Peroxisome Biogenesis in the Yeast Hansenula polymorpha: A Structural and Functional Analysis

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Peroxisome Biogenesis in the Yeast Hansenula polymorpha:

A Structural and Functional Analysis

IDA J. VAN DER KLEI AND MARTEN VEENHUIS

Department of Microbiology Groningen Biomolecular Sciences and Biotechnology Institute

University of Groningen Biological Centre

Kerklaan 30, 9751" Haren, the Netherlands

INTRODUCTION

Our knowledge of the molecular mechanisms of microbody biogenesis and function in yeasts is rapidly expanding, in particular following the isolation of various peroxisome-deficient (pel; pas, pay, peb) mutants. 1-5 Topics of current research inter- est include regulation of microbody maintenance and proliferation, sorting, folding and assembly of matrix proteins, synthesis and function of the microbody membrane, and microbody turnover. During the last decade, Hansenula polymorpha has emerged as an attractive model organism for these studies, because it is readily acces- sible by classical and molecular genetical techniques and has been extensively studied with respect to the physiologyhiochemistry and the ultrastructure of the organism.6 H. polymorphaper mutants have been valuable not only for the cloning of various PER genes but also allowed unique in vivo studies designed to elucidate the ad- vantage for the cell to compartmentalize certain metabolic pathways in peroxi~omes.~ Such studies are possible because the complete absence of peroxisomes does not affect the viability and growth of the organism in complex media. Hence, the specific role of peroxisomes can be studied under optimal physiological conditions as they are created in a glucose-limited chemostat to which a microbody- inducing compound is added (e.g., methanol, amines, purines and D-amino acids), thus combining maximal induction of microbody enzymes with controlled growth.

In this paper we summarize some important aspects of the biogenesis, function and turnover of peroxisomes in H. polymorpha.

INDUCTION OF PEROXISOMES IN H. POLYMORPHA

In yeasts, the induction and metabolic significance of microbodies (peroxisomes) is largely prescribed by environmental stimuli (e.g., growth conditions). In H. polymorpha, maximal peroxisome induction is obtained during growth of cells in a methanol-limited chemostat. Under these conditions the organelles may take up to 80 percent of the cytoplasmic volume; other substrates known to induce microbodies in

47

48 ANNALS NEW YORK ACADEMY OF SCIENCES

this organism are ethanol, primary amines, D-amino acids, and purines. A character- istic feature of these organelles is that they harbor the key enzymes involved in the metabolism of the above carbodnitrogen sources. Based on our present knowledge we are able to precisely adjust both the level of microbody induction as well as their protein composition by specific manipulations in growth conditions (TABLE 1).6

Upon induction, peroxisomes in H. polymorpha are believed to develop from al- ready existing organelles by growth and subsequent division. Cells from batch cul- tuxes in the exponential growth phase on glucose generally contain a single, small microbody which is maintained by a process of fission, generally taking place in the vicinity of the neck between mother cell and bud, followed by migration of one of the resulting organelles into the developing bud. Recently, we have identified a peroxiso- ma1 membrane protein of H. polyrnorpha (Per8p) which plays a key role in the multiplication of peroxisomes after their induction.8 Furthermore, the functional homo- logue of this “proliferation factor” from 5‘. cerevisiae (Pas4p) is known.g Detailed studies on per8 and pas4 deletion mutants indicated that the mechanisms of microbody multiplication after induction (proliferation, mediated by Per8p, Pas4p) and maintenance of the small organelle in fully repressed (glucose-grown) cells, which is independent of these proteins, are on that account separate processes. Probably, the mechanisms which control the propagation of peroxisomes in repressed cells are comparable to those involved in propagation of other cell organelles, which are considered to be cell cycle dependent.

The physiological role of the organelles in glucose-grown cells is not yet known. However, they may not be hlly redundant under these conditions, since growth of peroxisome-deficient mutants on the same media is slightly retarded compared to

TABLE 1. Peroxisome-inducing Growth Substrates, Corresponding Peroxisomal En- zymes, and Quantitation of Peroxisomal Volume Fraction in Hansenula polymorpha

Peroxisomal Carbon Source Nitrogen Source Major Enzymes Volume Fraction“

Glucose Ammonium sulfate Catalase

Ethanol Ammonium sulfate Isocitrate lyase

Methanol Ammonium sulfate Alcohol oxidase

D-Amino acid oxidase 0.1

Malate synthase 1 .o

Dihydroxyacetone synthase Catalase 19.9

48.4b Glucose Methylamine Amine oxidase

Glucose D- Alanine D-Amino acid oxidase

Glucose Urate Urate oxidase

“The data are collected from late exponential batch cultures unless indicated otherwise. The volume fraction is expressed as percentage of the cytoplasmic volume.

bContinuous culture, d = 0.03 h-I.

Catalase 2.3

Catalase 1.6

Catalase 0.6

VAN DER KLEI & VEENHUIS: PEROXISOME BIOGENESIS IN H. pobmorpha 49

wild type (WT) cells. On the other hand, there is no doubt that they serve as the initial target organelle (“mother organelle”) for newly synthesized matrix proteins induced after a shift of such cells to a peroxisome-inducing medium (FIG. 1).

In addition to these, alternative modes of peroxisome biogenesis may exist. Evi- dence for this came from an analysis of one of our Ts mutants ( p e ~ I 3 ~ @ ) , which allow one to manipulate the (re)appearance of peroxisomes by changing the temperature of cultivation.1° At permissive temperatures (5 37 “C), these mutants show WT properties in that they normally grow on methanol and contain intact peroxisomes. Howev- er, at the restrictive temperature (43 “C) they display a Per- phenotype and peroxisomes are filly lacking. In ~ e r - 1 3 ~ ~ ~ grown at 43 “C, peroxisomal “ghosts” or mem- branous remnants (for details see the section entitled “Peroxisomal remnants in PER deletion strains” below) were not detectable. After a shift of cells from the restrictive to the permissive temperature, peroxisomal profiles were first detected after ap- proximately 30 minutes. These newly formed organelles were small and comparable to peroxisomes in glucose-grown cells. Typically, only one organelle, or occasionally a few organelles, developed per cell. During firther cultivation these organelles grew and multiplied (as detailed above for a shift of WT cells from glucose to methanol:

protein import \

‘- ‘._-

degradation t glucose/ environmental

ammonium sulphate change

FIGURE 1. Schematic representation of peroxisome biogenesis and turnover in the yeast Hansenula polymorpha. Upon transfer of glucose-grown cells to methanol-containing media, peroxisomal matrix proteins are strongly induced. Protein import takes place into the small organelle, present in the glucose-grown cell, which increases in size and, when its mature size is reached, buds off a new small organelle. The new organelle is now the target for newly synthesized matrix protein. After a shift of methanol-grown cells to glucose- or ethanol-containing media, peroxisomes are selectively degraded by means of an autophagic process. At least one small organelle remains per cell.


see FIGURE 1). Unexpectedly, neither the matrix nor the membrane proteins, present in the cytosol prior to the shift of cells, are essential for peroxisome reassembly; invariably, only newly formed matrix proteins became incorporated in these new organelles.l0

In the same mutant, peroxisome assembly was also restored at restrictive temperatures by replacing ammonium sulphate by D-alanine as the sole nitrogen source.'* This effect appeared to be specific for o-alanine; in the presence of all other peroxisome-inducing substrates tested the Per- phenotype was maintained. We consider this an important observation because it demonstrates that the reassembly of peroxisomes in per mutants may be also dependent on cultivation conditions. On the mechanisms of this phenomenon we can only speculate, but these may reflect the possibility that in H. polymorpha alternative pathways for peroxisome biogenesis exist, either operative or induced by specific environmental stimuli and which-eventually partly- complement each other's function. Thus, during growth on D-alanine a specific protein factor may be induced that is capable of complementing the original ~ e r 1 3 ~ ' ~ mutation.

PEROXISOME DEGRADATION

In H. polymorpha, peroxisomes are actively degraded after a shift of methanol- grown cells to a new environment in which the organelles are redundant for growth13 (Frc. 1). A similar phenomenon is observed for organelles which have become functionally inactive, for example, as a result of chemical treatment, The degradation process is shown to be energy-dependent but independent of protein synthesis. In H. polymorpha, peroxisomes invariably were degraded individually by means of an autophagic process. The process is characterized by three distinct stages: (1) sequestration of the organelles destined for degradation from the cytosol by a number of mem- branous layers, (2) fusion of this compartment with-part of-the vacuole to acquire hydrolytic enzymes, and (3) proteolytic degradation of the microbody contents by these vacuolar enzymes. Degradation of individual peroxisomes is a rapid process and is generally completed within 30 minutes. The molecular mechanisms triggering this process are still unknown. Interestingly, peroxisomal matrix enzymes present in the cytosol of H. polymorphaper mutants me not degraded after exposure of the cells to glucose-excess condition^.'^ This suggests that the signals initiating peroxisome turnover are not directed towards the matrix proteins but instead, to the intact organelle. In line with this, in H. polymorpha Pim- mutants (mutants which are partial- ly defective in peroxisomal protein import), solely the few intact peroxisomes were susceptible to proteolytic degradation whereas the cytosolic part of the matrix proteins were

In order to obtain more insight into the molecular mechanisms of selective peroxisome degradation in H. polymorpha, we set out to generate mutants affected in this process (pdd mutants).ls Mutants were screened by a direct colony color assay, which allowed us to monitor the decrease of alcohol oxidase activity as a result of selective inactivation. In the collection of mutants obtained this way, all were impaired in either the first or the second stage of the selective autophagy of peroxisomes. At present, we have identified two complementation groups, PDDl and PDDZ. Mutations

VAN DER KLEI & VEENHUIS: PEROXISOME BIOGENESIS IN H. polymorpha 51

mapped in gene PDDl were affected in the initial step of peroxisome degradation (organelle sequestration). We cloned the PDDl gene, which encoded a protein of 117 kDa that appeared to be 40 percent identical to the S. cerevisiae VPS34 gene product, phosphatidylinositol(Pi)-3-kinase, a protein required for the sorting of soluble hydro- lases to the yeast vacuole.16 Judged from the morphological phenotype of pddl an identical role in peroxisome degradation is not immediately evident: at present, we are studying the specific role of Pddlp in the degradation process in detail. Pdd2p appeared to be essential for the second step in the degradation process (vacuolar fusion). Importantly, the pddl and pdd2 mutations showed genetic interactions which suggested that the corresponding gene products may physically or functionally inter- act with each other.

PEROXISOME-DEFICIENT MUTANTS OF H. POLYMORPHA

In 1989 the first peroxisome-deficient mutants of S. cerevisiae @as-mutants') and H. polymorpha (per-mutants2) were isolated in a combined effort together with the groups of Kunau (Bochum, Germany) and Cregg (Oregon, United States). All H. polymorpha mutants have been isolated from a collection of mutants which were impaired to grow on methanol (Mut- phenotype).

At present we have identified 28 complementation groups, namely 12 groups containing only constitutive per mutants (designated PERl-PER12) and 16 groups containing conditional (14 Ts and 2 Cs) mutants.

Extensive complementation analysis of the 12 constitutive PER genes revealed many cases of conditional non-complementation which were predominantly observed at lowered temperatures (cold sensitive non-complementation). These data strongly suggest the existence of hnctional and physical links between at least ten PER gene products essential for peroxisome biogenesis.

At present, nine PER genes have been cloned by means of functional complementation of selected per mutants using a H. polymorpha genomic bank and restoration of the Mut+ phenotype as selection criterion; the current information regarding these genes is summarized in TABLE 2.

PEROXISOMAL REMNANTS IN PER DELETION STRAINS

We have constructed deletion mutants of the nine PER genes indicated above and analyzed the presence of peroxisomal matrix and membrane proteins in cells incubat- ed on methanol. These studies revealed that all matrix proteins tested were normally synthesized and active in the cytosol. At high expression levels, alcohol oxidase formed large cytosolic crystalloids with which the bulk of the other peroxisomal enzymes, except catalase, were associated.2'

The peroxisomal membrane proteins tested were also normally synthesized. The location of these proteins was dependent on the PER gene which was deleted. In mutants which were affected in protein import (e.g., PERl), small vesicles were observed which did not contain matrix protein but were considered to be of peroxiso- ma1 origin since they were recognized by antibodies against Per8p, which is an inte-


TABLE 2. Genes Involved in Peroxisome Biogenesis from Hansenula polymorpha Location of Properties of Protein

Gene Homologuesa Gene Product Product/Putative Function

P E R I ‘ ~

PER2

PER3”

PER4

PER5

PER6 PER8

PER9

PER10

PER3 Pp

PAS2 sc PAS4 Pp PASl 0 Sc PAS8 Pp Human PXRl PAS1 Sc PAS1 Pp PASl Ct PAS8 Sc PASS Pp PAY4 YI -

PAS4 Sc

PAS3 sc PAS2 Pp -

Peroxisomal matrix

Peroxisomal membrane

Peroxisomal matrix + cytosol

Unknown

Unknown

Peroxisomes Peroxisomal membrane



74 kDa, protein contains both PTSl and PTS2 (KLx,QL)/matrix protein import

Ubiquitin binding protein

70 kDa, C-terminal TPR motif/ essential for PTS 1 -import

1 16 kDa, AAA-type ATPase

125 kDa AAA-type ATPase

65 kDa, N-terminal RV,QL 32 kDa, C-terminal Zn-finger-like

motifiperoxisome proliferation 52 kDa/membrane biogenesis

40 kDa, protein import

aAbbreviations: Ct = Candida tropicalis, Pp = Pichia pastoris, Sc = Saccharomyces cerevisiae, YI = Yarrowiu lipolytica. For references to the homologues see also Subramani.’”

gral membrane protein of peroxisomes of H. polymorpha. These vesicles still dis- played typical properties of intact peroxisomes in that they were inducible: for in- stance, they strongly proliferated in cells in which PER8 was overexpressed; also, they were susceptible to degradation when the induced cells were placed under glucose-excess conditions. On the other hand, in mutants affected in a gene encoding a peroxisomal membrane protein (e.g., PER9) these vesicles were not observed. In- stead, PMPs were found in small aggregates. At present, we investigate whether the membrane vesicles are essential to serve as a template for the reintroduction of peroxisomes, which occurs when the deletion mutants are transformed with a rescue plasmid carrying the gene, originally deleted.

PEROXISOMAL PROTEIN IMPORT

In H. polymorpha the size of what are considered to be “mature” peroxisomes is remarkably constant and predominantly prescribed by the prevailing growth conditions. We demonstrated that in vivo it is solely the few small organelles from the total peroxisomal population of the cells that are capable of importing matrix proteins.


Our data unequivocally demonstrated that a heterogeneity exists between peroxisomes within one cell with respect to their capacity to incorporate newly synthesized proteins. This was true not only for H. polymorpha,22 but also for other yeasts, for example, C. b ~ i d i n i i ~ ~ (see also FIG. 1). The mechanisms which control the protein import capacity are not yet fully clear. Waterham et al. l 8 argued that Perlp is involved in specifying the import capacity of individual peroxisomes, but preliminary experiments indicate that also other PER gene products play a role in this process (e.g., Per9p; see R. Baerends, H. Hut and I. J. van der Klei, in preparation). Our results furthermore suggest that this import capacity is correlated with the capacity of the organelles to multiply (by fission). One possible explanation for this is that special patches on the peroxisomal membrane exist which mediate import (i.e., contain “import sites”); based on the observed functional links between different PER genes, we speculate that various functions are concentrated in these regions (e.g., specifying import, fission, and degradation) which are donated to the newly formed organelle during fission. A schematic representation of this process is depicted in FIGURE 2. On the other hand, this temporal import capacity of peroxisomes in vivo is apparently in conflict with the general picture emerging from in vitro experiments that use semi- permeabilized cells or injection of proteins which do not show a selective import in

- - - - - - - 0 I

I I I I

/ / I

I

; I

I

: I

: I corporate additional matrix protein.

FIGURE 2. Model of peroxisome proliferation, explain- ing the heterogeneity among peroxisomes within one cell. The dots represent clusters/patches of proteins involves in

.( : peroxisome proliferation/protein import. When a small organelle buds off from the mature one, this cluster of proteins is incorporated in the newly formed organelle. Hence, the mature organelle now lacks components of the protein import machinery, resulting in the inability to in-

$. I

I

4 + I I


part of the peroxisomal p o p u l a t i ~ n . ~ ~ , ~ ~ One possible explanation for this discrepancy may be related to the fact that, in general, in these in vitro systems the amounts of proteins addedinjected largely exceed normal precursor concentrations.

IMPORT OF PEROXISOMAL MATRIX PROTEINS

As in other organisms, most matrix proteins in H. polymorpha also contain a PTS 1. In this organism, variants of the PTS 1 motif are found in the enzymes of methanol metabolism alcohol oxidase (AO) (-Am), dihydroxyacetone synthase (- NKL,) and catalase (-SKI).

At present, few PTS2 proteins have been encountered in H. polymorpha; besides thiolase, amine oxidase26 and PerlpLs also contain this motif. Interestingly, as for PTS 1, species-dependent variations may also exist for PTS2, exemplified by the fail- ure of baker's yeast peroxisomes to import I: brucei aldolase, which on the other hand is normally imported into the organelles of H. polymorpha.

The occurrence of different targeting signals for peroxisomal matrix proteins al- ready predicted the presence of separate import pathways for PTSl and PTS2 proteins. This was confirmed by the isolation of mutant yeast strains, specifically impaired in import of PTSl proteins (pas8 from I? p a s t o r i ~ , ~ ~ pas10 from S. cerevisiae,28 and per3 from H. po lymorph~'~) or PTS2 proteins (pas7 from S. ~ e r e v i s i a e ~ ~ andpebl from S. c e r e v i ~ i a e ~ ~ ) . Pas8p (Pp) PaslOp (Sc) and Per3p (Hp) are homologous proteins of about 65 to 69 kDa; Pas8p (Pp) is shown to bind to the PTSl se- quence (SKL), indicating that it finctions as the SKL receptor.27 This function of the various PasSp homologues is yet uncontroversial; on the other hand, the exact location of the SKL receptor is still a matter of debate. Different locations have been re- ported, ranging from completely cytosolic to a soluble peroxisomal matrix con- stituent. It remains to be seen whether these data represent species-specific differences or may have other causes, for example, differences in growth phase (exponential versus resting cells from the stationary growth phase) or are related to possible experimental errors. The presence of iso-enzymes is less likely based on the current data. In H. polymorpha, we believe that Per3p has a dual location and is present in both the cytosol and the peroxisomal matrix.19 Based on this location we propose that Per3p shuttles the protein to be imported into the organellar matrix, dissociates (probably mediated by the low internal pH of the organelle), with the eventual assembly/activation (compare FIGURE 4 for A 0 assembly) and subsequent release of Per3p from the matrix (FIGURE 3). A similar mechanism may be operative for the import of PTS2 proteins in baker's years29,30 (W. H. Kunau, personal communication). The finding that separate PTS-dependent import machineries exist raises the question whether they have common elements and how the translocation process fimctions. Studies in several groups showed that import does not necessarily depend on an un- folded conformation of the p ~ l y p e p t i d e ~ ~ ~ ~ ~ ; however, whether this is a common phenomenon in WT cells is not yet clear and remains to be elucidated.

The PTS2 import pathway of H. polymorpha has been studied in more detail by Faber et These studies showed that the PTS2 import machinery of H. polymorpha is inducible in nature. Evidence for this came from H. polyrnorpha transfor-


FIGURE 3. Schematic representation of the proposed pathway of PTSl protein import in H. polymorpha mediated by Per3p. When synthesis of a matrix precurser is complete, Per3p binds to its C-terminal tripeptide (PTSl). At this stage cytosolic chaperones (hsp70) might bind to the precurser proteins as well. Per3p undergoes a conformational change upon binding to PTSl, which allows interaction with the peroxisomal membrane andor a general receptor. After subsequent translocation of the Per3piprotein complex into the organellar matrix, Per3p dissociates (e.g., due to altered pH value) and crosses the peroxisomal membrane again. Upon reaching the cytosol Per3p can shuttle the next PTSl protein into the peroxisomal matrix.

mants, which carried an additional copy of the homologous A M 0 gene or genes encoding heterologous PTS2 proteins (watermelon glyoxysomal malate dehydrogenase and S. cerevisiae thiolase) under the control of the H. polymorpha A 0 promoter. These experiments demonstrated that import of PTS2 proteins was largely prevented in cells grown on ammonium sulfate, but was efficient-and complete-when the cells were grown under conditions in which the synthesis of A M 0 was induced (e.g., by primary a m i n e ~ ) . ~ ~ Most likely, a limiting import rate rather than a limiting stor- age capacity of the organelles accounts for the partial import of PTS2 proteins in ammonium sulfate-grown H. polymorpha. Studies on the S. cerevisiae PAS1 0, E! pastoris PAS8 and H. polymorpha PER3 deletion mutants indicated that solely PTS2


proteins were imported in peroxisomes; PTS 1 proteins were invariably in the cytosol independently of their expression levels. In contrast, in the S, cerevisiae pas7/pebl mutant, only thiolase is cytosolic and not detectable in peroxisomes in cells of a pas74ebl deletion strain. These results strongly suggest that the PTSl and PTS2 import machineries are indeed highly specific and cannot complement each other's functions.

In H. polymorpha, the PTSl import machinery is functional in fully repressed glucose-grown cells34 but strongly induced by methanol. Hence, the low-rate import of PTS2 proteins in repressed (ammonium sulfate-grown) cells of H. polymorpha is most probably due to the fact that the PTS2 import pathway is present at only basic levels which are too low to allow complete import of overexpressed PTS2 proteins. The fact that heterologous PTS2 proteins are also efficiently recognized and imported indicated that this pathway, like the PTS 1 pathway, is conserved between species. Both import machineries appear to be inducible and are in particular present at en- hanced levels in cells in which the synthesis of peroxisomes in induced by specific growth substrates.

ASSEMBLY OF PEROXISOMAL MATRIX PROTEINS

A major topic in our current studies on peroxisome biogenesis includes the assembly of matrix proteins, in particular the flavoprotein A 0 of H. polymorpha. A 0 is directed to peroxisomes by a PTSl (-ARF), a process which involves the function of Per3p (see the preceding section). Inside the organelle the protein obtains its enzymatically active conformation, which consists of an octamer of eight identical subunits, each of which has one FAD molecule noncovalently bound.3s

In WT cells the translocation of A0 into peroxisomes and its subsequent assembly into octamers are very efficient processes; the pool of cytosolic A 0 protein is generally undetectably low, while the peroxisomal protein is all in the octameric conformation.36,37 However, following overexpression of the A0 gene, part of the protein accu- mulates in the cytosol, where it forms inactive aggregates, which suggests that in the cytosol A 0 octamerizatiodassembly does not occur.

In contrast, peroxisomal amine oxidase (a Cu2+-containing dimeric enzyme) obtains its enzymatically active conformation both inside peroxisomes and in the cytosol upon overexpression in WT cells.33 Consequently, the tightly controlled process of A 0 assembly may not be generalized for all peroxisomal matrix proteins.

In order to unravel the molecular mechanisms of A 0 assembly and to identify the components involved, extensive studies have been carried out, both in vitro and in vivo. After in vitro denaturation of purified A 0 by strong denaturants (e.g., urea or guanidine chloride), renaturation invariably failed. With the use of these denaturants, the A0 octamers disassemble, the polypeptides unfold, and FAD dissociates. The major problem encountered in renaturation experiments is that A 0 protein aggregates are formed upon removal of the denaturants. From this work we speculated that specific (peroxisomal?) factors are required to assist the proper foldingiassembly of AO.

Recently, Evers et aL3* showed that in vitro reassembly of A 0 can be achieved using properly folded, FAD-containing A 0 monomers. These subunits are formed under relatively mild dissociation conditions, namely by incubation of purified oc-


tameric A 0 in 80% glycerol. Upon gradual dilution of the glycerol, these subunits spontaneously formed A 0 octamers again. Based on this latter finding we assume that the crucial step in A 0 assembly is the formation of FAD-containing A 0 subunits: once these subunits are formed spontaneous oligomerization into octamers takes place.

The folding andor FAD binding is probably assisted by specific peroxisomal factors (e.g., chaperones). These factors could represent peroxisomal counterparts of molecular chaperones (members of the hsp60 and hsp7O protein families), which are known to play an essential role in the biogenesis of other cell organelles. However, despite major efforts, we did not yet succeed in purifying peroxisomal homologues of these proteins from H. polymorpha peroxisomes in sufficient amounts to allow their further characterization. The isolation of a riboflavin(Rf)-auxotrophic mutant of H. polymorpha enabled us to study the role of FAD binding in A 0 assembly in vivo. In

cytosol peroxisom e edlaregate crystal

FIGURE 4. Alcohol oxidase assembly in peroxisomes of H. polyrnorpha. Monomeric A 0 (Sl), is synthesized in the cytosol and subsequently imported into peroxisomes. In the matrix it interacts with a peroxisomal factor, resulting in FAD binding. The FAD-containing monomer (S2) spontaneously forms octamers which crystallize. In the absence of FAD (in the rifl mutant), slow dissociation of FAD-lacking A 0 monomers from the peroxisomal factor leads to partial aggregation of the protein (filled black arrows).


this mutant (r$) the intracellular concentration of Rf, and thus of FAD, can be ma- nipulated by varying the Rf concentrations in the growth medium. With decreasing amounts of Rf in the growth medium, increasing amounts of FAD-less monomeric A 0 accumulated in the cytosol of rifl cells.39

The fact that the cytosolic FAD-less A 0 protein does not form octamers is in agreement with the in vitvo data that FAD binding is a prerequisite for oligomerization. Because the peroxisomal portion of the protein in rifr cells is properly assembled into FAD-containing octamers, we assume that FAD binding occurs immediately upon translocation of the protein into the peroxisomal matrix. In fact, we have evidence that A 0 translocation and assembly are tightly coupled processes.

Most probably the peroxisomal folding of A 0 polypeptides, including the proper exposure of the FAD binding fold, is assisted by a peroxisomal factor (FIG. 4). As soon as the FAD binding fold has assumed its appropriate conformation, FAD will associate to the monomeric protein, which then spontaneously octamerizes. Howev- er, when FAD association is prohibited because of FAD limitation, the peroxisomal factor involved in proper folding of the monomer may become saturated with non-assembled AO, as a result of which further import of this protein is blocked. Apparent- ly, this factor does not require the specific peroxisomal environment for function- ing?O This is indicated by the finding that A 0 (but also other peroxisomal matrix enzymes) are properly assembled and active in the cytosol ofper mutants. In these cells the putative peroxisomal factor is mislocated in the cytosol, where it can normally function in A 0 folding/FAD binding.

ACKNOWLEDGMENTS

We are indebted to Melchior Evers, Klaas Nico Faber, Vladimir Titorenko, Ilya Tolstorukov and Hans Waterham, who performed important parts of the work, de- scribed here. We thank Ineke Keizer, Klaas Sjollema and Jan Zagers for help in elec- tron microscopy.

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