distinct cannot yet be answered. However, the PAS7 gene will define the first component specific for thiolase import.
*Single letter code for Ser-Lys-Leu.
R. ERDMANN AND W-H. KUNAU
The existence of two import pathways for peroxisomal matrix enzymes, the thiolase and the SKLdependent pathway, raises the question whether mutants affected in the latter one would also reflect a type 111 pas phenotype as found in the thiolase import mutant pas7. Recent evidence suggests that this will not be the case. The predicted PAS6p contains a C-terminal SKL,39 suggesting that the protein performs its function in the peroxisomal matrix. The corresponding pas6 mutant shows the type I1 phenotype indicating a defect in peroxisome proliferation. Therefore, mislocalization of PAS6p as the consequence of an affected SKL-pathway would be expected to result in the type I1 phenotype. One can also envisage that so far unrecognized SKL-containing proteins exist whose mislocalization would result in the pas phenotype I. Consequently, mutants affected in components that are essential for peroxisomal import of SKL-containing proteins would be expected to resemble the pas phenotype I or 11. The same conclusions can be drawn for a defect in the pathway for PAS3p import. As PAS3p is essential for peroxisome formation,25 its mislocalization should result in peroxisome-deficiency, giving rise to a type I pas mutant.
COMPONENTS ESSENTIAL FOR PEROXISOME BIOGENESIS Until now 18 different pas complementation groups have been defined among the pas mutants found in our laboratory' and found by Van der Leij et al." (Table 1). As the first step to characterize the P A S gene products, wild-type genes corresponding to affected genetic loci of pas mutants have been cloned by functional c ~ m p l e m e n t a t i o n For . ~ ~ this purpose, advantage was taken from the fact that peroxisomal mutants, when transformed with the corresponding wild-type gene, regained the ability to grow on the oleic acid m e d i ~ m . ~ By ~ . ~this ' approach PAS1 to PAS7, PAS9, PAS11 and PAS12 have been cloned. Authenticity of the genes was confirmed by gene disruption and genetic analysis of the resulting null mutants. Gene disruptions also revealed that none of the cloned P A S genes is essential, therefore reflecting that peroxisomes, at least under certain growth conditions, are not required for cell viability. In order to identify PAS proteins in yeast, antisera have been raised so far against putative PAS1 to PAS3 gene products.
171
GENETICS OF PEROXISOMAL BIOGENESlS
Table 1 , Complementation groups of Surchuromyces cerevisiue mutants deficient in peroxisome assembly. Corresponding wild-type gene Cloned Sequenced
pus mutant ~
~
pus I * pu.P
/IU.S3*,t
pu.s4*,+ pu.s5*. i. pus6*,t
pu.s7*. t pus8*, + pu.sY* ptr.v/O*.i pu.s I I *
pu.s12* p“.’13* pus 14t pus /St pu.v 1 6 t pu” 17t
~~
+ + + + + + + + + + + + f
+ +
+ + + + + + f f
+ +
pus /(it
tlsolated in Amsterdam (see reference 10). *Isolated in Bochum (see also references 8 and 9).
PAS 1 PASlp has been identified as a rather hydrophilic low abundance protein of 117 kD that contains two putative ATP binding sites.47 A striking feature of PASlp is its partial sequence similarity to proteins associated with three different biological processes namely vesicle-mediated protein transport ( S e c 1 8 and ~ ~ ~NSF49),control of the cell cycle ( C d ~ 4 8 p , ~VCP,5’ ’ and p97-ATPaseS2) and expression of human immunodeficiency virus (TBP-1).53These proteins share a highly conserved domain of about 185 amino acids (Pasl-box) including a consensus sequence for ATP binding. This finding has led to the suggestion of a novel family of putative A T p a ~ e s . ~ ~ PAS2 PAS2p has been characterized as a low abundance protein of 21 k D with striking partial similarity to the family of ubiquitin-conjugating Ubiquitin-conjugating proteins (UBC) are part of the ubiquitin-conjugation ~ a s c a d e ~ ’which ~ ’ ~ transfers ubiquitin to protein substrates. One well-known function of the ubiquitin pathway is to mark proteins for degradation. To
date, the steadily expanding UBC family of S. cerevisiae comprises 10 members including PAS2p (UBC 10). Some of these fungal UBC are involved in fundamental cellular proce~ses,~’ e.g. the cell cycle. However, it is unclear how these complex functions are related to the ubiquitination signal. The connection between the ubiquitin pathway and peroxisome biogenesis raises the question whether protein degradation is involved in the assembly of this organelle. An attractive possibility is that PAS2p could catalyse, via ubiquitination, the degradative elimination of a factor inhibiting an essential step of peroxisome biogenesis. PAS3 PAS3p was identified as a 48 k D peroxisomal integral membrane protein, which is anchored in the membrane by its amino-terminus with the bulk of the protein exposed to the cytosol.2’ This coincides with the properties expected of a receptor protein of the peroxisomal membrane. The topology of the protein within the membrane suggested that the information for peroxisoma1 targeting resides in the N-terminus of the protein. This possibility was confirmed recently by the observation that the amino-terminal half of PAS3p contains all the information necessary to target a reporter protein to peroxisomes.’8 These results indicate the existence of a previously unrecognized N-terminal peroxisomal targeting signal in PAS3p. PAS4 The predicted PAS4p contains at its C-terminal part a cystein-rich region38 not matching the canonical zinc-fingers, but a recently reported zincfinger-like motif found in eight proteins, most of which have been proposed to interact with DNA because of their putative f ~ n c t i o n . ’In ~ this context it is interesting that a comparison of the morphology of the PAS4 null mutant and transformants expressing PAS4p from a multicopy vector suggests that the protein is specifically required for peroxisome proliferation and not for peroxisome biogenesis per se. The null mutant possesses a few small peroxisomes, and overexpression of PAS4p leads to a surprisingly large number of peroxisomes. Remarkably, the same zinc-finger-like motif is found in P A S ~ and P ~ PAF1.60 ~ The latter protein has been convincingly shown to be a peroxisomal
172
R. ERDMANN AND W-H. KUNAU
integral membrane protein of rat liver. These results seem to suggest that this motif is involved in protein-protein rather than protein-DNA interaction.
address experimentally a number of questions concerning the mechanisms of peroxisome biogenesis. The identification, intracellular localization, and characterization of the P A S gene products are prerequisites for detailed insights into the specific roles of these proteins and into the order of events COMPARISON OF YEAST AND HUMAN of the complex process of peroxisome biogenesis. PEROXISOMAL MUTANTS An intriguing question concerns the applicability Biochemical and morphological properties of the of the results obtained in S. cerevisiae to higher peroxisomal yeast mutants are very similar to those eukaryotes. Future studies will show whether, for of fibroblasts from patients affected by peroxisomal each yeast gene essential for peroxisome biogenesis, 2). For each of the three counterparts will be found in higher eukaryotes d i ~ e a s e s ’ ~ -(Table ~~ complementation groups of fox mutants there are and whether they can replace each other functionfibroblast cell lines from patients deficient for the ally. same p-oxidation activity. Moreover, the large Because of the ease with which a whole organelle number and different phenotypes of pas mutants with its large number of constituent proteins can be correspond to the situation found in cells of induced rapidly and repressed under defined condipatients with presumed defects in peroxisome tions, peroxisomes are an attractive model for the assembly. regulation of gene expression. The first studies related to this aspect have been (see also review by Einerhand et al.). CONCZUSIONS AND PERSPECTIVES Biogenesis of peroxisomes is a relatively new S. cerevisiae has proven to be a convenient organ- topic in the general field of intracellular protein ism for studying peroxisome function and biogene- transport. However, despite the late start, the prosis. The isolation of fox- and pas-mutants has mising results of the genetic approach in s. cerevopened the way for the identification of the corre- isiae and other yeasts make peroxisomes a valuable sponding wild-type genes by functional comple- model system, in addition to the well-established mentation. other subcellular compartments, to investigate proTo date, there are at least 18 known complemen- tein sorting in general and protein translocation tation groups of pas mutants. The sequences of 10 across membranes in particular. of the corresponding wild-type alleles have been determined and in some cases they give a clue to the possible function of the predicted gene product. ACKNOWLEDGEMENTS The PAS and FOX genes are important tools to We are grateful to members of our laboratory and Danny Schnell (Rockefeller University, New York) Table 2. Comparison of yeast pus mutants and peroxisomal for fruitful discussions and thank Henk Tabak and coworkers for communicating results before publidiseases cation. The original research described in this reYeast Peroxisomal view was supported in part by the Deutsche Deficiency mutant disease Forschungsgemeinschaft (grants Ku 329/11-3 and 11-4). R.E. is a recipient of a stipend from the Acyl-CoA for 1 Pseudo-neonatal Alexander von Humboldt-Stiftung. oxidase adrenoleucodystrophy Bifunctional enzyme
fox 2
pseudo-neonatal adrenoleucodystrophy
3-oxoacylCoA thiolase
.fox 3
Pseudo-Zellweger syndrome
Peroxisomes
pas 1-18
Catalase
cta
I
Zellweger syndrome Neonatal adrenoleucodystrophy Infantile Refsum syndrome (13 complementation groups) Acatalasaemia
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GENETICS OF PEROXISOMAL BIOGENESIS
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174 37. Borst, P. (1989). Peroxisome biogenesis revisited. Biochim. Biophys. Acta, 1008, 1-13. 38. Mertens, D. and Kunau, W-H. Unpublished. 39. Skaletz, A. and Kunau, W-H. Unpublished. 40. Marzioch, M. and Kunau, W-H. Unpublished. 41. Could, S. J., Keller, G. A,, Schneider, M., Howell, S. H., Garrard, L. J., Goodman, J. M., Distel, B., Tabak, H. F. and Subramani, S. (1990). Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J., 9, 85-90. 42. Miyazawa, S., Osumi, T., Hashimoto, T., Ohno, K., Miura, S. and Fujiki. Y. (1989). Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. Mol. Cell. Biol., 9, 83-91. 43. Swinkels, B. W., Could, S. J., Bodnar, A. G., Rachubinski, R. A. and Subramani, S. (1991). A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3ketoacyl-CoA thiolase. EMBO J., 10, 3255- 3262. 44. Osumi, T., Tsukamoto, T., Hata, S., Yokota, S., Miura, S., Fujiki, Y., Hijikata, M., Miyazawa, S. and Hashimoto, T. (1991). Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem. Biophys. Res. Commun., 181, 947-954. 45. Gietl. C. (1990). Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide. Proc. Nail. Acad. Sci. U.S.A.. 87, 5773-5777. 46. Struhl, K. (1983). The new yeast genetics. Nature, 305, 391-397. 47. Erdmann. R., Wibel, F. F., Flessau, A,, Rytka, J., Beyer, A., Frohlich, K. U. and Kunau, W-H. (1991). PASI, a yeast gene required for peroxisome biogenesis, encodes a member of a novel family of putative APTases, Cell, 64, 499- 510. 48. Eakle, K. A,, Bernstein, M. and Emr, S. D. (1988). Characterization o f a component of the yeast secretion machinery: identification of the SECl8 gene product. Mol. Cell. Biol., 8,4098-4109. 49. Wilson, D. W., Wilcox, C. A,, Flynn, G . C., Ellson, C., Kuang, W. J., Henzel, W. J., Block, M. R., Ullrich, A. and Rothman, J. E. (1989). A fusion protein required for vesicle-mediated transport in mammalian cells and yeast. Nu ture, 339, 35 5-359. 50. Frohlich, K. U., Fries, H. W., Riidiger, M.. Erdmann, R., Bothstein, D. and Mecke, D. (1991). Yeast cell cycle protein Cdc48p shows full-length homology to the mammalian protein. VCP and is a member of a protein family
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involved in secretion, peroxisome formation and gene expression. J . Cell. Biol., 114, 443-453. Koller, K. J. and Brownstein, M. J. (1987). Use of cDNA clone to identify a supposed precursor protein containing valosin. Nature, 325, 542-545. Peters, J. M., Walsh, M. J. and Franke, W. W. (1990). An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Secllp and NSF. E M B O J . , 9, 1757-1767. Nelbock, P., Dillon, P. J., Perkins, A., and Rosen, C. A. (1990). A cDNA for a protein that interacts with the human deficiency virus tat transactivator. Science, 145, 1650- 1653. Jentsch, S., Seufert, W., Sommer, T. and Reins, H. A. (1990). Ubiquitin-conjugating enzymes: novel regulators of eucaryotic cells. Trends Biochern. Sci., 15, 195-198. Jentsch, S., Seufert, W. and Hauser, H. P. (1991). Genetic analysis of the ubiquitin system. Biochirn. Biophys. Acta, 1089, 127-139. Wiebel, F. F. and Kunau, W.-H. (1992) PAS2, a yeast gene essential for peroxisome biogenesis is a putative ubiquitinconjugating enzyme. Nature, submitted. Finley, D., Chau, V. (1991). Ubiquitination. Ann. Reo. Cell Biol.,7, 25-69. Hohfeld, J. and Kunau, W-H. Unpublished. Freemont, P. S., Hanson, I. M. and Trowsdale, J. (1991). A novel cysteine-rich sequence motif. Cell, 64, 483-484. Tsukamoto, T., Satoshi, M. and Fujiki, Y. (1991). Restoration by a 35K membrane protein of peroxisome assembly in a mammalian cell mutant. Nature, 350, 77-81. Einerhand, A. W. C., Voorn-Brouwer, T. M., Erdmann, R., Kunau, W-H. and Tabak, H. F. (1991). Regulation of transcription of the gene coding for peroxisomal 3oxoacyl-CoA thiolase of Saccharornyces cereuisiae. Eur. J . Biochem., 200, 113-122. Simon, M., Adam, G., Rapatz, W., Spevak, W. and Ruis, H. (1991). The Saccharomyces cereaisiae ADRI gene is a positive regulator of transcription of genes encoding peroxisomal proteins. Mol. Cell. B i d , 11, 699-704. Sloots, J. A., Aitchison, J. D. and Rachubinski, R. A. (1991). Glucose-responsive and oleic acid-responsive elements in the gene encoding the peroxisomal trifunctional enzyme of Candida tropicalis. Gene, 105, 129-1 34. Simon, M., Binder, M., Adam, G., Hartig, A. and Ruis, H. (1992). Control of peroxisome proliferation in Saccharomvces cereiiyiae bv ADR1. S N F l (CATI. CCRII. and SNF4 (CAT3). Yea:st, 8, in press
10: 167-174 (1992)
A Genetic Approach to the Biogenesis of Peroxisomes in the Yeast Saccharomyces cerevisiae RALF ERDMANNt AND WOLF-H. KUNAU Instituf .fur Pliysiologische Chemie, Medizinische Fakultat der Ruhr- Universitat Bochum, 0-4630 Bochum, Federal Ropuhlir~of' Germany
INTRODUCTION
structure of protein components involved in peroxisome biogenesis in S. cerevisiae.
The budding yeast Succhurom,vces cerevisiue offers the advantages of combined genetic, molecular genetic and biochemical approaches to study cell PEROXISOMAL MUTANTS biological processes. S. cerevisiae mutants have been powerful tools to dissect the biogenesis of Like other yeasts, S. cerevisiue does not contain a subcellular structures, such as mitochondria,'.2 en- mitochondria1 P-oxidation system.' Consequentdoplasmic r e t i ~ u l u m ,g~~. ~l g ivacuoles,6 ,~ and most ly, for the utilization of fatty acids as carbon source, S. cerevisiue depends on a functional peroxisomal recently p e r o x i ~ o m e s . ~ - ' ~ /I-oxidation system. As a first step in identifying The presence of peroxisomes in S. cerevisiue was peroxisomal mutants, cells that were able to grow first demonstrated by Avers and Federman,' but on non-fermentable carbon sources such as glyin contrast to other yeasts and filamentous cerol and ethanol, but unable to grow on oleic acid fungi,' 2 . ' ' peroxisomes of S. cerevisiue have not as single carbon source were isolated (Figure 1). been characterized in great detail. This lack of This screening step defined oleic acid non-utilizing information is explained by the observation that under the growth conditions originally chosen, strains (onu-mutants) and therefore enriched for peroxisomes in S. cerevisiue are very rare and mutants affected in the degradation of fatty acids7 ~ r n a 1 l . lIn ~ several yeasts, proliferation of this or- The lack of a functional P-oxidation system in the ganelle can be induced by growth on distinct car- onu-mutants could be due to defects in individual bon and nitrogen sources1' but attempts to induce fi-oxidation enzyme activities or to defects in perthe proliferation of peroxisomes in S. cerevisiue oxisome biogenesis which in turn would prevent failed for a long time. It was not until Veenhuis et the proper assembly of a functional P-oxidation d.'" and Kunau et al.' demonstrated the inducibi- complex inside peroxisomes. By ultrastructural lity of peroxisomal P-oxidation as well as perox- analysis some of the onu-mutants were shown to be isome proliferation in yeasts by growth on oleic peroxisome-deficient. In order to identify these acid as the single carbon source that remarkable mutants by biochemical means, fi-oxidation enamounts of peroxisomes were also found in S. zyme activities as well as catalase activity were ccw~ci.siur~.'4.1This finding opened the way for the determined in whole cell extracts and subcellular development of a genetic screen to isolate peroxiso- fractions of onu-mutants. This screening step allowed the detection of mutants with individual ma1 mutants of S. ~erevisiue.~ ,fitty acid oxidation enzyme deficiencies In this report we review the first attempts to (fox-mutants)" as well as mutants with peroxisocharacterize peroxisome biogenesis in S. cerevisiue ma1 matrix enzymes mislocalized to the cytosol by a combined genetic and biochemical approach. (peroxisome assemblyor p ~ s - m u t a n t s )The . ~ misWe describe the different types of peroxisomal mutants and our current knowledge of the primary localization of peroxisomal matrix enzymes to the cytosol indicated a defect in peroxisome biogenesis. Used in this broad sense, peroxisome biogenesis t Present address: Laboratory of Cell Biology, Rockefeller Uni- includes all reactions required for import of peroxisomal proteins from the cytosol, reactions which versity, New York, New York 10021-6399, U.S.A.
'
0263 6484/92,'030167 08$09.00 \( 1992 by John Wiley & Sons, Ltd.
168
R. ERDMANN AND W-H. KUNAU
SCREENING PROTOCOL FOR PEROXISOMAL MUTANTS mutagenesis
++I1
COLONY SCREENING
screening for inability to
grow onoleote
-01e a te - 5 on - _uti Ii z i n g
strains
(zstra i nsl BIOCHEMICAL SCREEN I N G
mislocal i za t i o n of perox i s omal enzymes (25,00O*g)
deficiency o f a p-oxidation enzyme i
electron microscopy
+r
e l
MORPHOLOGICAL SCREENING
fox m u t a n t s
t y p e I- 111
Figure 1. Screening protocol for peroxisomal mutants
oxidase was cloned accidentally and identified by its homology to the acyl-CoA oxidase genes of C. tropicalis. The fox2 mutant showed concomitant loss of three P-oxidation enzyme activities, indicating the presence of a trifunctional fl-oxidation enzyme in S. cerevisiue. FOX2 has been cloned by functional complementation and the multidomain structure of the corresponding gene product is reflected in its primary sequence.I8 Thefox3 mutants are affected in 3-oxoacyl-CoA thiolase. As judged by sequence comparison, FOX3, which recently has been cloned by functional complementaFox MUTANTS ARE AFFECTED IN tion, indeed encodes yeast peroxisomal thiolase.” PEROXISOMAL ,!I-OXIDATION The thiolase deficiency and the onu-phenotype of The fox mutants fall into three complementation fox3 mutants indicate that unlike the situation in only one thiolase involved in groups. This result is consistent with biochemical mammalian results, indicating that peroxisomal fl-oxidation in peroxisomal 8-oxidation exists in S. cerevisiue. Other yeast mutants with defects in peroxisomal S. cerevisiue is performed by three gene product^.'^ All three corresponding genes have been cloned. enzymes have been isolated by different screening The FOXI or POXI” gene, encoding the yeast procedures and the corresponding wild-type genes peroxisomal P-oxidation entry enzyme acyl-CoA have been cloned by functional complementation: control peroxisome proliferation and those which provide the lipids for peroxisomal membranes. Further characterization of peroxisomal mutants was achieved by electronmicroscopical and immunocytochemical investigations. Recently, this screening protocol has been improved by introducing a positive selection step.” This resulted in the isolation of a number of additional pas mutants, among which five new complementation groups could be defined.
GENETICS OF PEROXISOMAL BIOGENESIS
CTAI,” encoding catalase A; A L ~ D H ~encoding ,’~ malate dehydrogenase 2 ; and CIT2,23 encoding citrate synthase 2. Fox mutants do contain peroxisomes which, apart from the deficiency of the corresponding b-oxidation enzyme, exhibit wildtype properties. Therefore, yeast mutants deficient in endogenous peroxisomal proteins might represent suitable systems to study homologous and heterologous expression and in uiuo import of peroxisomal proteins. For example, by taking advantage of a thiolase deficient fox3 mutant, very recently the peroxisomal targeting signal of yeast 3oxoacyl-CoA thiolase has been defined.24
PUS-MUTANTS ARE AFFECTED IN PEROXISOME ASSEMBLY Originally, pas mutants were defined by the (i) inability to grow on oleic acid (onu-phenotype), (ii) mislocalization of peroxisomal matrix enzymes to the cytosol and (iii) absence of detectable peroxisomes at the ultrastructural leveL7 However, detailed analysis of newly identified mutants revealed that mislocalization of peroxisomal proteins is not always due to the absence of peroxisomes. All the pas mutants so far characterized show the onuphenotype and the cytosolic mislocalization of one or all of the peroxisomal matrix enzymes tested. Based on these criteria three different subgroups of pas mutants, designated type I to I11 have been distinguished.
Type I pus Mutants are AfSected in Peroxisome Formation Type I pus mutants (pasl, pus2, pus3, p a d ) are characterized by the absence of morphologically detectable peroxisomes. The lack of peroxisomes was demonstrated electronmicroscopically using serial sections and cytochemical staining for catalase activity. Subcellular fractionation studies and immunogold labelling confirmed mislocalization of peroxisomal matrix enzymes to the ~ y t o s o l . ’ , ~ ~ Pus mutants of type I resemble the phenotype of fibroblasts from Zellweger patients, suffering from a human peroxisomal and peroxisome-deficient mutants of CHO-cell line^.^'-^^ This raises the question of whether peroxisomal membrane structures, called ‘ghosts’, that have been detected in both peroxisome-deficient cell types33-35 are also present in type I pus mutants.
169 The availability of antibodies against the peroxisoma1 integral membrane protein P A S ~ Pshould ~ ~ now open the possibility of detecting peroxisomal ghosts in pas mutants by a combined biochemical and immunocytochemical approach. Another intriguing question is whether or not these ghosts actually are precursors (pre-peroxisomes) of functionally and morphologically mature peroxisomes. In s. cerevisiae different lines of results strongly suggest the existence of such prestructures. Accumulated evidence indicates that peroxisomes are not formed de novo (for reviews see references 36 and 37). Complementation analysis demonstrated that crossing of two haploid type I pus mutants resulted in diploid cells with peroxisomes, Moreover, even transformations of type I pus mutants with plasmids carrying in their inserts single genes rescued the pus phenotype. This approach has been successfully used to clone the respective PAS genes. Thus, presuming that peroxisomes cannot be formed de nouo, these results imply the existence of peroxisomal remnants functioning as a starting point for peroxisome biogenesis. Because this argues for peroxisomal prestructures in complementable type I pus mutant, the absence of morphologically detectable peroxisomes may be due to defects directly or indirectly affecting the import of peroxisomal matrix proteins. If prestructures exist and are the prerequisite (conditio sine qua non) for functional complementation one can envisage defects resulting in their absence. Due to the lack of an initiative point for peroxisome formation, none of those pus mutants should be complementable by transformation. Such mutants have not yet been identified. Type II pus Mutants are Affected in Peroxisome Proliferation Type I1 pus mutants (pad, pus6) differ significantly from type I mutants by the presence of morphologically detectable peroxisomes. However, the number and size of peroxisomes of induced type I1 mutant cells are drastically decreased in comparison to wild-type ~ e l l s . ~The * , ~phenotype ~ of oleic acid-grown type I1 mutant cells resembles a glucose-repressed state rather than an induced state of wild-type cells. In type I1 mutants, peroxisomal matrix enzymes are present in wild-type amounts but, consistent with the pas phenotype, they are mainly mislocalized to the cytoplasm. Nevertheless, a small portion of peroxisomal matrix proteins is
170 correctly localized, indicating that peroxisomal import can occur. Therefore, mislocalization of peroxisomal proteins in these mutants is most likely to be due to the deficiency of sufficient host organelles for their uptake. These results and the observation that oleic acid-induced peroxisome proliferation is controlled by the level of PAS4p expression (see below) suggest that type I1 pas mutants are affected in peroxisome proliferation. A number of pas mutants with a similar phenotype have been isolated by Tabak and coworkers and are described in more detail in their review."
Type III pas Mutants are Characterized by the Misloculization of Individual Peroxisomal Proteins to the Cytoplasm Pas7, the only known mutant of type 111, shows mislocalization of 3-oxoacyl-CoA thiolase to the c y t ~ s o l .All ~ ~ other peroxisomal proteins tested were found within the organelle. Genetic and molecular biological results demonstrated that thiolase mislocalization in pas7 is not due to a defect in the thiolase itself. Therefore, an as yet undefined protein, essential for thiolase import into peroxisomes seems to be affected in the pas7 mutant. As FOX2p," carrying the known C-terminal peroxisomal targeting signal SKL*4 1 . 4 2 appears to be imported normally in the pas7 mutant, the occurrence of this mutant gives the first evidence for a SKL-independent peroxisomal import pathway. This conclusion is supported by recent findings, indicating that the targeting signals of peroxisomal thiolase of rat liver43.44 and S. cereai.siac~~~ resides at the amino terminus of the proteins. An amino terminal targeting signal has also been suggested for the peroxisomal malate dehydrogenase of the ~ a t e r m e l o n Also . ~ ~ the peroxisomal integral membrane protein PAS3p of S. cerevisiae appears to have an amino-terminal targeting signal.2s However, it is imported into peroxisomal membranes of p a ~ 7 , ~ ' suggesting the presence of a second SKL-independent peroxisoma1 import pathway in S. cereitsiae. Whether these import pathways converge or whether they are entirely distinct cannot yet be answered. However, the PAS7 gene will define the first component specific for thiolase import.
*Single letter code for Ser-Lys-Leu.
R. ERDMANN AND W-H. KUNAU
The existence of two import pathways for peroxisomal matrix enzymes, the thiolase and the SKLdependent pathway, raises the question whether mutants affected in the latter one would also reflect a type 111 pas phenotype as found in the thiolase import mutant pas7. Recent evidence suggests that this will not be the case. The predicted PAS6p contains a C-terminal SKL,39 suggesting that the protein performs its function in the peroxisomal matrix. The corresponding pas6 mutant shows the type I1 phenotype indicating a defect in peroxisome proliferation. Therefore, mislocalization of PAS6p as the consequence of an affected SKL-pathway would be expected to result in the type I1 phenotype. One can also envisage that so far unrecognized SKL-containing proteins exist whose mislocalization would result in the pas phenotype I. Consequently, mutants affected in components that are essential for peroxisomal import of SKL-containing proteins would be expected to resemble the pas phenotype I or 11. The same conclusions can be drawn for a defect in the pathway for PAS3p import. As PAS3p is essential for peroxisome formation,25 its mislocalization should result in peroxisome-deficiency, giving rise to a type I pas mutant.
COMPONENTS ESSENTIAL FOR PEROXISOME BIOGENESIS Until now 18 different pas complementation groups have been defined among the pas mutants found in our laboratory' and found by Van der Leij et al." (Table 1). As the first step to characterize the P A S gene products, wild-type genes corresponding to affected genetic loci of pas mutants have been cloned by functional c ~ m p l e m e n t a t i o n For . ~ ~ this purpose, advantage was taken from the fact that peroxisomal mutants, when transformed with the corresponding wild-type gene, regained the ability to grow on the oleic acid m e d i ~ m . ~ By ~ . ~this ' approach PAS1 to PAS7, PAS9, PAS11 and PAS12 have been cloned. Authenticity of the genes was confirmed by gene disruption and genetic analysis of the resulting null mutants. Gene disruptions also revealed that none of the cloned P A S genes is essential, therefore reflecting that peroxisomes, at least under certain growth conditions, are not required for cell viability. In order to identify PAS proteins in yeast, antisera have been raised so far against putative PAS1 to PAS3 gene products.
171
GENETICS OF PEROXISOMAL BIOGENESlS
Table 1 , Complementation groups of Surchuromyces cerevisiue mutants deficient in peroxisome assembly. Corresponding wild-type gene Cloned Sequenced
pus mutant ~
~
pus I * pu.P
/IU.S3*,t
pu.s4*,+ pu.s5*. i. pus6*,t
pu.s7*. t pus8*, + pu.sY* ptr.v/O*.i pu.s I I *
pu.s12* p“.’13* pus 14t pus /St pu.v 1 6 t pu” 17t
~~
+ + + + + + + + + + + + f
+ +
+ + + + + + f f
+ +
pus /(it
tlsolated in Amsterdam (see reference 10). *Isolated in Bochum (see also references 8 and 9).
PAS 1 PASlp has been identified as a rather hydrophilic low abundance protein of 117 kD that contains two putative ATP binding sites.47 A striking feature of PASlp is its partial sequence similarity to proteins associated with three different biological processes namely vesicle-mediated protein transport ( S e c 1 8 and ~ ~ ~NSF49),control of the cell cycle ( C d ~ 4 8 p , ~VCP,5’ ’ and p97-ATPaseS2) and expression of human immunodeficiency virus (TBP-1).53These proteins share a highly conserved domain of about 185 amino acids (Pasl-box) including a consensus sequence for ATP binding. This finding has led to the suggestion of a novel family of putative A T p a ~ e s . ~ ~ PAS2 PAS2p has been characterized as a low abundance protein of 21 k D with striking partial similarity to the family of ubiquitin-conjugating Ubiquitin-conjugating proteins (UBC) are part of the ubiquitin-conjugation ~ a s c a d e ~ ’which ~ ’ ~ transfers ubiquitin to protein substrates. One well-known function of the ubiquitin pathway is to mark proteins for degradation. To
date, the steadily expanding UBC family of S. cerevisiae comprises 10 members including PAS2p (UBC 10). Some of these fungal UBC are involved in fundamental cellular proce~ses,~’ e.g. the cell cycle. However, it is unclear how these complex functions are related to the ubiquitination signal. The connection between the ubiquitin pathway and peroxisome biogenesis raises the question whether protein degradation is involved in the assembly of this organelle. An attractive possibility is that PAS2p could catalyse, via ubiquitination, the degradative elimination of a factor inhibiting an essential step of peroxisome biogenesis. PAS3 PAS3p was identified as a 48 k D peroxisomal integral membrane protein, which is anchored in the membrane by its amino-terminus with the bulk of the protein exposed to the cytosol.2’ This coincides with the properties expected of a receptor protein of the peroxisomal membrane. The topology of the protein within the membrane suggested that the information for peroxisoma1 targeting resides in the N-terminus of the protein. This possibility was confirmed recently by the observation that the amino-terminal half of PAS3p contains all the information necessary to target a reporter protein to peroxisomes.’8 These results indicate the existence of a previously unrecognized N-terminal peroxisomal targeting signal in PAS3p. PAS4 The predicted PAS4p contains at its C-terminal part a cystein-rich region38 not matching the canonical zinc-fingers, but a recently reported zincfinger-like motif found in eight proteins, most of which have been proposed to interact with DNA because of their putative f ~ n c t i o n . ’In ~ this context it is interesting that a comparison of the morphology of the PAS4 null mutant and transformants expressing PAS4p from a multicopy vector suggests that the protein is specifically required for peroxisome proliferation and not for peroxisome biogenesis per se. The null mutant possesses a few small peroxisomes, and overexpression of PAS4p leads to a surprisingly large number of peroxisomes. Remarkably, the same zinc-finger-like motif is found in P A S ~ and P ~ PAF1.60 ~ The latter protein has been convincingly shown to be a peroxisomal
172
R. ERDMANN AND W-H. KUNAU
integral membrane protein of rat liver. These results seem to suggest that this motif is involved in protein-protein rather than protein-DNA interaction.
address experimentally a number of questions concerning the mechanisms of peroxisome biogenesis. The identification, intracellular localization, and characterization of the P A S gene products are prerequisites for detailed insights into the specific roles of these proteins and into the order of events COMPARISON OF YEAST AND HUMAN of the complex process of peroxisome biogenesis. PEROXISOMAL MUTANTS An intriguing question concerns the applicability Biochemical and morphological properties of the of the results obtained in S. cerevisiae to higher peroxisomal yeast mutants are very similar to those eukaryotes. Future studies will show whether, for of fibroblasts from patients affected by peroxisomal each yeast gene essential for peroxisome biogenesis, 2). For each of the three counterparts will be found in higher eukaryotes d i ~ e a s e s ’ ~ -(Table ~~ complementation groups of fox mutants there are and whether they can replace each other functionfibroblast cell lines from patients deficient for the ally. same p-oxidation activity. Moreover, the large Because of the ease with which a whole organelle number and different phenotypes of pas mutants with its large number of constituent proteins can be correspond to the situation found in cells of induced rapidly and repressed under defined condipatients with presumed defects in peroxisome tions, peroxisomes are an attractive model for the assembly. regulation of gene expression. The first studies related to this aspect have been (see also review by Einerhand et al.). CONCZUSIONS AND PERSPECTIVES Biogenesis of peroxisomes is a relatively new S. cerevisiae has proven to be a convenient organ- topic in the general field of intracellular protein ism for studying peroxisome function and biogene- transport. However, despite the late start, the prosis. The isolation of fox- and pas-mutants has mising results of the genetic approach in s. cerevopened the way for the identification of the corre- isiae and other yeasts make peroxisomes a valuable sponding wild-type genes by functional comple- model system, in addition to the well-established mentation. other subcellular compartments, to investigate proTo date, there are at least 18 known complemen- tein sorting in general and protein translocation tation groups of pas mutants. The sequences of 10 across membranes in particular. of the corresponding wild-type alleles have been determined and in some cases they give a clue to the possible function of the predicted gene product. ACKNOWLEDGEMENTS The PAS and FOX genes are important tools to We are grateful to members of our laboratory and Danny Schnell (Rockefeller University, New York) Table 2. Comparison of yeast pus mutants and peroxisomal for fruitful discussions and thank Henk Tabak and coworkers for communicating results before publidiseases cation. The original research described in this reYeast Peroxisomal view was supported in part by the Deutsche Deficiency mutant disease Forschungsgemeinschaft (grants Ku 329/11-3 and 11-4). R.E. is a recipient of a stipend from the Acyl-CoA for 1 Pseudo-neonatal Alexander von Humboldt-Stiftung. oxidase adrenoleucodystrophy Bifunctional enzyme
fox 2
pseudo-neonatal adrenoleucodystrophy
3-oxoacylCoA thiolase
.fox 3
Pseudo-Zellweger syndrome
Peroxisomes
pas 1-18
Catalase
cta
I
Zellweger syndrome Neonatal adrenoleucodystrophy Infantile Refsum syndrome (13 complementation groups) Acatalasaemia
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