Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530). Progress has been made in the elucidation of how these proteins are targeted to the organelle. In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly. However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited. The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles. Furthermore, the origin of the peroxisomal membrane is still enigmatic. Recent observations suggest the existence of two classes of peroxisomal membrane proteins. Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum.     

Peroxisome biogenesis: where Arf and coatomer might be involved

The present review summarizes recent observations on binding of Arf and COPI coat to isolated rat liver peroxisomes. The general structural and functional features of both Arf and coatomer were considered along with the requirements and dependencies of peroxisomal Arf and coatomer recruitment. Studies on the expression of mammalian Pex11 proteins, mainly Pex11alpha and Pex11beta, intimately related to the process of peroxisome proliferation, revealed a sequence of individual steps including organelle elongation/tubulation, formation of membrane and matrix protein patches segregating distinct proteins from each other, development of membrane constrictions and final membrane fission. Based on the similarities of the processes leading to cargo selection and concentration on Golgi membranes on the one hand and to the formation of peroxisomal protein patches on the other hand, an implication of Arf and COPI in distinct processes of peroxisomal proliferation is hypothesized. Alternatively, peroxisomal Arf/COPI might facilitate the formation of COPI-coated peroxisomal vesicles functioning in cargo transport and retrieval from peroxisomes to the ER. Recent observations suggesting transport of Pex3 and Pex19 during early steps of peroxisome biogenesis from the ER to peroxisomes inevitably propose such a retrieval mechanism, provided the ER to peroxisome pathway is based on transporting vesicles.     

Involvement of the endoplasmic reticulum in peroxisome formation

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.     

Peroxisomal membrane ascorbate peroxidase is sorted to a membranous network that resembles a subdomain of the endoplasmic reticulum

The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD(+) and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3'-dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted post-translationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase-peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CAT-pAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain.     

A subtle interplay between three Pex11 proteins shapes de novo formation and fission of peroxisomes

The organization of eukaryotic cells into membrane-bound compartments must be faithfully sustained for survival of the cell. A subtle equilibrium exists between the degradation and the proliferation of organelles. Commonly, proliferation is initiated by a membrane remodeling process. Here, we dissect the function of proteins driving organelle proliferation in the particular case of peroxisomes. These organelles are formed either through a growth and division process from existing peroxisomes or de novo from the endoplasmic reticulum (ER). Among the proteins involved in the biogenesis of peroxisomes, peroxins, members of the Pex11 protein family participate in peroxisomal membrane alterations. In the yeast Saccharomyces cerevisiae, the Pex11 family consists of three proteins, Pex11p, Pex25p and Pex27p. Here we demonstrate that yeast mutants lacking peroxisomes require the presence of Pex25p to regenerate this organelle de novo. We also provide evidence showing that Pex27p inhibits peroxisomal function and illustrate that Pex25p initiates elongation of the peroxisomal membrane. Our data establish that although structurally conserved each of the three Pex11 protein family members plays a distinct role. While ScPex11p promotes the proliferation of peroxisomes already present in the cell, ScPex25p initiates remodeling at the peroxisomal membrane and ScPex27p acts to counter this activity. In addition, we reveal that ScPex25p acts in concert with Pex3p in the initiation of de novo peroxisome biogenesis from the ER.     

Expression of the Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal

Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.     

Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal ER

Peroxisomal ascorbate peroxidase (APX) sorts indirectly via a subdomain of the ER (peroxisomal ER) to the boundary membrane of peroxisomes in tobacco Bright Yellow 2 cells. This novel subdomain characteristically appears as fluorescent reticular/circular compartments distributed variously in the cytoplasm. Further characterizations are presented herein. A peptide possessing the membrane targeting information for peroxisomal APX was fused to GFP (GFP-APX). Transiently expressed GFP-APX sorted to peroxisomes and to reticular/circular compartments; in both cases, the GFP moiety faced the cytosol. Of particular interest, both homotypic and heterotypic aggregates of peroxisomes, mitochondria, and/or plastids were formed. The latter two organelles comprised the circular portion of the reticular/circular compartments, apparently as a consequence of oligomerization (zippering) of the GFP moieties after insertion into the outer membranes of the affected organelles. These results, coupled with the accumulation of endogenous peroxisomal APX in cytoplasmic, noncircular compartment(s) following treatment with brefeldin A, indicate that authentic peroxisomal ER is composed only of a reticular compartment(s). Equally important, the data show that overexpressed, membrane-targeted GFP fusion proteins have a propensity to form organelle aggregates that may lead to misinterpretations of sorting pathways of trafficked proteins.     

PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intra- peroxisomal protein that is a member of the WD repeat family and is essential for the import of thiolase into peroxisomes

We have previously described mutant S. cerevisiae that are defective in peroxisome biogenesis (peb mutants) (Zhang, J. W., Y. Han, and P. B. Lazarow. 1993. J. Cell Biol. 123:1133-1147.). In some mutants, peroxisomes are undetectable. Other mutants contain normal-looking peroxisomes but fail to package subsets of peroxisomal proteins into the organelle (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359.). In peb1 (pas7) cells, for example, the peroxisomes contain proteins that are targeted by COOH-terminal tripeptides and contain acyl-CoA oxidase (which is probably targeted by internal oligopeptides), but fail to import thiolase (which is targeted by an NH(2)-terminal 16-amino acid sequence). These and other data suggest that there are three branches in the pathway for the import of proteins into peroxisomes, each of which contains a receptor for one type of peroxisomal topogenic information. Here, we report the cloning and characterization of the PEB1 gene, that encodes a 42,320-Da hydrophilic protein with no predicted transmembrane segment. The protein contains six WD repeats, a motif which has been found in 27 proteins involved in diverse cellular functions. The PEB1 gene product was tagged with the hemagglutinin epitope and found to rescue thiolase import in the peb1 null mutant. The epitope-tagged protein was shown to be inside of peroxisomes by immunofluorescence, digitonin permeabilization, equilibrium density centrifugation, immunoelectron microscopy, and proteinase K protection studies. The PEB1 gene product does not cleave the thiolase-targeting sequence. It may function to draw thiolase into peroxisomes.     

Arabidopsis peroxin 16 coexists at steady state in peroxisomes and endoplasmic reticulum

Homologs of peroxin 16 genes (PEX16) have been identified only in Yarrowia lipolytica, humans (Homo sapiens), and Arabidopsis (Arabidopsis thaliana). The Arabidopsis gene (AtPEX16), previously reported as the SSE1 gene, codes for a predicted 42-kD membrane peroxin protein (AtPex16p). Lin et al. (Y. Lin, J.E. Cluette-Brown, H.M. Goodman [2004] Plant Physiol 135: 814-827) reported that SSE1/AtPEX16 was essential for endoplasmic reticulum (ER)-dependent oil and protein body biogenesis in peroxisome-deficient maturing seeds and likely also was involved in peroxisomal biogenesis based on localization of stably expressed green fluorescent protein::AtPex16p in peroxisomes of Arabidopsis plants. In this study with Arabidopsis suspension-cultured cells, combined in vivo and in vitro experiments revealed a novel dual organelle localization and corresponding membrane association/topology of endogenous AtPex16p. Immunofluorescence microscopy with antigen affinity-purified IgGs showed an unambiguous, steady-state coexistence of AtPex16p in suspension cell peroxisomes and ER. AtPex16p also was observed in peroxisomes and ER of root and leaf cells. Cell fractionation experiments surprisingly revealed two immunorelated polypeptides, 42 kD (expected) and 52 kD (unexpected), in homogenates and microsome membrane pellets derived from roots, inflorescence, and suspension cells. Suc-gradient purifications confirmed the presence of both 42-kD and 52-kD polypeptides in isolated peroxisomes (isopycnic separation) and in rough ER vesicles (Mg2+ shifted). They were found peripherally associated with peroxisome and ER membranes but not as covalently bound subunits of AtPex16p. Both were mostly on the matrix side of peroxisomal membranes and unexpectedly mostly on the cytosolic side of ER membranes. In summary, AtPex16p is the only authentic plant peroxin homolog known to coexist at steady state within peroxisomes and ER; these data provide new insights in support of its ER-related, multifunctional roles in organelle biogenesis.     

Peroxisome biogenesis: recent advances

In recent years, it has become evident that peroxisomes form part of the endomembrane system. Peroxisomes can form from the ER via a maturation process and they can multiply by growth and division, whereby the ER provides membrane for growth and ongoing fission (Figure 1). Until very recently, it was widely accepted that most peroxisomal membrane proteins (PMPs) insert directly into peroxisomes, whereas a small subset of PMPs traffic via the ER. In this minireview, we focus mainly on PMP biogenesis, and highlight recent advances in peroxisomal matrix protein import, fission and segregation in yeast.     

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.     

Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations

Cooperation between cellular organelles such as mitochondria, peroxisomes and the ER is essential for a variety of important and diverse metabolic processes. Effective communication and metabolite exchange requires physical linkages between the organelles, predominantly in the form of organelle contact sites. At such contact sites organelle membranes are brought into close proximity by the action of molecular tethers, which often consist of specific protein pairs anchored in the membrane of the opposing organelles. Currently numerous tethering components have been identified which link the ER with multiple other organelles but knowledge of the factors linking the ER with peroxisomes is limited. Peroxisome-ER interplay is important because it is required for the biosynthesis of unsaturated fatty acids, ether-phospholipids and sterols with defects in these functions leading to severe diseases. Here, we characterize acyl-CoA binding domain protein 4 (ACBD4) as a tail-anchored peroxisomal membrane protein which interacts with the ER protein, vesicle-associated membrane protein-associated protein–B (VAPB) to promote peroxisome-ER associations.     

Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain

Peroxisomal ascorbate peroxidase (APX) (EC 1.11.1.11) was shown recently to sort through a subdomain of the ER (peroxisomal endoplasmic reticulum; pER), and in certain cases, alter the distribution and/or morphology of peroxisomes and pER when overexpressed transiently in Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells. Our goal was to gain insight into the dynamics of peroxisomal membrane protein sorting by characterizing the structure and formation of reorganized peroxisomes and pER. Specifically, we test directly the hypothesis that the observed phenomenon is due to the oligomerization of cytosol-facing, membrane-bound polypeptides. a process referred to as membrane "zippering". Results from differential detergent permeabilization experiments confirmed that peroxisomal APX is a C-terminal "tail-anchored" (Cmatrix-Ncytosol) membrane protein with a majority of the polypeptide facing the cytosol. Transient expression of several APX chimeras whose passenger polypeptides can form dimers or trimers resulted in the progressive formation of "globular" peroxisomes and circular pER membranes. Stable expression of the trimer-capable fusion protein yielded suspension cultures that reproducibly maintained a high degree of peroxisomal globules but relatively few detectable pER membranes. Electron micrographs revealed that the globules consisted of numerous individual peroxisomes, seemingly in direct contact with other peroxisomes and/or mitochondria. These peroxisomal clusters or aggregates were not observed in cells transiently expressing monomeric versions of APX. These findings indicate that the progressive, independent "zippering" of peroxisomes and pER is due to the post-sorting oligomerization of monomeric, cytosol-facing polypeptides that are integrally inserted into the membranes of "like" organelles. The dynamics of this process are discussed, especially with respect to the involvement of the microtubule cytoskeleton.     

Peroxisome Formation Requires the Endoplasmic Reticulum Channel Protein Sec61

In peroxisome formation, models of near‐autonomous peroxisome biogenesis with membrane protein integration directly from the cytosol into the peroxisomal membrane are in direct conflict with models whereby peroxisomes bud from the endoplasmic reticulum and receive their membrane proteins through a branch of the secretory pathway. We therefore reinvestigated the role of the Sec 61 complex, the protein‐conducting channel of the endoplasmic reticulum (ER) in peroxisome formation. We found that depletion or partial inactivation of Sec 61 in yeast disables peroxisome formation. The ER entry of the early peroxisomal membrane protein Pex 3 engineered with a glycosylation tag is reduced in sec61 mutant cells. Moreover, we were able to reconstitute Pex 3 import into ER membranes in vitro, and we identified a variant of a signal anchor sequence for ER translocation at the Pex 3 N‐terminus. Our findings are consistent with a Sec 61 requirement for peroxisome formation and a fundamental role of the ER in peroxisome biogenesis.     

Localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway

Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.     

Endoplasmic reticulum-directed Pex3p routes to peroxisomes and restores peroxisome formation in a Saccharomyces cerevisiae pex3Delta strain

Recent studies on the sorting of peroxisomal membrane proteins challenge the long-standing model in which peroxisomes are considered to be autonomous organelles that multiply by growth and division. Here, we present data lending support to the idea that the endoplasmic reticulum (ER) is involved in sorting of the peroxisomal membrane protein Pex3p, a protein required early in peroxisome biogenesis. First, we show that the introduction of an artificial glycosylation site into the N terminus of Pex3p leads to partial N-linked core glycosylation, indicative of insertion into the ER membrane. Second, when FLAG-tagged Pex3p is equipped with an ER targeting signal, it can restore peroxisome formation in pex3Delta cells. Importantly, FLAG antibodies that specifically recognize the processed Pex3p show that the signal peptide of the fusion protein is efficiently cleaved off and that the processed protein localizes to peroxisomes. In contrast, a Pex3p construct in which cleavage of the signal peptide is blocked by a mutation localizes to the ER and the cytosol and cannot complement pex3Delta cells. Together, these results strongly suggest that ER-targeted Pex3p indeed routes via the ER to peroxisomes, and we hypothesize that this pathway is also used by endogenous Pex3p.     

Peroxisome proliferation in Hansenula polymorpha requires Dnm1p which mediates fission but not de novo formation

We show that the dynamin-like proteins Dnm1p and Vps1p are not required for re-introduction of peroxisomes in Hansenula polymorpha pex3 cells upon complementation with PEX3-GFP. Instead, Dnm1p, but not Vps1p, plays a crucial role in organelle proliferation via fission. In H. polymorpha DNM1 deletion cells (dnm1) a single peroxisome is present that forms long extensions, which protrude into developing buds and divide during cytokinesis. Budding pex11.dnm1 double deletion cells lack these peroxisomal extensions, suggesting that the peroxisomal membrane protein Pex11p is required for their formation. Life cell imaging revealed that fluorescent Dnm1p-GFP spots fluctuate between peroxisomes and mitochondria. On the other hand Pex11p is present over the entire organelle surface, but concentrates during fission at the basis of the organelle extension in dnm1 cells.Our data indicate that peroxisome fission is the major pathway for peroxisome multiplication in H. polymorpha.     

Targeting signals in peroxisomal membrane proteins

Peroxisomal membrane proteins (PMPs) are encoded by the nuclear genome and translated on cytoplasmic ribosomes. Newly synthesized PMPs can be targeted directly from the cytoplasm to peroxisomes or travel to peroxisomes via the endoplasmic reticulum (ER). The mechanisms responsible for the targeting of these proteins to the peroxisomal membrane are still rather poorly understood. However, it is clear that the trafficking of PMPs to peroxisomes depends on the presence of cis-acting targeting signals, called mPTSs. These mPTSs show great variability both in the identity and number of requisite residues. An emerging view is that mPTSs consist of at least two functionally distinct domains: a targeting element, which directs the newly synthesized PMP from the cytoplasm to its target membrane, and a membrane-anchoring sequence, which is required for the permanent insertion of the protein into the peroxisomal membrane. In this review, we summarize our knowledge of the mPTSs currently identified.     

Peroxisome biogenesis

Peroxisome biogenesis conceptually consists of the (a) formation of the peroxisomal membrane, (b) import of proteins into the peroxisomal matrix and (c) proliferation of the organelles. Combined genetic and biochemical approaches led to the identification of 25 PEX genes-encoding proteins required for the biogenesis of peroxisomes, so-called peroxins. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes in the cytosol and posttranslationally imported into the organelle in an unknown fashion. The protein import into the peroxisomal matrix and the targeting and insertion of peroxisomal membrane proteins is performed by distinct machineries. At least three peroxins have been shown to be involved in the topogenesis of peroxisomal membrane proteins. Elaborate peroxin complexes form the machinery which in a concerted action of the components transports folded, even oligomeric matrix proteins across the peroxisomal membrane. The past decade has significantly improved our knowledge of the involvement of certain peroxins in the distinct steps of the import process, like cargo recognition, docking of cargo-receptor complexes to the peroxisomal membrane, translocation, and receptor recycling. This review summarizes our knowledge of the functional role the known peroxins play in the biogenesis and maintenance of peroxisomes. Ideas on the involvement of preperoxisomal structures in the biogenesis of the peroxisomal membrane are highlighted and special attention is paid to the concept of cargo protein aggregation as a presupposition for peroxisomal matrix protein import. Electronic Publication     

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.