Peroxisome development in yeast is associated with the formation of Pex3-dependent peroxisome-vacuole contact sites

Using electron and fluorescence microscopy techniques, we identified various physical contacts between peroxisomes and other cell organelles in the yeast Hansenula polymorpha.In exponential glucose-grown cells, which typically contain a single small peroxisome, contacts were only observed with the endoplasmic reticulum and the plasma membrane. Here we focus on a novel peroxisome-vacuole contact site that is formed when glucose-grown cells are shifted to methanol containing media, conditions that induce strong peroxisome development. At these conditions, the small peroxisomes rapidly increase in size, a phenomenon that is paralleled by the formation of distinct intimate contacts with the vacuole.Localization studies showed that the peroxin Pex3 accumulated in patches at the peroxisome-vacuole contact sites. In wild-type cells growing exponentially on medium containing glucose, peroxisome-vacuole contact sites were never observed. However, upon overproduction of Pex3 peroxisomes also associated to vacuoles at these growth conditions.Our observations strongly suggest a role for Pex3 in the formation of a novel peroxisome-vacuole contact site. This contact likely plays a role in membrane growth as it is formed solely at conditions of strong peroxisome expansion.     

Pex3 peroxisome biogenesis proteins function in peroxisome inheritance as class V myosin receptors

In Saccharomyces cerevisiae, peroxisomal inheritance from mother cell to bud is conducted by the class V myosin motor, Myo2p. However, homologues of S. cerevisiae Myo2p peroxisomal receptor, Inp2p, are not readily identifiable outside the Saccharomycetaceae family. Here, we demonstrate an unexpected role for Pex3 proteins in peroxisome inheritance. Both Pex3p and Pex3Bp are peroxisomal integral membrane proteins that function as peroxisomal receptors for class V myosin through direct interaction with the myosin globular tail. In cells lacking Pex3Bp, peroxisomes are preferentially retained by the mother cell, whereas most peroxisomes gather and are transferred en masse to the bud in cells overexpressing Pex3Bp or Pex3p. Our results reveal an unprecedented role for members of the Pex3 protein family in peroxisome motility and inheritance in addition to their well-established role in peroxisome biogenesis at the endoplasmic reticulum. Our results point to a temporal link between peroxisome formation and inheritance and delineate a general mechanism of peroxisome inheritance in eukaryotic cells.     

Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.     

Conserved function of pex11p and the novel pex25p and pex27p in peroxisome biogenesis

We describe the isolation and characterization of a homologous pair of proteins, Pex25p (YPL112c) and Pex27p (YOR193w), whose C-termini are similar to the entire Pex11p. All three proteins localize to the peroxisomal membrane and are likely to form homo-oligomers. Deletion of any of the three genes resulted in enlarged peroxisomes as revealed by fluorescence and electron microscopy. The partial growth defect on fatty acids of a pex25Δ mutant was not exacerbated by the additional deletion of PEX27; however, when PEX11 was deleted on top of that, growth was abolished on all fatty acids. Moreover, a severe peroxisomal protein import defect was observed in the pex11Δpex25Δpex27Δ triple mutant strain. This import defect was also observed when cells were grown on ethanol-containing medium, where peroxisomes are not required, suggesting that the function of the proteins in peroxisome biogenesis exceeds their role in proliferation. When Pex25p was overexpressed in the triple mutant strain, growth on oleic acid was completely restored and a massive proliferation of laminar membranes and peroxisomes was observed. Our data demonstrate that Pex11p, Pex25p, and Pex27p build a family of proteins whose members are required for peroxisome biogenesis and play a role in the regulation of peroxisome size and number.     

De novo peroxisome biogenesis in Penicillium chrysogenum is not dependent on the Pex11 family members or Pex16

We have analyzed the role of the three members of the Pex11 protein family in peroxisome formation in the filamentous fungus Penicillium chrysogenum. Two of these, Pex11 and Pex11C, are components of the peroxisomal membrane, while Pex11B is present at the endoplasmic reticulum. We show that Pex11 is a major factor involved in peroxisome proliferation. We also demonstrate that P. chrysogenum cells deleted for known peroxisome fission factors (all Pex11 family proteins and Vps1) still contain peroxisomes. Interestingly, we find that, unlike in mammals, Pex16 is not essential for peroxisome biogenesis in P. chrysogenum, as partially functional peroxisomes are present in a pex16 deletion strain. We also show that Pex16 is not involved in de novo biogenesis of peroxisomes, as peroxisomes were still present in quadruple Δpex11 Δpex11B Δpex11C Δpex16 mutant cells. By contrast, pex3 deletion in P. chrysogenum led to cells devoid of peroxisomes, suggesting that Pex3 may function independently of Pex16. Finally, we demonstrate that the presence of intact peroxisomes is important for the efficiency of ß-lactam antibiotics production by P. chrysogenum. Remarkably, distinct from earlier results with low penicillin producing laboratory strains, upregulation of peroxisome numbers in a high producing P. chrysogenum strain had no significant effect on penicillin production.     

A dual function for Pex3p in peroxisome formation and inheritance

Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation. We show that the peroxisome retention factor Inp1p interacts physically with Pex3p in vitro and in vivo, and split-GFP analysis shows that the site of interaction is the peroxisomal membrane. Furthermore, we have generated PEX3 alleles that support peroxisome formation but fail to support recruitment of Inp1p to peroxisomes, and as a consequence are affected in peroxisome segregation. We conclude that Pex3p functions as an anchor for Inp1p at the peroxisomal membrane, and that this function is independent of its role at the ER in peroxisome biogenesis.     

Extension of Yeast Chronological Lifespan by Methylamine

Background

Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast Hansenula polymorpha grown at conditions that require primary peroxisome metabolism for growth.

Methodology/Principal Findings

The chronological lifespan of H. polymorpha is strongly enhanced when cells are grown on methanol or ethanol, metabolized by peroxisome enzymes, relative to growth on glucose that does not require peroxisomes. The short lifespan of H. polymorpha on glucose is mainly due to medium acidification, whereas most likely ROS do not play an important role. Growth of cells on methanol/methylamine instead of methanol/ammonium sulphate resulted in further lifespan enhancement. This was unrelated to medium acidification. We show that oxidation of methylamine by peroxisomal amine oxidase at carbon starvation conditions is responsible for lifespan extension. The methylamine oxidation product formaldehyde is further oxidized resulting in NADH generation, which contributes to increased ATP generation and reduction of ROS levels in the stationary phase.

Conclusion/Significance

We conclude that primary peroxisome metabolism enhanced chronological lifespan of H. polymorpha. Moreover, the possibility to generate NADH at carbon starvation conditions by an organic nitrogen source supports further extension of the lifespan of the cell. Consequently, the interpretation of CLS analyses in yeast should include possible effects on the energy status of the cell.     

An ER‐peroxisome tether exerts peroxisome population control in yeast

Eukaryotic cells compartmentalize biochemical reactions into membrane‐enclosed organelles that must be faithfully propagated from one cell generation to the next. Transport and retention processes balance the partitioning of organelles between mother and daughter cells. Here we report the identification of an ER‐peroxisome tether that links peroxisomes to the ER and ensures peroxisome population control in the yeast Saccharomyces cerevisiae. The tether consists of the peroxisome biogenic protein, Pex3p, and the peroxisome inheritance factor, Inp1p. Inp1p bridges the two compartments by acting as a molecular hinge between ER‐bound Pex3p and peroxisomal Pex3p. Asymmetric peroxisome division leads to the formation of Inp1p‐containing anchored peroxisomes and Inp1p‐deficient mobile peroxisomes that segregate to the bud. While peroxisomes in mother cells are not released from tethering, de novo formation of tethers in the bud assists in the directionality of peroxisome transfer. Peroxisomes are thus stably maintained over generations of cells through their continued interaction with tethers.     

Give what you can and keep what you need!

EMBO J 32 18, 2439–2453 doi:10.1038/emboj.2013.170; published online July302013During cell division, peroxisomes are inherited to daughter cells but some are retained in the mother cells. Our knowledge on how peroxisome inheritance and retention is balanced and how this is regulated for each individual organelle remains incompletely understood. The new findings by Knoblach et al (2013) published in this issue of The EMBO Journal demonstrate that Inp1p functions as a bridging protein to connect ER-resident Pex3p and peroxisomal Pex3p, which anchors peroxisomes to the cortical ER for organelle retention in the mother cell. Asymmetric peroxisome division generates peroxisomes, which lack Inp1p but contain Inp2p instead, and only these peroxisomes are primed for myosin-driven transport to daughter cells.Peroxisomes are single membrane-bound organelles found in almost all eukaryotic cells. They harbour a wide spectrum of metabolic activities that vary among different species, developmental stages and cell types (Schlüter et al, 2010). Eukaryotic cells have evolved elaborate mechanisms to ensure the maintenance of peroxisomes. New peroxisomes can form either de novo by budding from the ER or by growth and division of pre-existing organelles (Lazarow and Fujiki, 1985; Hoepfner et al, 2005). Despite the fact that peroxisomes can form de novo, yeast favours to multiply peroxisomes by growth and division (Motley and Hettema, 2007). It therefore has to be ensured that both mother and daughter cells get their share of peroxisomes during cell division. Thus, some peroxisomes need to be retained in the mother cell, while other peroxisomes are directed for transport and inheritance to daughter cells. Both processes have to be balanced to ensure a successful distribution of the organelles between the mother cell and the newly formed bud.The molecular details of how an even peroxisome distribution of dividing cells are maintained have now been disclosed by Knoblach et al (2013), advancing an exciting scientific journey. This journey originally started by the finding that the partitioning of peroxisomes between mother cell and bud is dependent on actin filaments and the myosin motor protein Myo2p (Hoepfner et al, 2001). Inp1p and Inp2p were identified by the Rachubinski group and Inp2p turned out to function as the peroxisomal tether, which interacts with Myo2p and hooks the organelle onto the actin-track on the road to the bud (Fagarasanu et al, 2006). Inp1p was shown to be a peripheral peroxisomal membrane protein, which acts as a peroxisome-retention factor, tethering peroxisomes to putative anchoring structures within the mother cell and bud (Fagarasanu et al, 2005). Later on, Pex3p, a multi-functional protein of the peroxisomal life cycle, was identified as peroxisomal membrane anchor of Inp1p (Munck et al, 2009). Until now, it was therefore known that peroxisomes hook onto Inp1p by Pex3p and Inp1p connects peroxisomes to cortical structures of unknown nature. Thus, it was an open question how peroxisomes are trapped in the mother cell and which additional factors are required for this process.The work of Knoblach et al (2013) published in this issue of The EMBO Journal now unravelled this mystery, allowing for a more complete picture of the whole process of peroxisome retention and inheritance (Figure 1A). The authors show that peroxisomes are recruited to mitochondria that artificially expose Inp1p on their surface, clearly demonstrating that Inp1p acts as a peroxisome tether. Most importantly, they identified the mechanism of how peroxisomes are directed and anchored to the cell cortex: the ER acts as a membrane anchor for the retention of peroxisomes during cell division. In vitro binding assays revealed that Inp1p contains two independent binding sites for Pex3p, located at the C- and the N-terminal region of the protein, respectively. Since Pex3p exhibits a dual localization at the peroxisomal membrane and at the ER, Inp1p seems to bind to Pex3p of both compartments in vivo and thus link Pex3p molecules across two membranes. Indeed, it turned out that ER-located Pex3p recruits Inp1p to discrete foci in close proximity to the cortical ER. Using the split-GFP assay, the authors confirmed that Inp1p interacts not only with ER-bound Pex3p but also with Pex3p in the peroxisomal membrane. Thus, the core of the ER-peroxisome tether is generated by the Inp1p-mediated linkage of ER-bound Pex3p with peroxisomal Pex3p. The functional relevance of this ER-peroxisome tether is disclosed by the phenotype of peroxisome inheritance mutants. Accordingly, the Pex3p–V81E mutant, affected in the recruitment of Inp1p to the ER, is characterized by a defect of ER retention of peroxisomes, which drives all peroxisomes into the bud and leaves no peroxisomes in the mother cell (Figure 1B).Open in a separate windowFigure 1Peroxisome retention and inheritance (A) free peroxisomes in the mother cell (stage I) are anchored to cortical ER by a tethering complex consisting of two molecules Pex3p, one located at the ER and the other associated with the peroxisomal membrane and Inp1p, which connects the ER-bound and peroxisome-bound Pex3p (stage II). Accordingly, Inp1p contains two Pex3p-binding domains, allowing the protein to function as a bridge between the two Pex3p-containing organelles. Peroxisomes elongate and divide, and Inp2p is loaded onto peroxisomes with an asymmetric distribution (stage III). The peroxisomal population that lacks Inp2p is anchored to the cortical ER, whereas the population of cytosolic peroxisomes containing Inp2p is destined for the transport to the bud (stage IV). To this end, Inp2p interacts with Myo2p and thus triggers the movement of the peroxisome along actin cables to the bud. The process is completed when the peroxisome is released from Myo2p in the bud (stage I). In wild-type cells, the described retention and inheritance process leads to an equal distribution of peroxisomes between mother cell. The described molecular mechanism results in a regulated balance of retention and inheritance of peroxisomes, ensuring that both the mother cell and the newly formed bud gain their share of peroxisomes. (B) However, when the endogenous Pex3p is replaced by a Pex3p-mutant (Pex3p–V81E), which lost its strong binding capacity to Inp1p, peroxisomes are not anchored to the cortical ER anymore, with the consequence that during cells'' division the entire organelle population is transported to the bud and peroxisomes are not retained in the mother cell.To piece together the puzzle, a final gap had to be filled. How is the peroxisomal fraction remaining in the mother cell discriminated from those ferried to the bud during cell division? In budding wild-type cells, Inp1p exhibits a striking asymmetry along the cell division axis. Knoblach et al (2013) show that most peroxisomes of the mother cell contain Inp1p, while peroxisomes that are ferried towards the bud contain little or no Inp1p. Live-cell video microscopy of individual peroxisome revealed that Inp1p-containing peroxisomes were mostly immobile and retained in the mother cell, while highly mobile peroxisomes contained Inp2p and were predominantly found in the bud. The question remains of how peroxisomes lacking Inp1p but containing Inp2p are formed? To tackle this question, the authors took advantage of the fact that cells defective in peroxisome division contain single enlarged peroxisomes and project a tubular extension into the bud upon cell division (Kuravi et al, 2006). Remarkably, Knoblach et al (2013) show that Inp1p and Inp2p localized to opposite ends of the giant peroxisome. Inp1p was confined to the part of the peroxisome that was retained in the mother cell, while Inp2p enriched at the tubule that protruded into the bud.In summary, Knoblach et al (2013) discovered the ER as the site for peroxisome binding to the cell cortex that is responsible for the retention of peroxisomes in the mother cells during cell division and identified Inp1p as a molecular hinge connecting Pex3p of peroxisomal and ER membranes. Furthermore, peroxisome division is shown to result in an asymmetric distribution of inheritance factors with Inp1p-containing organelles remaining tethered to the ER in the mother cell, while Inp2p-containing peroxisomes hook onto myosin motor proteins for movement to the bud. These remarkable discoveries disclose the molecular mechanism of peroxisome retention and inheritance during cell division. Moreover, this study adds to other known functions of Pex3p, which besides its newly discovered role as ER-tether for peroxisomes is also known as an initiator of de novo formation of peroxisomes, a docking factor for the transport of peroxisomal membrane proteins and a tether for the regulated degradation of peroxisomes. This study adds more complexity to the network of regulated processes in peroxisome biogenesis that all merge at Pex3p, and will certainly provide the ground for further exploration.     

An electron microscopical study of the development of peroxisomes during formation and germination of ascospores in the methylotrophic yeast Hansenula polymorpha

Ascospore formation was studied in liquid cultures of the yeast Hansenula polymorpha, previously grown under conditions in which the synthesis of alcohol oxidase was repressed (glucose as growth substrate) or derepressed (methanol, glycerol and dihydroxyacetone as growth substrates and after growth on malt agar plates). In ascospores obtained from repressed cells, generally one small peroxisome was present. The organelle probably originated from the small peroxisome, originally present in the vegetative cells. They had no crystalline inclusions and cytochemical experiments indicated the presence of catalase, urate oxidase and amino acid oxidase activities in these organelles. In ascospores obtained from derepressed cells, generally 1–3 crystalline peroxisomes were observed. These organelles also originated from the peroxisomes originally present in the vegetative cells by means of fragmentation or division. They contained, in addition to the enzymes characteristic for peroxisomes in spores from repressed cells, also alcohol oxidase. The latter enzyme is probably responsible for the crystalline substructure of these peroxisomes.Peroxisomes had no apparent physiological function in the process of ascosporogenesis. A glyoxysomal function of the organelles during germination of the ascospores was also not observed. Germination of mature ascospores in media containing different sources of carbon and nitrogen showed that the function of the peroxisomes present in ascospores of Hansenula polymorpha is probably identical to that in vegetative haploid cells. They are involved in the oxidative metabolism of different carbon and nitrogen sources. Their enzyme profile is a reflection of that of peroxisomes of vegetative cells and their presence may enable the formation of cells which are optimally adapted to environmental conditions extant during spore germination.     

Pex3p initiates the formation of a preperoxisomal compartment from a subdomain of the endoplasmic reticulum in Saccharomyces cerevisiae

Peroxisomes are dynamic organelles that often proliferate in response to compounds that they metabolize. Peroxisomes can proliferate by two apparent mechanisms, division of preexisting peroxisomes and de novo synthesis of peroxisomes. Evidence for de novo peroxisome synthesis comes from studies of cells lacking the peroxisomal integral membrane peroxin Pex3p. These cells lack peroxisomes, but peroxisomes can assemble upon reintroduction of Pex3p. The source of these peroxisomes has been the subject of debate. Here, we show that the amino-terminal 46 amino acids of Pex3p of Saccharomyces cerevisiae target to a subdomain of the endoplasmic reticulum and initiate the formation of a preperoxisomal compartment for de novo peroxisome synthesis. In vivo video microscopy showed that this preperoxisomal compartment can import both peroxisomal matrix and membrane proteins leading to the formation of bona fide peroxisomes through the continued activity of full-length Pex3p. Peroxisome formation from the preperoxisomal compartment depends on the activity of the genes PEX14 and PEX19, which are required for the targeting of peroxisomal matrix and membrane proteins, respectively. Our findings support a direct role for the endoplasmic reticulum in de novo peroxisome formation.     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

The Intracellular Bacteria Chlamydia Hijack Peroxisomes and Utilize Their Enzymatic Capacity to Produce Bacteria-Specific Phospholipids

Chlamydia trachomatis is an obligate intracellular pathogen responsible for loss of eyesight through trachoma and for millions of cases annually of sexually transmitted diseases. The bacteria develop within a membrane-bounded inclusion. They lack enzymes for several biosynthetic pathways, including those to make some phospholipids, and exploit their host to compensate. Three-dimensional fluorescence microscopy demonstrates that small organelles of the host, peroxisomes, are translocated into the Chlamydia inclusion and are found adjacent to the bacteria. In cells deficient for peroxisome biogenesis the bacteria are able to multiply and give rise to infectious progeny, demonstrating that peroxisomes are not essential for bacterial development in vitro. Mass spectrometry-based lipidomics reveal the presence in C. trachomatis of plasmalogens, ether phospholipids whose synthesis begins in peroxisomes and have never been described in aerobic bacteria before. Some of the bacterial plasmalogens are novel structures containing bacteria-specific odd-chain fatty acids; they are not made in uninfected cells nor in peroxisome-deficient cells. Their biosynthesis is thus accomplished by the metabolic collaboration of peroxisomes and bacteria.     

Pexophagy-linked degradation of the peroxisomal membrane protein Pex3p involves the ubiquitin–proteasome system

Peroxisome autophagy, also known as pexophagy, describes the wholesale degradation of peroxisomes via the vacuole, when organelles become damaged or redundant. In the methylotrophic yeast Hansenula polymorpha, pexophagy is stimulated when cells growing on methanol are exposed to excess glucose. Degradation of the peroxisomal membrane protein Pex3p, a process that does not involve the vacuole, was shown to trigger pexophagy. In this contribution, we have characterised pexophagy-associated Pex3p degradation further. We show that Pex3p breakdown depends on ubiquitin and confirm that Pex3p is a target for ubiquitination. Furthermore, we identify a role for the peroxisomal E3 ligases Pex2p and Pex10p in Pex3p degradation, suggesting the existence of a ubiquitin-dependent pathway involved in removing proteins from the peroxisomal membrane.     

A Combined Approach of Quantitative Interaction Proteomics and Live-cell Imaging Reveals a Regulatory Role for Endoplasmic Reticulum (ER) Reticulon Homology Proteins in Peroxisome Biogenesis

Peroxisome biogenesis initiates at the endoplasmic reticulum (ER) and maturation allows for the formation of metabolically active organelles. Yet, peroxisomes can also multiply by growth and division. Several proteins, called peroxins, are known to participate in these processes but little is known about their organization to orchestrate peroxisome proliferation. Here, we demonstrate that regulation of peroxisome proliferation relies on the integrity of the tubular ER network. Using a dual track SILAC-based quantitative interaction proteomics approach, we established a comprehensive network of stable as well as transient interactions of the peroxin Pex30p, an integral membrane protein. Through association with merely ER resident proteins, in particular with proteins containing a reticulon homology domain, and with other peroxins, Pex30p designates peroxisome contact sites at ER subdomains. We show that Pex30p traffics through the ER and segregates in punctae to which peroxisomes specifically append, and we ascertain its transient interaction with all subunits of the COPI coatomer complex suggesting the involvement of a vesicle-mediated transport. We establish that the membrane protein Pex30p facilitates the connection of peroxisomes to the ER. Taken together, our data indicate that Pex30p-containing protein complexes act as focal points from which peroxisomes can form and that the tubular ER architecture organized by the reticulon homology proteins Rtn1p, Rtn2p and Yop1p controls this process.All nucleated cells contain essential round-shaped organelles called peroxisomes, whose function is mainly associated with lipid metabolism (1). Depending on the cellular requirements, the size, number, and protein content of these single membrane-bound organelles can vary widely. Although peroxisomes are dispensable for unicellular species such as yeasts, they are essential for the development of multicellular organisms (2, 3). In human, mutations in PEX genes lead to defects in peroxisome function or formation and are associated with the development of lethal pathologies (4). These PEX genes code for proteins, called peroxins, which are involved in peroxisome assembly and maintenance (5).Two major routes seem to lead to peroxisome formation, namely, de novo biogenesis and growth/division of pre-existing peroxisomes. The division pathway operates with proteins of the Pex11 family and requires fission factors shared with mitochondria (6). Studies in yeast and mammalian cells revealed that through the action of the protein Pex3p peroxisome precursors can also originate from the endoplasmic reticulum (ER)¹ and, via import of membrane and matrix proteins, mature into fully functional organelles (7, 8). Furthermore, several peroxisomal membrane proteins were shown to migrate to peroxisomes via the ER (7, 9, 10). The molecular mechanism underlying the biogenic pathway of peroxisome formation has not been clarified so far. Recent data based on cell-free vesicle-budding reactions, however, demonstrated that several peroxisomal proteins traffic from the ER to peroxisomes in a COPII vesicle-independent manner (11). These observations point to the existence of vesicular events to mediate the transport of peroxisomal membrane proteins from the ER. In fact, analysis of secretory mutant yeast cells already suggest that part of the ER-associated secretory machinery is involved in peroxisome biogenesis (12).The de novo biogenesis of peroxisomes and the growth/division pathways are usually seen as independent routes; however, these events may be coordinated and, thus, intimately linked. Indeed, peroxisomes need to acquire membrane components to proliferate and it has been proposed that their binding to the cell cortex or to the cytoskeleton allows their partitioning and segregation during cell division (13–15).Among the proteins required for assembly of peroxisomes, the membrane proteins Pex23p and Pex24p play essential roles in the yeast Yarrowia lipolytica (16, 17). Homologs of these two proteins in Saccharomyces cerevisiae are Pex30p, Pex31p, and Pex32p, all containing at least one transmembrane domain and a dysferlin domain as common structural motifs, as well as Pex28p and Pex29p. In S. cerevisiae, these proteins seem to negatively control peroxisomal size and number (18, 19). Interestingly, Pex30p seems to exhibit species-specific differences in the regulation of peroxisome proliferation. While the lack of Pex30p in S. cerevisiae leads to an increase in the number of normal-sized peroxisomes (18), in Pichia pastoris its absence correlates with the appearance of fewer and clustered peroxisomes (20). Although peroxisomes are highly versatile organelles, under given conditions their total number per cell remains fairly constant owing to the delicate balance of proliferation, inheritance and degradation (21, 22). The question is: what are the molecular mechanisms responsible for the spatiotemporal organization of these events?Here, we present data obtained from a dual approach based on quantitative interaction proteomics using stable isotope labeling with amino acids in cell culture (SILAC) (23, 24) and live-cell imaging, revealing for the first time the dynamic interaction network around Pex30p and its function in the organization of ER-to-peroxisome membrane associations. We report the existence of a macromolecular membrane protein complex that acts as a hub for the regulation of peroxisome proliferation and movement. Our data suggest a direct role for the tubular cortical ER and the reticulon homology proteins Rtn1p, Rtn2p, and Yop1p in the regulation of peroxisome biogenesis. Furthermore, as an initially cortical-ER localized protein that interacts with reticulon homology proteins, Pex30p is shown in this work to establish contacts between ER tubules and peroxisomes and to specifically traffic through the ER. In summary, our data reveal a central role for Pex30p in the formation of ER-to-peroxisomes associations that appear to be involved in the coordination of peroxisome biogenesis and maintenance.     

The Arabidopsis Chloroplast Division Protein DYNAMIN-RELATED PROTEIN5B Also Mediates Peroxisome Division

Peroxisomes are highly dynamic organelles involved in various metabolic pathways. The division of peroxisomes is regulated by factors such as the PEROXIN11 (PEX11) proteins that promote peroxisome elongation and the dynamin-related proteins (DRPs) and FISSION1 (FIS1) proteins that function together to mediate organelle fission. In Arabidopsis thaliana, DRP3A/DRP3B and FIS1A (BIGYIN)/FIS1B are two pairs of homologous proteins known to function in both peroxisomal and mitochondrial division. Here, we report that DRP5B, a DRP distantly related to the DRP3s and originally identified as a chloroplast division protein, also contributes to peroxisome division. DRP5B localizes to both peroxisomes and chloroplasts. Mutations in the DRP5B gene lead to peroxisome division defects and compromised peroxisome functions. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we further demonstrate that DRP5B can interact or form a complex with itself and with DRP3A, DRP3B, FIS1A, and most of the Arabidopsis PEX11 isoforms. Our data suggest that, in contrast with DRP3A and DRP3B, whose orthologs exist across plant, fungal, and animal kingdoms, DRP5B is a plant/algal invention to facilitate the division of their organelles (i.e., chloroplasts and peroxisomes). In addition, our results support the notion that proteins involved in the early (elongation) and late (fission) stages of peroxisome division may act cooperatively.     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Lumenal peroxisomal protein aggregates are removed by concerted fission and autophagy events

We demonstrated that in the yeast Hansenula polymorpha peroxisome fission and degradation are coupled processes that are important to remove intra-organellar protein aggregates. Protein aggregates were formed in peroxisomes upon synthesis of a mutant catalase variant. We showed that the introduction of these aggregates in the peroxisomal lumen had physiological disadvantages as it affected growth and caused enhanced levels of reactive oxygen species. Formation of the protein aggregates was followed by asymmetric peroxisome fission to separate the aggregate from the mother organelle. Subsequently, these small, protein aggregate-containing organelles were degraded by autophagy. In line with this observation we showed that the degradation of the protein aggregates was strongly reduced in dnm1 and pex11 cells in which peroxisome fission is reduced. Moreover, this process was dependent on Atg1 and Atg11.     

In the yeast Hansenula polymorpha, peroxisome formation from the ER is independent of Pex19p, but involves the function of p24 proteins

The peroxin Pex19p is important for the formation of functional peroxisomal membranes. Here we show that Hansenula polymorpha Pex19p is also required for peroxisome inheritance. Peroxisome inheritance is partly defective when Pex19p farnesylation is blocked, whereas deletion of PEX19 resulted in a severe defect in partitioning of peroxisomal structures. Time lapse imaging revealed that in newly formed buds, which had not inherited a peroxisome from the mother cell, new peroxisomes are formed that derive from the nuclear envelope/endoplasmic reticulum. This process was impaired upon deletion of EMP24 and ERP3, genes that encode p24 proteins. p24 Proteins are components of coated vesicles that mediate trafficking between the endoplasmic reticulum and Golgi apparatus. In an H. polymorpha wild-type background, deletion of EMP24 and ERP3 resulted in a strong reduction of organelle number in conjunction with an increase in the size of individual peroxisomes. This observation suggests that p24 proteins also play a role in peroxisome development in wild-type H. polymorpha cells.     

Peroxisomal Membrane Proteins Insert into the Endoplasmic Reticulum

We show that a comprehensive set of 16 peroxisomal membrane proteins (PMPs) encompassing all types of membrane topologies first target to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. These PMPs insert into the ER membrane via the protein import complexes Sec61p and Get3p (for tail-anchored proteins). This trafficking pathway is representative for multiplying wild-type cells in which the peroxisome population needs to be maintained, as well as for mutant cells lacking peroxisomes in which new peroxisomes form after complementation with the wild-type version of the mutant gene. PMPs leave the ER in a Pex3p-Pex19p–dependent manner to end up in metabolically active peroxisomes. These results further extend the new concept that peroxisomes derive their basic framework (membrane and membrane proteins) from the ER and imply a new functional role for Pex3p and Pex19p.