Báo cáo khoa học: Peroxisomes as dynamic organelles: peroxisome abundance in yeast docx

MINIREVIEW Peroxisomes as dynamic organelles: peroxisome abundance in yeast Ruchi Saraya, Marten Veenhuis and Ida J. van der Klei Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kluyver Centre for Genomics of Industrial Fermentation, Haren, The Netherlands Introduction Eukaryotic cells are characterized by the presence of specific compartments, the organelles. The advantages of compartmentalization may include the creation of unique microenvironments with specific (bio)chemical properties to improve the efficiency of certain pro- cesses or to provide additional pathways for regula- tion. To form and maintain these compartments, highly complex mechanisms exist in eukaryotic cells. Peroxisomes represent an important class of organ- elles that are present in almost all eukaryotes [1]. Their function and significance varies with the organism in which they occur, their developmental stage and envi- ronmental conditions. They are generally involved in the metabolism of reactive compounds, such as hydro- gen peroxide or glyoxylate [1]. In yeast, peroxisomes are predominantly involved in the metabolism of various unusual carbon and nitrogen sources, such as oleic acid, methanol, d- amino acids and purines [2]. Upon transfer of glucose- grown yeast cells to media containing these com- pounds, the number and size of peroxisomes shows a strong increase. The biogenesis of peroxisomes depends on the func- tions of unique genes (termed PEX genes). At present, over 30 PEX genes have been identified, most of which are involved in the process of matrix protein import [3]. Two PEX genes (PEX3 and PEX19) have been impli- cated in the targeting and insertion of peroxisomal membrane proteins. The remaining PEX genes are involved in regulating organelle size and numbers [4]. Conceptually, peroxisome abundance is a result of the rate of development (fission, de novo synthesis) rel- ative to the rate of (autophagic) degradation and reduction via the segregation of organelles to daughter Keywords de novo synthesis; fission; organelle inheritance; peroxisomes; yeast Correspondence I. J. van der Klei, Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), PO Box 14, NL-9750 AA Haren, The Netherlands Fax: +31 (0)50 363 8280 Tel: +31 (0)50 363 2179 E-mail: I.J.van.der.klei@rug.nl (Received 2 March 2010, revised 23 April 2010, accepted 17 May 2010) doi:10.1111/j.1742-4658.2010.07740.x Peroxisomes are cell organelles that are present in almost all eukaryotic cells and involved in a large range of metabolic pathways. The organelles are highly dynamic in nature: their number and enzyme content is highly variable and continuously adapts to prevailing environmental conditions. This review summarizes recent relevant developments in research on pro- cesses that are involved in the regulation of peroxisome abundance and maintenance. These processes include fission of the organelles, formation of new peroxisomes from the endoplasmic reticulum, autophagic degradation and segregation ⁄ inheritance during cell division. Abbreviations DRP, dynamin-related protein; PEX, peroxisome gene. FEBS Journal 277 (2010) 3279–3288 ª 2010 The Authors Journal compilation ª 2010 FEBS 3279 cells during cell division (Fig. 1). The extent to which these processes control peroxisome numbers in a spe- cific cell is still largely unknown. In this review we summarize the current knowledge of the various pro- cesses regulating peroxisome abundance in yeast. Modes of peroxisome formation For decades, the classical view on peroxisome prolifer- ation was that the organelles are autonomous and rep- licate by fission. This growth-and-fission model was supported by the finding that peroxisomal membrane and matrix proteins are synthesized on free polysomes and post-translationally incorporated into pre-existing organelles. In this view the close connection of growing peroxisomes with the endoplasmic reticulum was inter- preted to mean the endoplasmic reticulum served as a source of membrane lipids. More recently, evidence has accumulated in support of a model suggesting that peroxisomes can arise de novo from the endoplasmic reticulum. This phenomenon was in observed par- ticularly in specific peroxisome-deficient (pex) yeast mutants (which lack any peroxisome membrane rem- nants) upon re-introduction of the corresponding gene [5–8]. Recently, it became clear that the two machiner- ies – de novo synthesis from the endoplasmic reticulum and fission of pre-existing peroxisomes – may occur simultaneously, especially in higher eukaryotes and in the yeast Yarrowia lipolytica [9–12]. However, in wild-type strains of the yeasts Saccharomyces cerevisiae and Hansenula polymorpha this is probably not true, because in these species peroxisomes seem to prolifer- ate exclusively by fission [13,14]. In these yeast species de novo formation is only observed during conditions when the cells lack peroxisomes, in which, by defini- tion, peroxisomes cannot be formed by fission of a pre-existing organelle. Peroxisome formation from the endoplasmic reticulum De novo peroxisome formation has predominantly been studied in cells with a defect in PEX3. Yeast pex3 cells lack any peroxisome membrane structures, but intact peroxisomes re-appear after re-introduction of the cor- responding deleted gene. Upon re-introduction of the PEX3 gene in pex3 cells, the Pex3 produced is first sorted to the perinuclear or cortical endoplasmic retic- ulum, after which it colocalizes with Pex19 in specific compartments, termed pre-peroxisomes. In this sce- nario, Pex3 is suggested to be essential for the forma- tion of this initial vesicular subcompartment where it serves as a docking site for Pex19–peroxisomal mem- brane protein complexes that are essential to direct Fig. 1. Hypothetical model of peroxisome abundance. In wild-type yeast cells peroxisome numbers may be maintained by a balance between four processes. (1) Peroxisome formation from the endoplasmic reticulum (involving Pex3), during which a pre-peroxisomal struc- ture is formed that grows by importing newly synthesized peroxisomal membrane and matrix proteins to form a mature peroxisome. (2) Per- oxisome fission (involving Pex11 and DRPs), during which a mature peroxisome first elongates, and then divides, to form a new small peroxisome that can grow to form a mature peroxisome. (3) Peroxisome inheritance (involving Myo2, Inp2, Inp1, Pex3 and Pex19), in which peroxisomes are faithfully inherited into the newly formed bud (3a) and ⁄ or are retained in the mother cell (3b) during cell division. (4) Peroxi- some degradation, when redundant ⁄ exhausted organelles are degraded in the vacuole. Peroxisome abundance in yeast R. Saraya et al. 3280 FEBS Journal 277 (2010) 3279–3288 ª 2010 The Authors Journal compilation ª 2010 FEBS various peroxisomal membrane proteins to this newly formed structure. Once a functional peroxisomal matrix protein import complex is formed, these pre- peroxisomal structures will import matrix proteins (see the review by Wolf et al. [15] in this miniseries), grow and subsequently multiply by fission. However, the molecular details of this pathway, for instance the principles of Pex3 docking and subsequent vesicle for- mation from the endoplasmic reticulum (which is pre- dominantly resolved by advanced live-cell imaging techniques), are still an enigma. The endoplasmic reticulum, as a template for de novo peroxisome formation during complementation of yeast pex3 cells, is not debated. During this process, Pex3 is first targeted to the endoplasmic reticulum and, at a later stage, is present at the peroxisomal mem- brane. However, whether Pex3 invariably traffics via the endoplasmic reticulum to peroxisomes (i.e. also in wild-type cells), is still uncertain. In fact, recent data in mammalian cells [16,17] indicated that newly synthe- sized Pex3 protein can also directly sort to pre-existing peroxisomes. In wild-type cells, peroxisomal membrane proteins other than Pex3 have also been suggested to travel via the endoplasmic reticulum to pre-existing organelles. However, the transient localization of certain peroxi- somal membrane proteins at the endoplasmic reticu- lum (e.g. Pichia pastoris Pex30 and Pex31 [18] may also be related to other processes. For instance, a vesicular transport pathway has been suggested to exist which transports endoplasmic reticulum-derived lipids, together with certain peroxisomal membrane proteins, to peroxisomes. However, recent data indicate that phopholipids are probably directly transferred from the endoplasmic reticulum to peroxisomes without vesicular transport [19]. Hence, the physiological role of the localization of certain peroxisomal membrane proteinss at the endoplasmic reticulum needs further analysis. The peroxisome fission machinery Based on studies of the function of Pex11b in mam- malian cells, the process of peroxisome fission has been proposed to involve four, partially overlapping, consecutive steps, namely (a) the insertion of Pex11b into the membrane, (b) the elongation of peroxisomes, (c) the segregation of Pex11b and the formation of Pex11b-enriched patches and (d) the division of per- oxisomes [20,21] (see also Fig. 2). Pex11b (or its homolog in other organisms) is important for the ini- tial stages of peroxisome fission (steps a–c), whereas the organelle fission machinery is responsible for the final step (d). The yeast homolog of mammalian Pex11b is Pex11. Upon overexpression of S. cerevisiae PEX11, elongated clusters of peroxisomes were observed and the cyto- plasm of the cells was crowded with peroxisomes [22,23]. In contrast, deletion of PEX11 resulted in a strong decrease in peroxisome numbers, which was paralleled by a strong increase in size. Very similar observations have been made in many other organisms (e.g. filamentous fungi, trypanosomes and human cells; reviewed previously [21]), indicating that the role of Pex11 in peroxisome elongation is highly conserved. Of all the PEX genes known, the expression levels of PEX11 are enhanced most when peroxisome prolif- eration is induced. This is true upon shifting S. cerevi- siae cells from glucose- to oleic acid-containing media [24,25], as well as for H. polymorpha cells shifted from glucose to methanol [26]. Hence, modulating Pex11 levels is an important mode to vary peroxisome abun- dance. Unexpectedly, mammalian Pex11b is not induced by peroxisome proliferators. AB C Fig. 2. Morphological stages of peroxisome fission. Ultrathin sections of KMnO 4 -fixed cells grown in a methanol-limited chemostat at D = 0.12 h )1 , demonstrating the three stages involved during peroxisome inheritance: (A) elongation into the bud; (B) separation of a small organelle; and (C) the actual fission and migration of a small organelle into the bud. The bar represents 0.5 lm. R. Saraya et al. Peroxisome abundance in yeast FEBS Journal 277 (2010) 3279–3288 ª 2010 The Authors Journal compilation ª 2010 FEBS 3281 Recently, Knoblach & Rachubinski [27] showed that in vivo S. cerevisiae Pex11 exists in two isoforms, namely a phosphorylated form and a dephosphorylated form. Interestingly, studies using PEX11 phosphomim- icking mutants indicated that strains producing only constitutively dephosphorylated Pex11 show a pheno- type similar to that of pex11 cells, whereas strains producing constitutive phosphorylated Pex11 show enhanced peroxisome proliferation, similar to that of Pex11-overproducing cells. This suggests that Pex11 phosphorylation may include a mechanism to regulate Pex11 activation ⁄ inactivation. A recent study of 249 S. cerevisiae kinase- and phos- phatase-deletion strains [28] indeed indicated that phosphorylation processes are crucial in regulating per- oxisome abundance. In particular, deletion of PHO85, a cyclin-dependent kinase, had a strongly negative effect on peroxisome numbers. Interestingly, overex- pression of PHO85 results in hyperphosphorylation of Pex11 and peroxisome proliferation [27]. The second class of proteins essential for peroxisome fission is the family of dynamin-related proteins (DRPs). DRPs are large GTPases that are involved in membrane fission and fusion events. In S. cerevisiae, two DRPs – Vps1 and Dnm1 – play a role in peroxi- some fission. Dnm1 also plays a role in mitochondrial fission. Dnm1 is, in particular, essential for peroxisome fission during conditions of peroxisome induction by oleate [29], whereas Vps1 functions in peroxisome rep- lication under repressing conditions (e.g. in the pres- ence of glucose). Dnm1 is recruited to peroxisomes via two homologus proteins, Mdv1 and Caf4, which are associated with the peroxisomal membrane via the tail- anchored protein, Fis1 [30]. Mdv1 is a WD repeat pro- tein, which is absent in higher eukaryotes. Caf4 is an Mdv1 paralog in S. cerevisiae that is absent in other organisms. In S. cerevisiae, Vps1 is involved in peroxisome fis- sion [29]; however, in H. polymorpha, Vps1 does not play a role in this process [14]. In this respect, H. poly- morpha seems to be more similar to mammalian and plant cells, where a single DRP (Dlp1 or DRP3A, respectively) is involved in peroxisome fission. Interest- ingly, in Arabidopsis thaliana, it has been shown that the DRP 5B is responsible for the fission of chlorop- lasts as well as of peroxisomes [31]. Additionally in A. thaliana it has been shown that three out of five PEX11 isoforms (PEX11c, PEX11d and PEX11e) are important in the recruitment of Fis1b to the peroxi- some membrane for the replication of pre-existing per- oxisomes [32]. Similarly, in mammals, Fis1 interacts with Pex11b [33]. As for other peroxisomal membrane proteins, Pex19, a peroxin important for peroxisomal membrane biogenesis, is also important for the target- ing of Fis1 to peroxisomes in mammals [34]. Remarkably, the Fis1–DRP organelle fission machinery was initially identified as being responsible for mitochondrial fission [30]. Indeed, Fis1 and Dnm1 show a dual localization on peroxisomes and mito- chondria. In contrast to peroxisomal Fis1, no proteins involved in Fis1 targeting to mitochondria have yet been identified. Why both organelles share the same fission machin- ery is unknown, but this may serve as a mechanism to coordinate mitochondrial and peroxisome fission (e.g. during the cell cycle). Fluorescence microscopy studies in H. polymorpha revealed that green fluorescent pro- tein (GFP)-conjugated Dnm1 is not evenly distributed over the cytosol, but is present as multiple spots that contain many GFP–Dnm1 molecules. Interestingly, Mdv1 co-localizes with these Dnm1 spots. Live cell imaging revealed that these spots dynamically associate and disassociate from mitochondria and peroxisomes, stressing the fact that the same protein molecules are involved in the fission of both organelles [35]. Peroxisome fission in H. polymorpha is fully blocked upon the deletion of DNM1 [14]. These cells contain a single, enlarged peroxisome, which forms a long exten- sion that protrudes into the developing bud. These extensions are not observed in dnm1 pex11 cells, which is in agreement with the model in which Pex11 plays a role in peroxisome elongation. Notably, as in mamma- lian cells [20], Pex11 is concentrated at the base of these peroxisome extensions in dnm1 cells, indicating that also in yeast the third step in peroxisome fission is the segregation of Pex11 and the formation of Pex11- enriched patches. Other proteins implemented in peroxisome development and abundance Besides Pex3, Pex11 and Fis1 ⁄ DRPs as key compo- nents in determining organelle development and abun- dance, other proteins have been identified as regulators of these processes. These include components that were initially identified in the secretory pathway and various recently identified peroxins, and are discussed in more detail below. Components of the secretory pathway Several proteins known to play a role in the secretory pathway and localized to membranes of compartments involved in this pathway (e.g. endoplasmic reticulum, Golgi, COP vesicles) have been suggested to play a Peroxisome abundance in yeast R. Saraya et al. 3282 FEBS Journal 277 (2010) 3279–3288 ª 2010 The Authors Journal compilation ª 2010 FEBS role in peroxisome abundance. These proteins may be important for de novo peroxisome formation or for the delivery of endoplasmic reticulum-derived lipids to the peroxisomal membrane. A recent study indicated a possible role for S. cerevi- siae SEC39, SEC21 and DSL in the trafficking of per- oxisomal membrane proteins from the endoplasmic reticulum to the peroxisome [36]. S. cerevisiae ARF1 and ARF3 were also proposed to work antagonistically during peroxisome proliferation [37]. Emp24 is a protein of the p24 family of proteins and localizes to the Golgi apparatus, endoplasmic reticulum and COP vesicles [38]. However, a detailed proteomics study in S. cerevisiae suggested that Emp24 is also localized to peroxisomes [39]. Moreover, in the yeast H. polymorpha, Emp24 was localized to peroxi- somes and the endoplasmic reticulum [40]. Interest- ingly, deletion of EMP24 in H. polymorpha resulted in a strong reduction in peroxisome number. Unexpect- edly, this was not caused by a defect in the formation of peroxisomes from the endoplasmic reticulum, but by a defect in peroxisome fission. Possibly, p24 pro- teins are required to bring various components involved in peroxisome fission together at the peroxi- somal membrane to allow organelle elongation at the initial stage of peroxisome fission. A similar function has recently been suggested for caveolin-1 at peroxisomes in mammalian cells [41]. Caveolin-1 is crucial for the formation of caveolae, subtypes of microdomains ⁄ rafts that are morphologi- cally recognizable as flask-like invaginations in the plasma membrane. Recent localization studies in rat hepatocytes revealed that caveolin-1 is also enriched in the peroxisomal membrane. A function for this protein at the peroxisomal membrane, however, has not yet been established. Peroxins Besides Pex11, two other members of the S. cerevisiae Pex11 family – Pex25 and Pex27 – play a role in per- oxisome proliferation [23]. Data obtained from the analysis of overexpression strains suggest that both peripheral membrane proteins function in organelle fis- sion, in particular under conditions when proliferation of the organelles is repressed. Also, proteins of the Pex24 protein family (Pex24, Pex28 and Pex29) are involved in regulating peroxisome numbers. All three proteins are components of the peroxisomal mem- brane, of which Pex24, but not Pex28 and Pex29, is induced by growth conditions that promote peroxi- some proliferation (i.e. oleate). Remarkably, deletion of PEX28 and PEX29 in S. cerevisiae is accompanied by increased numbers of reduced-size organelles [42]. In addition, three other oleate-inducible baker’s yeast proteins (Pex30, Pex31 and Pex32), which show homol- ogy towards Y. lipolytica Pex23, have been shown to be involved in regulating peroxisome numbers [43]. Peroxisome inheritance During vegetative reproduction of wild-type yeast cells, organelle replication is essential for maintaining the organelle population in the mother cells during multi- ple rounds of budding. Upon division, part of the organelle population is administered to the bud. In the methylotrophic yeast H. polymorpha, this is accompa- nied by asymmetrical peroxisome fission and subse- quent migration of the newly formed, small organelle to the developing bud. The number of organelles migrating to the bud is dependent on the culture con- ditions [44] (Fig. 3). In yeast, peroxisome inheritance requires the func- tion of Inp1, Inp2, the class V myosin motor (Myo2) and the actin skeleton [45–47]. Of these, Inp1 has been identified as the peroxisome-specific retention factor, connecting peroxisomes that are retained in the mother cells to a yet-unknown anchoring structure. Similarly, Inp1 is also implemented in the retention of peroxi- somes in developing buds [45,48]. Unexpectedly, in the absence of Pex11, peroxisome retention is also defec- tive in H. polymorpha, despite the fact that Inp1 is properly localized to peroxisomes [48]. Hence, Pex11 may have a second function in organelle retention in addition to its role in peroxisome fission. Recently, a function in peroxisome inheritance was also attributed to Pex3 [49]. In an elegant study, Munck et al. [49] demonstrated that Pex3 also func- tions in peroxisome retention. The authors showed that Pex3 interacted directly with Inp1 at the peroxi- somal membrane and suggested a role for Pex3 to recruit Inp1 to the peroxisomal membrane. Impor- tantly, the Inp1-binding region in the Pex3 protein could be separated from the regions involved in membrane formation during the de novo synthesis of peroxisomes [49]. Hence, Pex3 is a multifunctional pro- tein in peroxisome biology, implemented in formation of the peroxisome membrane and organelle inheritance. Inp2 is a peroxisomal membrane protein that acts as the peroxisomal receptor for Myo2 and attaches the globular tail of Myo2 to the peroxisome, thus allowing transport of the organelle to the bud [46]. Recently, the region of Myo2 involved in Inp2 binding was iden- tified using mutant variants of Myo2 [50]. These stud- ies also showed that Inp2 is a phosphoprotein whose level of phosphorylation is coupled to the cell cycle. R. Saraya et al. Peroxisome abundance in yeast FEBS Journal 277 (2010) 3279–3288 ª 2010 The Authors Journal compilation ª 2010 FEBS 3283 Chang et al. [51] recently suggested that Inp2 is unique for baker’s yeast and related species and pro- vided evidence that Y. lipolytica Pex3 and its paralog, Pex3B, function as the peroxisome-specific receptors of Myo2. A similar function was attributed to baker’s yeast Pex3. However, in a subsequent study, Saraya et al. [52] demonstrated that Inp2, although weekly conserved, is also present and functional in other yeast species, including H. polymorpha. The finding that H. polymorpha Inp2 interacted with Myo2 points to a conserved function for this protein as a binding factor for Myo2. Remarkably, in H. polymorpha, Myo2–Inp2 binding was dependent on Pex19. This is consistent with the view that Pex19 may have a stabilizing role in the interaction between Inp2 and Myo2, and also is in line with the observed defect in peroxisome inheritance in H. polymorpha pex19 cells [53]. Constitutive peroxisome degradation Peroxisomal membrane proteins are generally post- translationally incorporated into the organelle mem- brane. This implies that the main quality-control systems for these proteins reside outside the organelle (i.e. in the cytosol). However, peroxisomes do contain a few specific proteases that are implemented in the removal of exhausted or nonfunctional matrix pro- teins. Although different protease activities have been detected in peroxisomes [54], so far only one gene encoding a peroxisomal protease, a Lon protease, has been identified in yeast, in contrast to mammals where up to three proteases have been identified [55]. Peroxisomal Lon of H. polymorpha degrades short- lived or nonfunctional components of the peroxisomal lumen and therefore may participate in a housekeep- ing process aimed at maintaining a functional peroxi- some population. In the absence of Lon, protein aggregates may accumulate in the organelle lumen. Such protein aggregates are probably devastating for organelle function and require removal of the entire organelle to maintain cell vitality. Recent studies in human cells suggested that the peroxisomal Lon pro- tease is involved in accurate sorting, processing and activation of the peroxisomal enzyme acyl CoA oxidase [56]. Redundant organelles are removed by selective per- oxisome autophagy (see the review in this miniseries by Oku & Sakai [57] for details). However, constitutive removal of peroxisomes is observed in H. polymorpha when cultured under conditions that promote organelle proliferation. Hence, under conditions of peroxisome induction, development and degradation of the organ- elles occurs simultaneously. The data from Bener Aksam et al. [55] suggest that constitutive peroxisome degradation suppresses the negative effects of deletion of LON. This is indicated by the observation that in an ATG1 deletion background, in which peroxisome turnover is inhibited, deletion of the gene encoding AB Fig. 3. Peroxisome inheritance numbers vary with environmental conditions. In budding cells of methanol-limited cultures of Hansenula poly- morpha cells grown at high dilution rates (A; D = 0.12 h )1 ), generally only a single peroxisome is inherited to the bud, whereas several small organelles are inherited to buds in cultures grown at low dilution rates (B; D = 0.03 h )1 ). Electron micrographs of thin sections are shown. Cells are fixed in KMnO 4 . The bar represents 1 lm. Peroxisome abundance in yeast R. Saraya et al. 3284 FEBS Journal 277 (2010) 3279–3288 ª 2010 The Authors Journal compilation ª 2010 FEBS peroxisomal Lon resulted in a decrease of cell viability. This is consistent with the view that timely removal of these organelles is essential for cell viability. Untimely removal of peroxisomes may result in detrimental effects (i.e. the accumulation of reactive oxygen spe- cies, finally resulting in cell death) [55]. Constitutive degradation of peroxisomes in H. polymorpha is an autophagic process and thus requires the function of ATG genes. However, the precise sequence of events that mediate this constitutive degradation process is still unknown and awaits further elucidation. One pos- sibility is that, similarly to mitochondria, fission pro- cesses may be involved that allow separation of dysfunctional, aggregate-containing parts, which are specifically recognized for degradation [58]. Perspectives Peroxisomes are extremely flexible and dynamic organ- elles. Several cues are known that cause rapid changes in their abundance. During recent years much progress has been made in the identification and analysis of genes involved in changing organelle abundance. How- ever, except for the proteins of the Fis1 ⁄ DRP organelle fission machinery, the function of most other proteins is still very speculative. One problem that may have been underestimated so far is that – unlike for genes involved in peroxisome protein import – the underlying mechanism of mutants displaying aberrant organelle numbers may be related to two, basically opposite, machineries. Obviously, a protein import defect results in cytosolic mislocaliza- tion of matrix proteins. However, alterations in orga- nelle abundance may, in fact, reflect either defects in organelle formation or, alternatively, in organelle turn- over by autophagy. Also, mutations that affect the rate of the two opposite machineries of organelle formation and degradation to the same extent, will result in an unaltered steady-state number of peroxisomes. Thus, the mere organelle steady-state number, which is gen- erally used to determine peroxisome abundance, is not sufficiently informative about the actual rates of the different processes that determine organelle abundance in a separate cell. To understand in more detail the underlying reasons for the presence of certain phenotypes there is an urgent need to develop better techniques to establish the phenotype of mutants more precisely. Using live cell imaging techniques the rates of the processes that affect peroxisome abundance should be quantitatively determined in vivo. Such data could eventually be used to develop mathematical models describing the kinetics of these processes. Organelle fission and de novo synthesis could be studied using photoactivatable proteins or the HaloTag technology, as successfully used in mammalian cells [10,17]. The rate of degradation can easily be deter- mined biochemically by determining protein half lives. One way to determine the involvement of a gene in de novo synthesis is to study the effect of mutations on functional complementation of a pex3 mutant with the PEX3 gene. Using this approach we showed that DNM1, VPS1 and EMP24 are not required for peroxi- some re-introduction from the endoplasmic reticulum in H. polymorpha pex3 cells [14,40]. Interestingly, no mutations have so far been described that result in a defect in pex3 mutant complementation. Why such genes ⁄ mutations have not yet been identified is unknown. Possibly these genes are also involved in other endoplasmic reticulum-related process and hence the mutations may be lethal. 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Saraya et al. 3288 FEBS Journal 277 (2010) 3279–3288 ª 2010 The Authors Journal compilation ª 2010 FEBS . form a mature peroxisome. (3) Peroxisome inheritance (involving Myo2, Inp2, Inp1, Pex3 and Pex19), in which peroxisomes are faithfully inherited into the newly. MINIREVIEW Peroxisomes as dynamic organelles: peroxisome abundance in yeast Ruchi Saraya, Marten Veenhuis and Ida