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Peroxisome biogenesis: advances and conundrums
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Peroxisome biogenesis: advances and conundrums Paul B Lazarow Investigations of peroxisome biogenesis in diverse organisms reveal new details of this unique process and its evolutionary conservation. Interactions among soluble receptors and the membrane peroxins that catalyze protein translocation are being mapped. Ubiquitination is observed. A receptor enters the organelle carrying folded cargo and recycles back to the cytosol. Tiny peroxisome remnants — vesicles and tubules — are discovered in pex3 mutants that lack the organelle. When the mutant is transfected with a good PEX3 gene, these protoperoxisomes acquire additional membrane peroxins and then import the matrix enzymes to reform peroxisomes. Thus, de novo formation need not be postulated. Dynamic imaging of yeast reveals dynamin-dependent peroxisome division and regulated actin-dependent segregation of the organelle before cell division. These results are consistent with biogenesis by growth and division of pre-existing peroxisomes. Addresses Mount Sinai School of Medicine, 1190 Fifth Avenue, Box 1007, New York, NY 10029-6574, USA e-mail: [email protected]
Here, we briefly summarize what is well established and discuss recent discoveries that add rich detail to our understanding of protein translocation into peroxisomes and point toward requisite future research. Other discoveries discussed herein illuminate peroxisome inheritance and clarify the controversies.
Peroxisome movement and segregation: differences between mammals, yeast and plants Dynamic observations of fluorescing peroxisomes in cultured mammalian cells reveal two types of movement. Most peroxisomes move slowly and randomly. A minority show fast, directional movement along microtubules [5,6] that depends upon cytoplasmic dynein [7]. The fast component is regulated by a signaling cascade: stimulation of Chinese hamster ovary (CHO) cells with extracellular ATP and lysophosphatidic acid together cause an arrest of peroxisome movement but not that of other organelles [8]. Segregation of peroxisomes to daughter cells at mitosis appears to be stochastic in mammalian cells [6].
Current Opinion in Cell Biology 2003, 15:489–497 This review comes from a themed issue on Membranes and organelles Edited by Alice Dautry-Varsat and Alberto Luini 0955-0674/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00082-6
Abbreviations CHO Chinese hamster ovary COP coat protein GFP green fluorescent protein PTS peroxisome targeting sequence
In Saccharomyces cerevisiae, in contrast, peroxisome movement does not use microtubules, but depends on the actin cytoskeleton and the actin-associated motor protein Myo2p [9]. Moreover, in this yeast, which typically possesses less than a dozen peroxisomes rather than the hundreds found in mammalian cells, the segregation of peroxisomes into the budding daughter cell is strictly regulated and depends upon an intact actin cytoskeleton (Figure 1). If a bud fails to receive at least one peroxisome, cytokinesis is significantly retarded [9]. This careful cellular surveillance ensuring that all daughter cells receive at least one peroxisome reminds us of similar surveillance for mitochondria and vacuoles [10]. It is consistent with the idea that peroxisomes form solely from pre-existing peroxisomes.
Introduction The major features of peroxisome biogenesis resemble those of mitochondria and chloroplasts, but the details are entirely different [1]. Like the latter two organelles, peroxisomes grow by the post-translational import of newly synthesized proteins into pre-existing organelles; these divide to form daughter peroxisomes, which segregate at cell division [2]. However, the proteins that catalyze peroxisome assembly (termed ‘peroxins’) and the molecular mechanisms of this assembly are unique (reviewed in depth in [3,4]). Controversies have concerned the suggestion that the organelle sometimes forms de novo and that essential membrane proteins could be donated by the endoplasmic reticulum (ER). www.current-opinion.com
In plants (onion epidermal cells or various cell types of Arabidopsis), peroxisome movement is also actin-based [11–13]. These species differences in the mechanism of peroxisome motility (which apply to the motility of other organelles as well) represent one of the exceptions to the generally remarkable evolutionary conservation of peroxisome biogenesis.
Peroxisome division involves dynamin The process of peroxisome division in S. cerevisiae depends, at least in part, on Vps1p, one of the three dynamin-like proteins in this organism [9]. Some Dvps1 cells contain a single large peroxisome and others contain Current Opinion in Cell Biology 2003, 15:489–497
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Figure 1
Dynamics of peroxisome segregation into S. cerevisiae buds in (a) wild-type cells and (b) in cells without dynamin Vps1p. Dividing yeast labeled with GFP–PTS1 were photographed at the times indicated (in minutes). (c) Co-localization of peroxisomes (red) and actin cables (green) in a budding wild-type cell. Videos from which these images were taken may be viewed at www.jcb.org/cgi/content/full/jcb.200107028/DC1. For full details, see [9]. Reproduced from The Journal of Cell Biology 2001, 155:979-990 [9] by copyright permission of the Rockefeller University Press. Bar ¼ 5 mm.
clusters and strings of small, interconnected peroxisomes that look as if they were caught in abortive fission. Nevertheless, peroxisomes do manage to divide at cytokinesis in Dvps1 cells, driving the conclusion that there is also a dynamin-independent peroxisome fission, perhaps based on mechanical pulling [9]. Peroxisome budding has also been observed in Arabidopsis [13]. The division and proliferation of peroxisomes is further regulated by Pex11p. Overexpression of Pex11p causes proliferation of small peroxisomes and/or elaboration of tubular peroxisomes in organisms as diverse as yeast [14] and trypanosomes [15], whereas deficiency of Pex11p results in a few large organelles. Rat liver Pex11p binds coat protein (COP) via a dilysine motif at its carboxyl terminus [16], as does trypanosome Pex11p, but this motif is not present in all Pex11p proteins and its mutational disruption does not abolish the function of the trypanosome protein [17]. Current Opinion in Cell Biology 2003, 15:489–497
Different mechanisms for peroxisome membrane elaboration and matrix protein import The insertion of newly synthesized proteins into the peroxisome membrane occurs by mechanisms distinct from those involved in the import of soluble enzymes into the organelle interior or ‘matrix’ (Figure 2). As a result, when the import of matrix proteins is defective, membrane ‘ghosts’ of peroxisomes can persist in cells and segregate at cell division. For instance, ghosts are present in skin fibroblasts of Zellweger syndrome neonates (which do not make a normal peroxisome after ovum fertilization) [18]. Three peroxins, Pex3p, Pex16p and Pex19p, are required to elaborate and maintain the membrane. One of these, Pex19p, is a soluble, mostly cytosolic, sometimes farnesylated protein that assists in bringing newly synthesized membrane proteins from their site of synthesis on free www.current-opinion.com
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Figure 2
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Peroxisome biogenesis by growth and division. Numbers refer to PEX gene products. (a–e) Steady-state physiological formation. (a) Synthesis of peroxisomal proteins on free polyribosomes. (b) Import of matrix proteins via a branched pathway. This requires cytosolic peroxins (including Pex5p [blue], the PTS1 receptor and Pex7p [orange], the PTS2 receptor), as well as peroxisome membrane peroxins (PMPs) 1, 2, 4, 6, 8–10, 12–15, 17 and 22–24. Yellow ovals represent the still mysterious translocation machinery. (c) Insertion of membrane proteins. mPTS, targeting sequence to the peroxisome membrane. Requires cytosolic Pex19p (green) and membrane-bound Pex3p and Pex16p but not the other PEX gene products. (d) Insertion of membrane phospholipids, which are synthesized in the ER (see text for transport alternatives). (e) Peroxisome division. (f) Peroxisome abundance and size are regulated by the organism in response to nutrients, drugs, etc. (g,h) Pathological transitions owing to mutations or genetic complementation. (g) Mutations that cause loss of matrix protein import result in mostly empty membrane ghosts. Restoration of the wild-type gene allows the ghosts to re-acquire their normal content of proteins. (h) Mutations in PEX genes 3, 16 or 19 cause the inability to assemble most membrane proteins, and consequently loss of import of matrix proteins. This results in small, hard-to-detect, vesicular or tubular protoperoxisomes. Genetic complementation allows re-formation of ghosts followed by re-import of matrix proteins. Modified and reproduced with permission from the Annual Review of Cell and Developmental Biology, volume 17 [4] ß 2001 Annual Reviews (http://www.annualreviews.org).
polyribosomes through the cytosol to the peroxisomes, perhaps or in part by recognizing targeting information within these proteins. Pex3p and Pex16p are integral membrane proteins of the peroxisome membrane that are involved in insertion of new membrane proteins and/or in subsequent steps in the maintenance of the membrane. www.current-opinion.com
If any of these three peroxins is defective, neither normal peroxisomes nor membrane ghosts are present. Many other peroxins are required for matrix proteins to reach the peroxisome interior. The matrix proteins are directed to peroxisomes by one of several types of Current Opinion in Cell Biology 2003, 15:489–497
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peroxisome targeting sequences (PTS), of which two are well characterized: a carboxy-terminal tripeptide PTS1 (canonical sequence ¼ SKL) and an amino-terminal nanopeptide PTS2 (consensus sequence ¼ RLXXXXXHL, where X can be any amino acid). Soluble cytosolic receptors for each of these, Pex5p and Pex7p, respectively, bring newly synthesized protein cargo to peroxisomes and then recycle back to the cytosol to pick up more cargo. Pex5p does this job on its own, but Pex7p requires a species-specific auxiliary protein to help it: Pex18p or Pex21p in S. cerevisiae [19], Pex20p in Yarrowia lipolytica [20] and Neurospora crassa [21], or the longer of two splice isoforms of Pex5p in mammals [22,23]. These non-orthologous proteins possess a conserved sequence region that probably represents a common Pex7p binding site, suggesting the evolutionary conservation of a functional module rather than an entire protein [20,23].
Ubiquitination Unexpectedly, the auxiliary proteins, Pex18p and Pex21p, turn over with a half-life of less than 10 min [24]. This rapid degradation is tightly coupled to their function because mutations that block peroxisome assembly also arrest turnover. The stabilized Pex18p is in a cytosolic complex with the PTS2 receptor. Moreover, mono- and di-ubiquitinated forms of Pex18p are observed, predominantly in association with the peroxisomes [24]. Recent evidence suggests that Pex4p, a peroxisomemembrane-bound ubiquitin-conjugating enzyme, adds the second but not the first of these ubiquitins (PE Purdue and PB Lazarow, unpublished data). Three peroxins embedded in the peroxisome membrane contain RING fingers (Pex2p, Pex10p and Pex12p) and are candidates for E3 ubiquitin ligases. These and other data suggest the possibility that sequential ubiquitinations occur at, and perhaps regulate, discrete steps during docking of a Pex18p–PTS2 receptor–cargo complex on the surface of the peroxisome, delivery of the cargo to the matrix and recycling of the receptor, followed by degradation of Pex18p.
A receptor enters peroxisomes The receptors dock at a complex of three interacting membrane peroxins: Pex13p, Pex14p and Pex17p. The details of the receptors’ interactions with individual members of this complex are under intensive investigation [25–29]. Initially, it was thought that the receptors recycle right back into the cytosol after dropping their cargo at this docking site (a ‘simple shuttle’). However, recent experiments have clearly shown that Pex5p enters human peroxisomes during the course of its normal function and then re-emerges to the cytosol to carry out further rounds of import (an ‘extended shuttle’) [30]. The proteolytic cleavage of a Pex5p fusion protein was demonstrated within peroxisomes and the processed Pex5p was detected back out in the cytosol. Some question remains Current Opinion in Cell Biology 2003, 15:489–497
as to whether the Pex5p fusion protein went all the way into the peroxisome interior or merely poked itself far enough in to be acted upon by the interior protease.
Translocation remains mysterious What remains mysterious and challenges future experimentation is exactly what happens after docking. Several additional peroxins, as well as some mentioned above, are known to be required. It is clear that protein translocation into peroxisomes differs profoundly from transport into mitochondria, which threads unfolded polypeptide chains through a narrow channel. By contrast, peroxisomes can import folded and homooligomeric proteins [31], heterooligomers [32,33] and even 4–9 nm gold beads coated with albumin bearing PTS1s and microinjected into cultured cells [34]. Oligomer import is usually facultative, not obligatory; Candida boidinii peroxisomes import one enzyme as a pre-formed dimer and another, alcohol oxidase, as a monomer that is octamerized inside [35]. The transport of large cargo accompanied by a receptor somewhat resembles protein traffic into the nucleus. However, nothing resembling a nuclear pore complex has ever been observed in a peroxisome membrane. Although peroxisomes import large protein cargo, the peroxisome membrane is tight enough to require a variety of transporters for the passage of substrates and products of peroxisome metabolism (reviewed in [36]). It had been thought that the peroxisome is impermeable to protons and supports a pH difference between the interior of the organelle and the cytosol [37,38]; however, a recent experiment using a pH-sensitive variant of green fluorescent protein (GFP) casts doubt on this [39]. In any event, the protein-translocation mechanism must accommodate large cargo but prevent the passage of small organic molecules. Freeze-fracture studies of the peroxisome membrane have not been informative in this regard; no channel-forming proteins have been identified among the peroxins. One interesting speculation suggests that cargo of newly synthesized proteins might enter peroxisomes via membrane invaginations, a sort of peroxisomal pinocytosis [31]. The presence of internal membrane structures or fragments in certain peroxisome biogenesis mutants (e.g. [40]) lends indirect support to this hypothesis. It is also possible that there are additional peroxins crucial to translocation that have not yet been identified, despite the many clever screens and selections that have been carried out to find informative mutants. Normal peroxisomes are not necessary for yeast to grow on glucose, and the mutant isolations have exploited this fact. Searches for temperature-sensitive mutants have not been described. Lethal mutants may have been missed. Transcriptome profiling identified a novel peroxin, Pex25p, which affects peroxisome size and maintenance [41], and www.current-opinion.com
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proteomic analyses [42] also show promise for identifying new components.
arily diverse organisms an ER contribution of proteins appears doubtful.
What is the contribution of the endoplasmic reticulum to normal peroxisome biogenesis?
Do peroxisomes sometimes arise de novo?
Phospholipids
The phospholipids of the peroxisome membrane are mainly phosphatidylcholine and phosphatidylethanolamine, with a tendency toward somewhat longer-chain fatty acids than in other membranes [43]. These phospholipids are synthesized on the ER and transported to the peroxisome membrane by an unknown mechanism (see in [2]). This is also the case for mitochondria, most of the phospholipids of which are made in the ER and perhaps move from ER to mitochondria at sites of close apposition [44,45]. This type of transfer could also apply to peroxisomes, and this suggestion is supported by old morphological data on the adjacency of ER and peroxisomes [46]. Alternatively, we have speculated that specialized vesicles might carry phospholipids from ER to peroxisomes [4]. Such vesicles might contain one or a few proteins that specify the destination. Interestingly, small vesicles that are distinct from mature peroxisomes have been identified in Pichia pastoris, which bear two peroxins, Pex1p and Pex6p [47]. These peroxins are members of the functionally diverse AAA family of ATPases, some of the members of which are involved in membrane-fusion events. It is tempting to speculate that these vesicles might ferry phospholipids to the peroxisomes. Proteins
There have been occasional reports during the past decade that certain peroxisome membrane proteins can be found in the ER. This led to proposals that the ER might contribute a subset of the peroxisome membrane proteins via budding vesicles that either fuse with peroxisomes [48] or mature into peroxisomes [49]. These ‘new’ or ‘immature’ peroxisome vesicles were suggested to contribute to normal peroxisome biogenesis [48,49], and this became a topic of considerable discussion and debate within the peroxisome community. Leaving aside temporarily the exceptional case of Y. lipolytica [49], the evidence for other organisms consisted mainly of finding certain peroxisome membrane proteins in the ER in experimentally engineered situations. Careful review of the data, which space considerations preclude repeating here, suggests that most such cases were artefacts of protein overexpression, or artefacts of cutting and splicing of membrane proteins [4]. For example, in wild-type S. cerevisiae, newly synthesized endogenous Pex15p inserts directly into peroxisome membranes in a Pex3pand Pex19p-dependent fashion (as shown in Figure 2), without visiting the ER, but overexpression of Pex15p leads to its accumulation in ER membranes [40]. Thus, under normal physiological circumstances, in evolutionwww.current-opinion.com
But what happens in mutants that lack peroxisomes when resumption of peroxisome biogenesis is induced experimentally? In the many mutants in which matrix protein import is defective (Figure 2), membrane ghosts are present that provide the structural basis to reform normal peroxisomes when the missing peroxin is restored by genetic complementation. This can be rapid (half-time of 10 min) if cells of different complementation groups are fused and no new protein synthesis is needed, or a matter of hours if gene expression is required [50]. However, three mutants (pex3, pex16 and pex19) have defects in peroxisome membrane assembly and lack ghosts as well as peroxisomes. Nonetheless, they reform peroxisomes when the missing gene is restored by transfection. This process occurs slowly and stepwise: first membranes appear which subsequently fill with matrix enzymes in a process that may take days (e.g. [51,52]). In these cases, it has been suggested that the organelle forms de novo [53,54]. But is the evidence for de novo formation compelling and how might this occur?
Protein translocation through the endoplasmic reticulum is not required to form peroxisomes If peroxisomes were ever to form de novo, the ER would be an obvious potential source of new membrane vesicles, which need only contain the crucial peroxins that act first to elaborate the organelle [48,49]. These are the integral membrane proteins Pex3p and Pex16p, which, together with the soluble Pex19p, are required for the assembly of other peroxisomal membrane proteins, which in turn allow the import of matrix proteins (Figure 2). With this in mind, the biogenesis of these two proteins has been intensively investigated in normal human fibroblasts: they never were observed in ER; the first place they could be detected after their biosynthesis was in the peroxisomes; this transport to peroxisomes was unaffected by inhibitors of COPI or COPII [52,55,56]. Moreover, the reformation of normal peroxisomes in pex3 and pex16 mutants upon rescue with the appropriate wild-type gene was unaffected by inhibitors of COPI or COPII [52,55]. Perhaps the putative peroxisome-forming vesicles bud out of the ER before the COPI- or COPII-mediated steps? This was tested by using mutant S. cerevisiae defective in SEC61, an essential component of the ER translocon, or its homolog SSH1. These mutations prevent proteins from entering the ER. Peroxisome biogenesis proceeds normally in these mutants [57]. If the ER does not provide new membrane to reform peroxisomes, where do de novo peroxisomes come from? Vesicles of indeterminate origin have been postulated [54]. Current Opinion in Cell Biology 2003, 15:489–497
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The existence of protoperoxisomes makes de novo formation and new vesicles unnecessary The evidence for the absence of typical peroxisome membrane ghosts in pex3, pex16 and pex19 mutants is compelling: ghosts have been sought aggressively in these mutants with a battery of antisera against membrane proteins that are found in ghosts in other mutants, and the results have been consistently negative [40,55,58]. However, these data do not preclude the existence of peroxisomal membrane remnants, perhaps smaller or with a more limited repertoire of proteins, that might have been missed by the methods used. Recent experiments provide strong indications for the existence of such structures, which we have termed ‘protoperoxisomes’ [4]. Tiny peroxisomal vesicles and tubules were first discovered in the P. pastoris Dpex19 mutant by deconvolution microscopy and an antibody that recognizes endogenous Pex3p [59]. These structures were then visualized by
electron microscopy (Figure 3). There is evidence of similar protoperoxisomes in several pex3 mutants. The first 40–50 amino acids of Pex3p, fused in front of GFP, are sufficient to anchor GFP to the surface of peroxisomes in a variety of species. This construct serves as a useful probe for peroxisomal membranes because it has never been found to insert in any other endomembrane, even when overexpressed (nor has wild-type Pex3p, which may instead lead to the presence of many small peroxisomes when overexpressed [60]). This GFP construct labels wild-type peroxisomes in CHO cells and also detects small vesicles in a CHO pex3 mutant (see Figure 6d of [51]). Such vesicles were found similarly in pex3 mutants of S. cerevisiae (K Huang and PB Lazarow, unpublished data) and Hansenula polymorpha [61]. In the latter case, the GFP vesicles were shown to contain a second peroxisome membrane protein, Pex14p. Moreover, the vesicles had a characteristic property of wild-type H. polymorpha peroxisomes in that they were susceptible to selective degradation when cells were shifted to growth on glucose.
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Protoperoxisomes in Dpex3 and Dpex19 mutants of P. pastoris. (a,b) Electron microscopy of (a) wild type and (b) Dpex19 cells. m, mitochondrion; n, nucleus; p, peroxisomes; v, vacuole. The dashed white box in (b) indicates the tiny vesicles and tubules in the mutant, identified as peroxisomal by deconvolution microscopy. Reproduced from [59], with permission by the American Society for Cell Biology. (c,d) Immunofluorescence with antibodies against Pex8p to identify peroxisome membranes (green) and MitoTracker to label mitochondria (red): (c) wild type, (d) Dpex3. Bar ¼ 1 mm. Reproduced with permission from [62], ß Blackwell Publishing Ltd. Current Opinion in Cell Biology 2003, 15:489–497
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Recently, the use of previously unavailable antibodies against endogenous Pex8p, Pex12p, Pex14p and Pex17p has provided compelling evidence for peroxisome remnants in the P. pastoris Dpex3 mutant. Vesicles and tubular, torpedo-shaped peroxisomal structures were observed with deconvolution microscopy (Figure 3) and characterized by isopycnic and flotation gradient centrifugation [62]. These ‘protoperoxisomes’ can provide the structural basis to reform the normal peroxisome compartment (see Figure 2) and have been shown to do so in the case of H. polymorpha [61]. Thus, there is no need for de novo formation in these species and mutants. This methodology has not yet been tried in pex19 mutants in other species, nor in pex16 mutants. Peroxisomal remnants could easily have been missed in these cells. The role of protoperoxisomes in reforming peroxisomes following gene rescue is analogous to the role of promitochondria in reforming mitochondria when anaerobically grown yeast receive oxygen [63].
Evolutionary conservation There is a remarkable degree of evolutionary conservation in the mechanisms of peroxisome assembly and many peroxins are well-conserved among yeasts and mammals and in other diverse species including plants [64], Caenorhabditis elegans [65] and trypanosomes [66]. This has allowed the formulation of a unified, species-independent PEX gene nomenclature [67]. It has permitted many genes responsible for inherited human disorders of peroxisome biogenesis to be identified by virtue of their similarity to yeast genes [54]. Inevitably there are some species differences, but for the most part these have been minor. One striking exception is Y. lipolytica [49,68]. Normal peroxisome biogenesis in this organism is said to begin by the COP-dependent budding of a vesicle from the ER that contains core-glycosylated Pex2p and Pex16p. This vesicle is then reported to undergo a series of maturation steps that result in its gradual conversion to a mature peroxisome. Pex16p and Pex19p have different functions in this organism than in others (e.g. normal-looking peroxisomes exist in Dpex19 [69]). The species differences are not confined to peroxisome biogenesis; for example, four distinct secretory pathways are reported to diverge at the level of the ER in Y. lipolytica [49]. Certain defects in secretion impair peroxisome biogenesis. Conversely, defects in the PTS1 receptor inhibit secretion, apparently from inside peroxisomes. These curious features have been observed so far in one organism in one laboratory.
branes are believed to come from other membranes, not assembled from scratch. The apparent absence of peroxisomal structures in the pex3, pex16 and pex19 mutants was the motive to postulate de novo formation. The discovery of protoperoxisomes in P. pastoris Dpex19 and in Dpex3 mutants of several species removes the motive, at least in those species. An alternative, the trafficking of crucial proteins through the ER and vesicle budding, has been refuted, at least in human fibroblasts. Protoperoxisomes are remnants of once-functional peroxisomes, before a mutation; they are not ‘new’ and not ‘young’. At least in S. cerevisiae, careful regulation ensures that a minimum of one peroxisome is segregated into each daughter yeast cell. These results support the sufficiency of the simple model that peroxisomes form by growth and division of pre-existing peroxisomes. There is a superficial similarity between the model of Figure 2 and some proposed models of de novo formation. Recovery of peroxisomes after gene rescue definitely occurs stepwise. The key point is that the several types of peroxisomal structures shown in Figure 2 (including ghosts and remnants) can be interconverted and thus need not be made de novo. The field lacks a simple model to account for the facts of protein translocation into peroxisomes. Exciting new results stimulate the imagination: complexes of proteins in the membrane, a PTS receptor that enters the organelle and recycles back to the cytosol, stepwise ubiquitinations, large cargo. . . This is a challenge for future research.
Acknowledgements The work of the author was supported by the National Institutes of Health, grant DK19394. Owing to space constraints, this review discusses selected recent highlights – apologies to colleagues whose work was omitted. Few papers before 2000 are cited; they may be found in previous reviews. I thank S Subramani and HF Tabak for permission to reproduce their figures.
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17. Maier AG, Schulreich S, Bremser M, Clayton C: Binding of coatomer by the PEX11 C-terminus is not required for function. FEBS Lett 2000, 484:82-86.
34. Walton PA, Hill PE, Subramani S: Import of stably folded proteins into peroxisomes. Mol Biol Cell 1995, 6:675-683.
18. Santos MJ, Imanaka T, Shio H, Small GM, Lazarow PB: Peroxisomal membrane ghosts in Zellweger syndromeaberrant organelle assembly. Science 1988, 239:1536-1538. 19. Purdue PE, Yang X, Lazarow PB: Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway. J Cell Biol 1998, 143:1859-1869. 20. Einwachter H, Sowinski S, Kunau WH, Schliebs W: Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway. EMBO Rep 2001, 2:1035-1039. A conserved structural motif enables diverse proteins to fulfil a similar auxiliary function in PTS2 protein import in several species. 21. Sichting M, Schell-Steven A, Prokisch H, Erdmann R, Rottensteiner H: Pex7p and Pex20p of Neurospora crassa function together in PTS2-dependent protein import into peroxisomes. Mol Biol Cell 2003, 14:810-821. 22. Otera H, Harano T, Honsho M, Ghaedi K, Mukai S, Tanaka A, Kawai A, Shimizu N, Fujiki Y: The mammalian peroxin Pex5pL, the longer isoform of the mobile peroxisome targeting signal (PTS) type 1 transporter, translocates the Pex7p.PTS2 protein complex into peroxisomes via its initial docking site, Pex14p. J Biol Chem 2000, 275:21703-21714.
35. Stewart MQ, Esposito RD, Gowani J, Goodman JM: Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments. J Cell Sci 2001, 114:2863-2868. Proteins can fold before or after translocation into peroxisomes. 36. Tabak HF, Braakman I, Distel B: Peroxisomes: simple in function but complex in maintenance. Trends Cell Biol 1999, 9:447-453. 37. Nicolay K, Veenhuis M, Douma AC, Harder W: A 31P NMR study of the internal pH of yeast peroxisomes. Arch Microbiol 1987, 147:37-41. 38. Dansen TB, Wirtz KW, Wanders RJ, Pap EH: Peroxisomes in human fibroblasts have a basic pH. Nat Cell Biol 2000, 2:51-53. 39. Jankowski A, Kim JH, Collins RF, Daneman R, Walton P, Grinstein S: In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin. J Biol Chem 2001, 276:48748-48753. 40. Hettema EH, Girzalsky W, Van Den Berg M, Erdmann R, Distel B: Saccharomyces cerevisiae Pex3p and Pex19p are required for proper localization and stability of peroxisomal membrane proteins. EMBO J 2000, 19:223-233.
23. Dodt G, Warren D, Becker E, Rehling P, Gould SJ: Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p. J Biol Chem 2001, 276:41769-41781.
41. Smith JJ, Marelli M, Christmas RH, Vizeacoumar FJ, Dilworth DJ, Ideker T, Galitski T, Dimitrov K, Rachubinski RA, Aitchison JD: Transcriptome profiling to identify genes involved in peroxisome assembly and function. J Cell Biol 2002, 158:259-271.
24. Purdue PE, Lazarow PB: Pex18p is constitutively degraded during peroxisome biogenesis. J Biol Chem 2001, 276:47684-47689.
42. Fukao Y, Hayashi M, Nishimura M: Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana. Plant Cell Physiol 2002, 43:689-696.
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43. Schneiter R, Brugger B, Sandhoff R, Zellnig G, Leber A, Lampl M, Athenstaedt K, Hrastnik C, Eder S, Daum G et al.: Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane. J Cell Biol 1999, 146:741-754. 44. Achleitner G, Gaigg B, Krasser A, Kainersdorfer E, Kohlwein SD, Perktold A, Zellnig G, Daum G: Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur J Biochem 1999, 264:545-553. 45. Voelker DR: Interorganelle transport of aminoglycerophospholipids. Biochim Biophys Acta 2000, 1486:97-107. 46. Novikoff AB, Shin WY: The endoplasmic reticulum in the Golgi zone and its relations to microbodies, Golgi apparatus and autophagic vacuoles in rat liver cells. J Microsc 1964, 3:187-206. 47. Faber KN, Heyman JA, Subramani S: Two AAA family peroxins, PpPex1p and PpPex6p, interact with each other in an ATPdependent manner and are associated with different subcellular membranous structures distinct from peroxisomes. Mol Cell Biol 1998, 18:936-943. 48. Erdmann R, Veenhuis M, Kunau WH: Peroxisomes: organelles at the crossroads. Trends Cell Biol 1997, 7:400-407. 49. Titorenko VI, Rachubinski RA: The endoplasmic reticulum plays an essential role in peroxisome biogenesis. Trends Biochem Sci 1998, 23:231-233. 50. Brul S, Wiemer EA, Westerveld A, Strijland A, Wanders RJ, Schram AW, Heymans HS, Schutgens RB, van-den-Bosch H, Tager JM: Kinetics of the assembly of peroxisomes after fusion of complementary cell lines from patients with the cerebrohepato-renal (Zellweger) syndrome and related disorders. Biochem Biophys Res Commun 1988, 152:1083-1089. 51. Ghaedi K, Tamura S, Okumoto K, Matsuzono Y, Fujiki Y: The peroxin Pex3p initiates membrane assembly in peroxisome biogenesis. Mol Biol Cell 2000, 11:2085-2102. 52. South ST, Sacksteder KA, Li XL, Liu YF, Gould SJ: Inhibitors of COPI and COPII do not block PEX3-mediated peroxisome synthesis. J Cell Biol 2000, 149:1345-1359. 53. Fujiki Y: Peroxisome biogenesis and peroxisome biogenesis disorders. FEBS Lett 2000, 476:42-46.
COP-mediated vesicle traffic in the early secretory pathway is not required to make peroxisomes. 57. South ST, Baumgart E, Gould SJ: Inactivation of the endoplasmic reticulum protein translocation factor, Sec61p, or its homolog, Ssh1p, does not affect peroxisome biogenesis. Proc Natl Acad Sci U S A 2001, 98:12027-12031. Protein traffic into the ER is not required to make peroxisomes. 58. Shimozawa N, Suzuki Y, Zhang Z, Imamura A, Kondo N, Kinoshita N, Fujiki Y, Tsukamoto T, Osumi T, Imanaka T et al.: Genetic basis of peroxisome-assembly mutants of humans, Chinese hamster ovary cells, and yeast: Identification of a new complementation group of peroxisome-biogenesis disorders apparently lacking peroxisomal-membrane ghosts. Am J Hum Genet 1998, 63:1898-1903. 59. Snyder WB, Faber KN, Wenzel TJ, Koller A, Lu¨ ers GH, Rangell L, Keller GA, Subramani S: Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris. Mol Biol Cell 1999, 10:1745-1761. 60. Baerends RJS, Rasmussen SW, Hilbrands RE, Van der Heide M, Faber KN, Reuvekamp PTW, Kiel JAKW, Cregg JM, van der Klei IJ, Veenhuis M: The Hansenula polymorpha PER9 gene encodes a peroxisomal membrane protein essential for peroxisome assembly and integrity. J Biol Chem 1996, 271:8887-8894. 61. Faber KN, Haan GJ, Baerends RJ, Kram AM, Veenhuis M: Normal peroxisome development from vesicles induced by truncated Hansenula polymorpha Pex3p. J Biol Chem 2002, 277:11026-11033. 62. Hazra PP, Suriapranata I, Snyder WB, Subramani S: Peroxisome remnants in pex3delta cells and the requirement of Pex3p for interactions between the peroxisomal docking and translocation subcomplexes. Traffic 2002, 3:560-574. 63. Criddle RS, Schatz G: Promitochondria of anaerobically grown yeast. I. Isolation and biochemical properties. Biochemistry 1969, 8:322-334. 64. Olsen LJ: The surprising complexity of peroxisome biogenesis. Plant Mol Biol 1998, 38:163-189. 65. Thieringer H, Moellers B, Dodt G, Kunau WH, Driscoll M: Modeling human peroxisome biogenesis disorders in the nematode Caenorhabditis elegans. J Cell Sci 2003, 116:1797-1804. 66. Parsons M, Furuya T, Pal S, Kessler P: Biogenesis and function of peroxisomes and glycosomes. Mol Biochem Parasitol 2001, 115:19-28.
54. Gould SJ, Valle D: Peroxisome biogenesis disorders – genetics and cell biology. Trends Genet 2000, 16:340-345.
67. Distel B, Erdmann R, Gould SJ, Blobel G, Crane DI, Cregg JM, Dodt G, Fujiki Y, Goodman JM, Just WW et al.: A unified Nomenclature for peroxisome biogenesis factors. J Cell Biol 1996, 135:1-3.
55. South ST, Gould SJ: Peroxisome synthesis in the absence of preexisting peroxisomes. J Cell Biol 1999, 144:255-266.
68. Titorenko VI, Rachubinski RA: Dynamics of peroxisome assembly and function. Trends Cell Biol 2001, 11:22-29.
56. Voorn-Brouwer T, Kragt A, Tabak HF, Distel B: Peroxisomal membrane proteins are properly targeted to peroxisomes in the absence of COPI- and COPII-mediated vesicular transport. J Cell Sci 2001, 114:2199-2204.
69. Lambkin GR, Rachubinski RA: Yarrowia lipolytica cells mutant for the peroxisomal peroxin Pex19p contain structures resembling wild-type peroxisomes. Mol Biol Cell 2001, 12:3353-3364.
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Current Opinion in Cell Biology 2003, 15:489–497
Peroxisome biogenesis: advances and conundrums Paul B Lazarow Investigations of peroxisome biogenesis in diverse organisms reveal new details of this unique process and its evolutionary conservation. Interactions among soluble receptors and the membrane peroxins that catalyze protein translocation are being mapped. Ubiquitination is observed. A receptor enters the organelle carrying folded cargo and recycles back to the cytosol. Tiny peroxisome remnants — vesicles and tubules — are discovered in pex3 mutants that lack the organelle. When the mutant is transfected with a good PEX3 gene, these protoperoxisomes acquire additional membrane peroxins and then import the matrix enzymes to reform peroxisomes. Thus, de novo formation need not be postulated. Dynamic imaging of yeast reveals dynamin-dependent peroxisome division and regulated actin-dependent segregation of the organelle before cell division. These results are consistent with biogenesis by growth and division of pre-existing peroxisomes. Addresses Mount Sinai School of Medicine, 1190 Fifth Avenue, Box 1007, New York, NY 10029-6574, USA e-mail: [email protected]
Here, we briefly summarize what is well established and discuss recent discoveries that add rich detail to our understanding of protein translocation into peroxisomes and point toward requisite future research. Other discoveries discussed herein illuminate peroxisome inheritance and clarify the controversies.
Peroxisome movement and segregation: differences between mammals, yeast and plants Dynamic observations of fluorescing peroxisomes in cultured mammalian cells reveal two types of movement. Most peroxisomes move slowly and randomly. A minority show fast, directional movement along microtubules [5,6] that depends upon cytoplasmic dynein [7]. The fast component is regulated by a signaling cascade: stimulation of Chinese hamster ovary (CHO) cells with extracellular ATP and lysophosphatidic acid together cause an arrest of peroxisome movement but not that of other organelles [8]. Segregation of peroxisomes to daughter cells at mitosis appears to be stochastic in mammalian cells [6].
Current Opinion in Cell Biology 2003, 15:489–497 This review comes from a themed issue on Membranes and organelles Edited by Alice Dautry-Varsat and Alberto Luini 0955-0674/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00082-6
Abbreviations CHO Chinese hamster ovary COP coat protein GFP green fluorescent protein PTS peroxisome targeting sequence
In Saccharomyces cerevisiae, in contrast, peroxisome movement does not use microtubules, but depends on the actin cytoskeleton and the actin-associated motor protein Myo2p [9]. Moreover, in this yeast, which typically possesses less than a dozen peroxisomes rather than the hundreds found in mammalian cells, the segregation of peroxisomes into the budding daughter cell is strictly regulated and depends upon an intact actin cytoskeleton (Figure 1). If a bud fails to receive at least one peroxisome, cytokinesis is significantly retarded [9]. This careful cellular surveillance ensuring that all daughter cells receive at least one peroxisome reminds us of similar surveillance for mitochondria and vacuoles [10]. It is consistent with the idea that peroxisomes form solely from pre-existing peroxisomes.
Introduction The major features of peroxisome biogenesis resemble those of mitochondria and chloroplasts, but the details are entirely different [1]. Like the latter two organelles, peroxisomes grow by the post-translational import of newly synthesized proteins into pre-existing organelles; these divide to form daughter peroxisomes, which segregate at cell division [2]. However, the proteins that catalyze peroxisome assembly (termed ‘peroxins’) and the molecular mechanisms of this assembly are unique (reviewed in depth in [3,4]). Controversies have concerned the suggestion that the organelle sometimes forms de novo and that essential membrane proteins could be donated by the endoplasmic reticulum (ER). www.current-opinion.com
In plants (onion epidermal cells or various cell types of Arabidopsis), peroxisome movement is also actin-based [11–13]. These species differences in the mechanism of peroxisome motility (which apply to the motility of other organelles as well) represent one of the exceptions to the generally remarkable evolutionary conservation of peroxisome biogenesis.
Peroxisome division involves dynamin The process of peroxisome division in S. cerevisiae depends, at least in part, on Vps1p, one of the three dynamin-like proteins in this organism [9]. Some Dvps1 cells contain a single large peroxisome and others contain Current Opinion in Cell Biology 2003, 15:489–497
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Figure 1
Dynamics of peroxisome segregation into S. cerevisiae buds in (a) wild-type cells and (b) in cells without dynamin Vps1p. Dividing yeast labeled with GFP–PTS1 were photographed at the times indicated (in minutes). (c) Co-localization of peroxisomes (red) and actin cables (green) in a budding wild-type cell. Videos from which these images were taken may be viewed at www.jcb.org/cgi/content/full/jcb.200107028/DC1. For full details, see [9]. Reproduced from The Journal of Cell Biology 2001, 155:979-990 [9] by copyright permission of the Rockefeller University Press. Bar ¼ 5 mm.
clusters and strings of small, interconnected peroxisomes that look as if they were caught in abortive fission. Nevertheless, peroxisomes do manage to divide at cytokinesis in Dvps1 cells, driving the conclusion that there is also a dynamin-independent peroxisome fission, perhaps based on mechanical pulling [9]. Peroxisome budding has also been observed in Arabidopsis [13]. The division and proliferation of peroxisomes is further regulated by Pex11p. Overexpression of Pex11p causes proliferation of small peroxisomes and/or elaboration of tubular peroxisomes in organisms as diverse as yeast [14] and trypanosomes [15], whereas deficiency of Pex11p results in a few large organelles. Rat liver Pex11p binds coat protein (COP) via a dilysine motif at its carboxyl terminus [16], as does trypanosome Pex11p, but this motif is not present in all Pex11p proteins and its mutational disruption does not abolish the function of the trypanosome protein [17]. Current Opinion in Cell Biology 2003, 15:489–497
Different mechanisms for peroxisome membrane elaboration and matrix protein import The insertion of newly synthesized proteins into the peroxisome membrane occurs by mechanisms distinct from those involved in the import of soluble enzymes into the organelle interior or ‘matrix’ (Figure 2). As a result, when the import of matrix proteins is defective, membrane ‘ghosts’ of peroxisomes can persist in cells and segregate at cell division. For instance, ghosts are present in skin fibroblasts of Zellweger syndrome neonates (which do not make a normal peroxisome after ovum fertilization) [18]. Three peroxins, Pex3p, Pex16p and Pex19p, are required to elaborate and maintain the membrane. One of these, Pex19p, is a soluble, mostly cytosolic, sometimes farnesylated protein that assists in bringing newly synthesized membrane proteins from their site of synthesis on free www.current-opinion.com
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Figure 2
ER-to-peroxisome phospholipid transfer (f) Induction/repression
Post-translational targeting/import
(d) PTS1
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11, 25 (b)
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7, 18, 20, 21 PTS2
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Protoperoxisomes Current Opinion in Cell Biology
Peroxisome biogenesis by growth and division. Numbers refer to PEX gene products. (a–e) Steady-state physiological formation. (a) Synthesis of peroxisomal proteins on free polyribosomes. (b) Import of matrix proteins via a branched pathway. This requires cytosolic peroxins (including Pex5p [blue], the PTS1 receptor and Pex7p [orange], the PTS2 receptor), as well as peroxisome membrane peroxins (PMPs) 1, 2, 4, 6, 8–10, 12–15, 17 and 22–24. Yellow ovals represent the still mysterious translocation machinery. (c) Insertion of membrane proteins. mPTS, targeting sequence to the peroxisome membrane. Requires cytosolic Pex19p (green) and membrane-bound Pex3p and Pex16p but not the other PEX gene products. (d) Insertion of membrane phospholipids, which are synthesized in the ER (see text for transport alternatives). (e) Peroxisome division. (f) Peroxisome abundance and size are regulated by the organism in response to nutrients, drugs, etc. (g,h) Pathological transitions owing to mutations or genetic complementation. (g) Mutations that cause loss of matrix protein import result in mostly empty membrane ghosts. Restoration of the wild-type gene allows the ghosts to re-acquire their normal content of proteins. (h) Mutations in PEX genes 3, 16 or 19 cause the inability to assemble most membrane proteins, and consequently loss of import of matrix proteins. This results in small, hard-to-detect, vesicular or tubular protoperoxisomes. Genetic complementation allows re-formation of ghosts followed by re-import of matrix proteins. Modified and reproduced with permission from the Annual Review of Cell and Developmental Biology, volume 17 [4] ß 2001 Annual Reviews (http://www.annualreviews.org).
polyribosomes through the cytosol to the peroxisomes, perhaps or in part by recognizing targeting information within these proteins. Pex3p and Pex16p are integral membrane proteins of the peroxisome membrane that are involved in insertion of new membrane proteins and/or in subsequent steps in the maintenance of the membrane. www.current-opinion.com
If any of these three peroxins is defective, neither normal peroxisomes nor membrane ghosts are present. Many other peroxins are required for matrix proteins to reach the peroxisome interior. The matrix proteins are directed to peroxisomes by one of several types of Current Opinion in Cell Biology 2003, 15:489–497
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peroxisome targeting sequences (PTS), of which two are well characterized: a carboxy-terminal tripeptide PTS1 (canonical sequence ¼ SKL) and an amino-terminal nanopeptide PTS2 (consensus sequence ¼ RLXXXXXHL, where X can be any amino acid). Soluble cytosolic receptors for each of these, Pex5p and Pex7p, respectively, bring newly synthesized protein cargo to peroxisomes and then recycle back to the cytosol to pick up more cargo. Pex5p does this job on its own, but Pex7p requires a species-specific auxiliary protein to help it: Pex18p or Pex21p in S. cerevisiae [19], Pex20p in Yarrowia lipolytica [20] and Neurospora crassa [21], or the longer of two splice isoforms of Pex5p in mammals [22,23]. These non-orthologous proteins possess a conserved sequence region that probably represents a common Pex7p binding site, suggesting the evolutionary conservation of a functional module rather than an entire protein [20,23].
Ubiquitination Unexpectedly, the auxiliary proteins, Pex18p and Pex21p, turn over with a half-life of less than 10 min [24]. This rapid degradation is tightly coupled to their function because mutations that block peroxisome assembly also arrest turnover. The stabilized Pex18p is in a cytosolic complex with the PTS2 receptor. Moreover, mono- and di-ubiquitinated forms of Pex18p are observed, predominantly in association with the peroxisomes [24]. Recent evidence suggests that Pex4p, a peroxisomemembrane-bound ubiquitin-conjugating enzyme, adds the second but not the first of these ubiquitins (PE Purdue and PB Lazarow, unpublished data). Three peroxins embedded in the peroxisome membrane contain RING fingers (Pex2p, Pex10p and Pex12p) and are candidates for E3 ubiquitin ligases. These and other data suggest the possibility that sequential ubiquitinations occur at, and perhaps regulate, discrete steps during docking of a Pex18p–PTS2 receptor–cargo complex on the surface of the peroxisome, delivery of the cargo to the matrix and recycling of the receptor, followed by degradation of Pex18p.
A receptor enters peroxisomes The receptors dock at a complex of three interacting membrane peroxins: Pex13p, Pex14p and Pex17p. The details of the receptors’ interactions with individual members of this complex are under intensive investigation [25–29]. Initially, it was thought that the receptors recycle right back into the cytosol after dropping their cargo at this docking site (a ‘simple shuttle’). However, recent experiments have clearly shown that Pex5p enters human peroxisomes during the course of its normal function and then re-emerges to the cytosol to carry out further rounds of import (an ‘extended shuttle’) [30]. The proteolytic cleavage of a Pex5p fusion protein was demonstrated within peroxisomes and the processed Pex5p was detected back out in the cytosol. Some question remains Current Opinion in Cell Biology 2003, 15:489–497
as to whether the Pex5p fusion protein went all the way into the peroxisome interior or merely poked itself far enough in to be acted upon by the interior protease.
Translocation remains mysterious What remains mysterious and challenges future experimentation is exactly what happens after docking. Several additional peroxins, as well as some mentioned above, are known to be required. It is clear that protein translocation into peroxisomes differs profoundly from transport into mitochondria, which threads unfolded polypeptide chains through a narrow channel. By contrast, peroxisomes can import folded and homooligomeric proteins [31], heterooligomers [32,33] and even 4–9 nm gold beads coated with albumin bearing PTS1s and microinjected into cultured cells [34]. Oligomer import is usually facultative, not obligatory; Candida boidinii peroxisomes import one enzyme as a pre-formed dimer and another, alcohol oxidase, as a monomer that is octamerized inside [35]. The transport of large cargo accompanied by a receptor somewhat resembles protein traffic into the nucleus. However, nothing resembling a nuclear pore complex has ever been observed in a peroxisome membrane. Although peroxisomes import large protein cargo, the peroxisome membrane is tight enough to require a variety of transporters for the passage of substrates and products of peroxisome metabolism (reviewed in [36]). It had been thought that the peroxisome is impermeable to protons and supports a pH difference between the interior of the organelle and the cytosol [37,38]; however, a recent experiment using a pH-sensitive variant of green fluorescent protein (GFP) casts doubt on this [39]. In any event, the protein-translocation mechanism must accommodate large cargo but prevent the passage of small organic molecules. Freeze-fracture studies of the peroxisome membrane have not been informative in this regard; no channel-forming proteins have been identified among the peroxins. One interesting speculation suggests that cargo of newly synthesized proteins might enter peroxisomes via membrane invaginations, a sort of peroxisomal pinocytosis [31]. The presence of internal membrane structures or fragments in certain peroxisome biogenesis mutants (e.g. [40]) lends indirect support to this hypothesis. It is also possible that there are additional peroxins crucial to translocation that have not yet been identified, despite the many clever screens and selections that have been carried out to find informative mutants. Normal peroxisomes are not necessary for yeast to grow on glucose, and the mutant isolations have exploited this fact. Searches for temperature-sensitive mutants have not been described. Lethal mutants may have been missed. Transcriptome profiling identified a novel peroxin, Pex25p, which affects peroxisome size and maintenance [41], and www.current-opinion.com
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proteomic analyses [42] also show promise for identifying new components.
arily diverse organisms an ER contribution of proteins appears doubtful.
What is the contribution of the endoplasmic reticulum to normal peroxisome biogenesis?
Do peroxisomes sometimes arise de novo?
Phospholipids
The phospholipids of the peroxisome membrane are mainly phosphatidylcholine and phosphatidylethanolamine, with a tendency toward somewhat longer-chain fatty acids than in other membranes [43]. These phospholipids are synthesized on the ER and transported to the peroxisome membrane by an unknown mechanism (see in [2]). This is also the case for mitochondria, most of the phospholipids of which are made in the ER and perhaps move from ER to mitochondria at sites of close apposition [44,45]. This type of transfer could also apply to peroxisomes, and this suggestion is supported by old morphological data on the adjacency of ER and peroxisomes [46]. Alternatively, we have speculated that specialized vesicles might carry phospholipids from ER to peroxisomes [4]. Such vesicles might contain one or a few proteins that specify the destination. Interestingly, small vesicles that are distinct from mature peroxisomes have been identified in Pichia pastoris, which bear two peroxins, Pex1p and Pex6p [47]. These peroxins are members of the functionally diverse AAA family of ATPases, some of the members of which are involved in membrane-fusion events. It is tempting to speculate that these vesicles might ferry phospholipids to the peroxisomes. Proteins
There have been occasional reports during the past decade that certain peroxisome membrane proteins can be found in the ER. This led to proposals that the ER might contribute a subset of the peroxisome membrane proteins via budding vesicles that either fuse with peroxisomes [48] or mature into peroxisomes [49]. These ‘new’ or ‘immature’ peroxisome vesicles were suggested to contribute to normal peroxisome biogenesis [48,49], and this became a topic of considerable discussion and debate within the peroxisome community. Leaving aside temporarily the exceptional case of Y. lipolytica [49], the evidence for other organisms consisted mainly of finding certain peroxisome membrane proteins in the ER in experimentally engineered situations. Careful review of the data, which space considerations preclude repeating here, suggests that most such cases were artefacts of protein overexpression, or artefacts of cutting and splicing of membrane proteins [4]. For example, in wild-type S. cerevisiae, newly synthesized endogenous Pex15p inserts directly into peroxisome membranes in a Pex3pand Pex19p-dependent fashion (as shown in Figure 2), without visiting the ER, but overexpression of Pex15p leads to its accumulation in ER membranes [40]. Thus, under normal physiological circumstances, in evolutionwww.current-opinion.com
But what happens in mutants that lack peroxisomes when resumption of peroxisome biogenesis is induced experimentally? In the many mutants in which matrix protein import is defective (Figure 2), membrane ghosts are present that provide the structural basis to reform normal peroxisomes when the missing peroxin is restored by genetic complementation. This can be rapid (half-time of 10 min) if cells of different complementation groups are fused and no new protein synthesis is needed, or a matter of hours if gene expression is required [50]. However, three mutants (pex3, pex16 and pex19) have defects in peroxisome membrane assembly and lack ghosts as well as peroxisomes. Nonetheless, they reform peroxisomes when the missing gene is restored by transfection. This process occurs slowly and stepwise: first membranes appear which subsequently fill with matrix enzymes in a process that may take days (e.g. [51,52]). In these cases, it has been suggested that the organelle forms de novo [53,54]. But is the evidence for de novo formation compelling and how might this occur?
Protein translocation through the endoplasmic reticulum is not required to form peroxisomes If peroxisomes were ever to form de novo, the ER would be an obvious potential source of new membrane vesicles, which need only contain the crucial peroxins that act first to elaborate the organelle [48,49]. These are the integral membrane proteins Pex3p and Pex16p, which, together with the soluble Pex19p, are required for the assembly of other peroxisomal membrane proteins, which in turn allow the import of matrix proteins (Figure 2). With this in mind, the biogenesis of these two proteins has been intensively investigated in normal human fibroblasts: they never were observed in ER; the first place they could be detected after their biosynthesis was in the peroxisomes; this transport to peroxisomes was unaffected by inhibitors of COPI or COPII [52,55,56]. Moreover, the reformation of normal peroxisomes in pex3 and pex16 mutants upon rescue with the appropriate wild-type gene was unaffected by inhibitors of COPI or COPII [52,55]. Perhaps the putative peroxisome-forming vesicles bud out of the ER before the COPI- or COPII-mediated steps? This was tested by using mutant S. cerevisiae defective in SEC61, an essential component of the ER translocon, or its homolog SSH1. These mutations prevent proteins from entering the ER. Peroxisome biogenesis proceeds normally in these mutants [57]. If the ER does not provide new membrane to reform peroxisomes, where do de novo peroxisomes come from? Vesicles of indeterminate origin have been postulated [54]. Current Opinion in Cell Biology 2003, 15:489–497
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The existence of protoperoxisomes makes de novo formation and new vesicles unnecessary The evidence for the absence of typical peroxisome membrane ghosts in pex3, pex16 and pex19 mutants is compelling: ghosts have been sought aggressively in these mutants with a battery of antisera against membrane proteins that are found in ghosts in other mutants, and the results have been consistently negative [40,55,58]. However, these data do not preclude the existence of peroxisomal membrane remnants, perhaps smaller or with a more limited repertoire of proteins, that might have been missed by the methods used. Recent experiments provide strong indications for the existence of such structures, which we have termed ‘protoperoxisomes’ [4]. Tiny peroxisomal vesicles and tubules were first discovered in the P. pastoris Dpex19 mutant by deconvolution microscopy and an antibody that recognizes endogenous Pex3p [59]. These structures were then visualized by
electron microscopy (Figure 3). There is evidence of similar protoperoxisomes in several pex3 mutants. The first 40–50 amino acids of Pex3p, fused in front of GFP, are sufficient to anchor GFP to the surface of peroxisomes in a variety of species. This construct serves as a useful probe for peroxisomal membranes because it has never been found to insert in any other endomembrane, even when overexpressed (nor has wild-type Pex3p, which may instead lead to the presence of many small peroxisomes when overexpressed [60]). This GFP construct labels wild-type peroxisomes in CHO cells and also detects small vesicles in a CHO pex3 mutant (see Figure 6d of [51]). Such vesicles were found similarly in pex3 mutants of S. cerevisiae (K Huang and PB Lazarow, unpublished data) and Hansenula polymorpha [61]. In the latter case, the GFP vesicles were shown to contain a second peroxisome membrane protein, Pex14p. Moreover, the vesicles had a characteristic property of wild-type H. polymorpha peroxisomes in that they were susceptible to selective degradation when cells were shifted to growth on glucose.
Figure 3
∆pex19
Wild type (a)
0.7 µm
(b)
p v
p
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m n m 1.0 µm ∆pex3
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(d)
Current Opinion in Cell Biology
Protoperoxisomes in Dpex3 and Dpex19 mutants of P. pastoris. (a,b) Electron microscopy of (a) wild type and (b) Dpex19 cells. m, mitochondrion; n, nucleus; p, peroxisomes; v, vacuole. The dashed white box in (b) indicates the tiny vesicles and tubules in the mutant, identified as peroxisomal by deconvolution microscopy. Reproduced from [59], with permission by the American Society for Cell Biology. (c,d) Immunofluorescence with antibodies against Pex8p to identify peroxisome membranes (green) and MitoTracker to label mitochondria (red): (c) wild type, (d) Dpex3. Bar ¼ 1 mm. Reproduced with permission from [62], ß Blackwell Publishing Ltd. Current Opinion in Cell Biology 2003, 15:489–497
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Recently, the use of previously unavailable antibodies against endogenous Pex8p, Pex12p, Pex14p and Pex17p has provided compelling evidence for peroxisome remnants in the P. pastoris Dpex3 mutant. Vesicles and tubular, torpedo-shaped peroxisomal structures were observed with deconvolution microscopy (Figure 3) and characterized by isopycnic and flotation gradient centrifugation [62]. These ‘protoperoxisomes’ can provide the structural basis to reform the normal peroxisome compartment (see Figure 2) and have been shown to do so in the case of H. polymorpha [61]. Thus, there is no need for de novo formation in these species and mutants. This methodology has not yet been tried in pex19 mutants in other species, nor in pex16 mutants. Peroxisomal remnants could easily have been missed in these cells. The role of protoperoxisomes in reforming peroxisomes following gene rescue is analogous to the role of promitochondria in reforming mitochondria when anaerobically grown yeast receive oxygen [63].
Evolutionary conservation There is a remarkable degree of evolutionary conservation in the mechanisms of peroxisome assembly and many peroxins are well-conserved among yeasts and mammals and in other diverse species including plants [64], Caenorhabditis elegans [65] and trypanosomes [66]. This has allowed the formulation of a unified, species-independent PEX gene nomenclature [67]. It has permitted many genes responsible for inherited human disorders of peroxisome biogenesis to be identified by virtue of their similarity to yeast genes [54]. Inevitably there are some species differences, but for the most part these have been minor. One striking exception is Y. lipolytica [49,68]. Normal peroxisome biogenesis in this organism is said to begin by the COP-dependent budding of a vesicle from the ER that contains core-glycosylated Pex2p and Pex16p. This vesicle is then reported to undergo a series of maturation steps that result in its gradual conversion to a mature peroxisome. Pex16p and Pex19p have different functions in this organism than in others (e.g. normal-looking peroxisomes exist in Dpex19 [69]). The species differences are not confined to peroxisome biogenesis; for example, four distinct secretory pathways are reported to diverge at the level of the ER in Y. lipolytica [49]. Certain defects in secretion impair peroxisome biogenesis. Conversely, defects in the PTS1 receptor inhibit secretion, apparently from inside peroxisomes. These curious features have been observed so far in one organism in one laboratory.
branes are believed to come from other membranes, not assembled from scratch. The apparent absence of peroxisomal structures in the pex3, pex16 and pex19 mutants was the motive to postulate de novo formation. The discovery of protoperoxisomes in P. pastoris Dpex19 and in Dpex3 mutants of several species removes the motive, at least in those species. An alternative, the trafficking of crucial proteins through the ER and vesicle budding, has been refuted, at least in human fibroblasts. Protoperoxisomes are remnants of once-functional peroxisomes, before a mutation; they are not ‘new’ and not ‘young’. At least in S. cerevisiae, careful regulation ensures that a minimum of one peroxisome is segregated into each daughter yeast cell. These results support the sufficiency of the simple model that peroxisomes form by growth and division of pre-existing peroxisomes. There is a superficial similarity between the model of Figure 2 and some proposed models of de novo formation. Recovery of peroxisomes after gene rescue definitely occurs stepwise. The key point is that the several types of peroxisomal structures shown in Figure 2 (including ghosts and remnants) can be interconverted and thus need not be made de novo. The field lacks a simple model to account for the facts of protein translocation into peroxisomes. Exciting new results stimulate the imagination: complexes of proteins in the membrane, a PTS receptor that enters the organelle and recycles back to the cytosol, stepwise ubiquitinations, large cargo. . . This is a challenge for future research.
Acknowledgements The work of the author was supported by the National Institutes of Health, grant DK19394. Owing to space constraints, this review discusses selected recent highlights – apologies to colleagues whose work was omitted. Few papers before 2000 are cited; they may be found in previous reviews. I thank S Subramani and HF Tabak for permission to reproduce their figures.
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