Determinants of the assembly,integrity and maintenance of the endoplasmic reticulum‐peroxisome tether

Organelle tethering and intercommunication are crucial for proper cell function. We previously described a tether between peroxisomes and the endoplasmic reticulum (ER) that acts in peroxisome population control in the yeast, Saccharomyces cerevisiae. Components of this tether are Pex3p, an integral membrane protein of both peroxisomes and the ER and Inp1p, a connector that links peroxisomes to the ER. Here, we report the analysis of random Inp1p mutants that enabled identification of regions in Inp1p required for the assembly and maintenance of the ER‐peroxisome tether. Interaction analysis between Inp1p mutants and known Inp1p‐binding proteins demonstrated that Pex3p and Inp1p do not constitute the sole components of the ER‐peroxisome tether. Deletion of these Inp1p interactors whose steady‐state localization is outside of ER‐peroxisome tethers affected peroxisome dynamics. Our findings are consistent with the presence of regulatory cues that act on ER‐peroxisome tethers and point to the existence of membrane contact sites between peroxisomes and organelles other than the ER.      

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.     

Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae

Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Little is known about how peroxisomes are inherited. Inp1p is a peripheral membrane protein of peroxisomes of Saccharomyces cerevisiae that affects both the morphology of peroxisomes and their partitioning during cell division. In vivo 4-dimensional video microscopy showed an inability of mother cells to retain a subset of peroxisomes in dividing cells lacking the INP1 gene, whereas cells overexpressing INP1 exhibited immobilized peroxisomes that failed to be partitioned to the bud. Overproduced Inp1p localized to both peroxisomes and the cell cortex, supporting an interaction of Inp1p with specific structures lining the cell periphery. The levels of Inp1p vary with the cell cycle. Inp1p binds Pex25p, Pex30p, and Vps1p, which have been implicated in controlling peroxisome division. Our findings are consistent with Inp1p acting as a factor that retains peroxisomes in cells and controls peroxisome division. Inp1p is the first peroxisomal protein directly implicated in peroxisome inheritance.     

Pex19p contributes to peroxisome inheritance in the association of peroxisomes to Myo2p

During budding of yeast cells peroxisomes are distributed over mother cell and bud, a process that involves the myosin motor protein Myo2p and the peroxisomal membrane protein Inp2p. Here, we show that Pex19p, a peroxin implicated in targeting and complex formation of peroxisomal membrane proteins, also plays a role in peroxisome partitioning. Binding studies revealed that Pex19p interacts with the cargo-binding domain of Myo2p. We identified mutations in Myo2p that specifically reduced binding to Pex19p, but not to Inp2p. The interaction between Myo2p and Pex19p was also reduced by a mutation that blocked Pex19p farnesylation. Microscopy revealed that the Pex19p-Myo2p interaction is important for peroxisome inheritance, because mutations that affect this interaction hamper peroxisome inheritance in vivo. Together these data suggest that both Inp2p and Pex19p are required for proper association of peroxisomes to Myo2p.     

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.     

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.     

Sharing the wealth: peroxisome inheritance in budding yeast

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful partitioning of cellular components during cell division. The budding yeast Saccharomyces cerevisiae has to actively deliver about half of its organelles to the growing bud, while retaining the remaining organelles in the mother cell. Until lately, little was known about the inheritance of peroxisomes. Recent studies have identified the peroxisomal proteins Inp1p and Inp2p as two key regulators of peroxisome inheritance that perform antagonistic functions. Inp1p is required for the retention of peroxisomes in mother cells, whereas Inp2p promotes the bud-directed movement of these organelles. Inp1p anchors peroxisomes to the cell cortex by interacting with specific structures lining the cell periphery. On the other hand, Inp2p functions as the peroxisome-specific receptor for the class V myosin, Myo2p, thereby linking peroxisomes to the translocation machinery that propels peroxisome movement. Tight coordination between Inp1p and Inp2p ensures a fair and harmonious spatial segregation of peroxisomes upon cell division.     

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 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.     

The peroxisomal membrane protein Inp2p is the peroxisome-specific receptor for the myosin V motor Myo2p of Saccharomyces cerevisiae

The faithful inheritance of organelles by daughter cells is essential to maintain the benefits afforded to eukaryotic cells by compartmentalization of biochemical functions. In Saccharomyces cerevisiae, the class V myosin, Myo2p, is involved in transporting different organelles, including the peroxisome, along actin cables to the bud. We identified Inp2p as the peroxisome-specific receptor for Myo2p. Cells lacking Inp2p fail to partition peroxisomes to the bud but are unaffected in the inheritance of other organelles. Inp2p is a peroxisomal membrane protein, preferentially enriched in peroxisomes delivered to the bud. Inp2p interacts directly with the globular tail of Myo2p. Cells overproducing Inp2p often transfer their entire populations of peroxisomes to buds. The levels of Inp2p oscillate with the cell cycle. Organelle-specific receptors like Inp2p explain how a single motor can move different organelles in distinct and specific patterns. To our knowledge, Inp2p is the first peroxisomal protein implicated in the vectorial movement of peroxisomes.     

Contribution of the endoplasmic reticulum to peroxisome formation

How peroxisomes are formed in eukaryotic cells is unknown but important for insight into a variety of diseases. Both human and yeast cells lacking peroxisomes due to mutations in PEX3 or PEX19 genes regenerate the organelles upon reintroduction of the corresponding wild-type version. To evaluate how and from where new peroxisomes are formed, we followed the trafficking route of newly made YFP-tagged Pex3 and Pex19 proteins by real-time fluorescence microscopy in Saccharomyces cerevisiae. Remarkably, Pex3 (an integral membrane protein) could first be observed in the endoplasmic reticulum (ER), where it concentrates in foci that then bud off in a Pex19-dependent manner and mature into fully functional peroxisomes. Pex19 (a farnesylated, mostly cytosolic protein) enriches first at the Pex3 foci on the ER and then on the maturing peroxisomes. This trafficking route of Pex3-YFP is the same in wild-type cells. These results demonstrate that peroxisomes are generated from domains in the ER.     

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.     

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.     

Pex20p functions as the receptor for non‐PTS1/non‐PTS2 acyl‐CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica

Most soluble proteins targeted to the peroxisomal matrix contain a C‐terminal peroxisome targeting signal type 1 (PTS1) or an N‐terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl‐CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co‐receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2‐containing protein 3‐ketoacyl‐CoA thiolase (Pot1p) but also the non‐PTS1/non‐PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C‐terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C‐terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non‐PTS1/non‐PTS2 Aox isozymes into peroxisomes.     

Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) beta-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA beta-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in beta-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.     

Peroxisomal peripheral membrane protein YlInp1p is required for peroxisome inheritance and influences the dimorphic transition in the yeast Yarrowia lipolytica

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful inheritance of organelles by daughter cells in order to maintain the benefits afforded by the compartmentalization of biochemical functions. Little is known about the inheritance of peroxisomes, organelles of lipid metabolism. We have analyzed peroxisome dynamics and inheritance in the dimorphic yeast Yarrowia lipolytica. Most peroxisomes are anchored at the periphery of cells of Y. lipolytica. In vivo video microscopy showed that at cell division, approximately half of the anchored peroxisomes in the mother cell are dislodged individually from their static positions and transported to the bud. Peroxisome motility is dependent on the actin cytoskeleton. YlInp1p is a peripheral peroxisomal membrane protein that affects the partitioning of peroxisomes between mother cell and bud in Y. lipolytica. In cells lacking YlInp1p, most peroxisomes were transferred to the bud, with only a few remaining in the mother cell, while in cells overexpressing YlInp1p, peroxisomes were preferentially retained in the mother cell, resulting in buds nearly devoid of peroxisomes. Our results are consistent with a role for YlInp1p in anchoring peroxisomes in cells. YlInp1p has a role in the dimorphic transition in Y. lipolytica, as cells lacking the YlINP1 gene more readily convert from the yeast to the mycelial form in oleic acid-containing medium, the metabolism of which requires peroxisomal activity, than does the wild-type strain. This study reports the first analysis of organelle inheritance in a true dimorphic yeast and identifies the first protein required for peroxisome inheritance in Y. lipolytica.     

Structural, functional and genetic aspects of peroxisome biogenesis

Intracellular organelles, peroxisomes, occur in cells of most eukaryotic species. Human severe congenital disorders are associated with defective assembly and functioning of peroxisomes, which partly explains the attention of researchers paid to peroxisome biogenesis. It has been shown that peroxisomes are involved in the realization of eukaryotic developmental programs (in particular, neuroblast differentiation and postembryonic development). Cytobiochemical and electron-microscopic studies of mutations involving peroxisomes showed that the primary role in peroxisome biogenesis is played by synthesis of proteins (peroxins) and their transport and incorporation into peroxisome membranes. More than 30 peroxin-encoding genes have been examined. These genes are synthesized on free polysomes and transported into peroxisomes by means of specific signaling peptides, PTS1, PTS2, and PTS3. The import of matrix proteins depends on at least two shuttle receptor proteins, Pex5p and Pex7p. Some proteins regulating peroxisome proliferation in cells have been identified.     

Requirement for microtubules and dynein motors in the earliest stages of peroxisome biogenesis

Our aim was to determine the role of microtubules in the biogenesis of peroxisomes. Fusion experiments between human PEX16- and PEX1-mutant cells in the presence of nocodazol implied that microtubules were not required for import of proteins into the peroxisomal matrix after cell fusion complementation. We further studied the importance of microtubules in the early stages of peroxisome biogenesis following the microinjection complementation of PEX16-mutant cells. In the absence of nocodazol, nuclear microinjection of plasmids expressing EGFP-SKL and Pex16p in PEX16-mutant cells resulted in the accumulation of EGFP-SKL into newly formed peroxisomes. However, pretreatment of the cells with nocodazol, prior to microinjection, resulted in the inhibition of complementation of the PEX16 mutant and the cytosolic location of the EGFP-SKL. In addition, coexpression of a dominant-negative CC1 subunit of the dynein/dynactin motor complex resulted in the inability to complement PEX16-mutant cells. Both of these treatments resulted in the cytosolic localization of expressed Pex16p. Our results demonstrate that the formation of peroxisomes via the preperoxisomal compartment is dependent upon microtubules and minus-end-directed motor proteins and that the inhibition described above occurs at a step that precedes the association of Pex16p with the vesicles that would otherwise become the peroxisomes.     

Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane.

We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae. pex15Delta cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants. PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p). Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail-anchored type II (Ncyt-Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy-terminus. Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane. The lumenal carboxy-terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O-glycosylation. Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N-terminus of mature invertase. This resulted in core N-glycosylation of the hybrid protein. The lumenal C-terminal tail of Pex15p is essential for targeting to the peroxisomal membrane. Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein. These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.     

Structural,functional and genetic aspects of peroxisome biogenesis

Intracellular organelles, peroxisomes, occur in cells of most eukaryotic species. Human severe congenital disorders are associated with defective assembly and functioning of peroxisomes, which partly explains the attention of researchers paid to peroxisome biogenesis. It has been shown that peroxisomes are involved in the realization of eukaryotic developmental programs (in particular, neuroblast differentiation and postembryonic development). Cytobiochemical and electron-microscopic studies of peroxisomal mutations showed that the primary role in peroxisome biogenesis is played by synthesis of specific proteins (peroxins) and their transport and incorporation into peroxisome membranes. More than 30 peroxin-encoding genes have been examined. These proteins are synthesized on free polysomes and transported into peroxisomes by means of specific signaling peptides, PTS1, PTS2, and PTS3. The import of matrix proteins depends on at least two shuttle receptor proteins, Pex5p and Pex7p. Some proteins regulating peroxisome proliferation in cells have been identified.Translated from Genetika, Vol. 41, No. 2, 2005, pp. 149–165.Original Russian Text Copyright © 2005 by Kurbatova, Dutova, Trotsenko.