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.     

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.     

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.     

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.     

A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Delta mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Delta cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.     

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.     

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.     

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.     

Reevaluation of the role of Pex1 and dynamin-related proteins in peroxisome membrane biogenesis

A recent model for peroxisome biogenesis postulates that peroxisomes form de novo continuously in wild-type cells by heterotypic fusion of endoplasmic reticulum–derived vesicles containing distinct sets of peroxisomal membrane proteins. This model proposes a role in vesicle fusion for the Pex1/Pex6 complex, which has an established role in matrix protein import. The growth and division model proposes that peroxisomes derive from existing peroxisomes. We tested these models by reexamining the role of Pex1/Pex6 and dynamin-related proteins in peroxisome biogenesis. We found that induced depletion of Pex1 blocks the import of matrix proteins but does not affect membrane protein delivery to peroxisomes; markers for the previously reported distinct vesicles colocalize in pex1 and pex6 cells; peroxisomes undergo continued growth if fission is blocked. Our data are compatible with the established primary role of the Pex1/Pex6 complex in matrix protein import and show that peroxisomes in Saccharomyces cerevisiae multiply mainly by growth and division.     

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.     

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.     

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.     

Pex19p, a Farnesylated Protein Essential for Peroxisome Biogenesis

We report the identification and molecular characterization of Pex19p, an oleic acid-inducible, farnesylated protein of 39.7 kDa that is essential for peroxisome biogenesis in Saccharomyces cerevisiae. Cells lacking Pex19p are characterized by the absence of morphologically detectable peroxisomes and mislocalization of peroxisomal matrix proteins to the cytosol. The human HK33 gene product was identified as the putative human ortholog of Pex19p. Evidence is provided that farnesylation of Pex19p takes place at the cysteine of the C-terminal CKQQ amino acid sequence. Farnesylation of Pex19p was shown to be essential for the proper function of the protein in peroxisome biogenesis. Pex19p was shown to interact with Pex3p in vivo, and this interaction required farnesylation of Pex19p.     

Yeast pex1 cells contain peroxisomal ghosts that import matrix proteins upon reintroduction of Pex1

Pex1 and Pex6 are two AAA-ATPases that play a crucial role in peroxisome biogenesis. We have characterized the ultrastructure of the Saccharomyces cerevisiae peroxisome-deficient mutants pex1 and pex6 by various high-resolution electron microscopy techniques. We observed that the cells contained peroxisomal membrane remnants, which in ultrathin cross sections generally appeared as double membrane rings. Electron tomography revealed that these structures consisted of one continuous membrane, representing an empty, flattened vesicle, which folds into a cup shape. Immunocytochemistry revealed that these structures lack peroxisomal matrix proteins but are the sole sites of the major peroxisomal membrane proteins Pex2, Pex10, Pex11, Pex13, and Pex14. Upon reintroduction of Pex1 in Pex1-deficient cells, these peroxisomal membrane remnants (ghosts) rapidly incorporated peroxisomal matrix proteins and developed into peroxisomes. Our data support earlier views that Pex1 and Pex6 play a role in peroxisomal matrix protein import.     

Peroxisome biogenesis and peroxisome biogenesis disorders

Peroxisome assembly in mammals requires more than 15 genes. Two isoforms of the peroxisome targeting signal type 1 (PTS1) receptor, Pex5pS and Pex5pL, are identified in mammals. Pex5pS and Pex5pL bind PTS1 proteins. Pex5pL, but not Pex5pS, directly interacts with the PTS2 receptor, Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5p carrying the cargos, PTS1 and PTS2, docks with the initial site Pex14p in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p and Pex12p, whereby the matrix proteins are imported. The peroxins, Pex3p, Pex16p and Pex19p, function in the assembly of peroxisomal membrane vesicles that precedes the import of matrix proteins. Hence, peroxisomes may form de novo and do not have to arise from pre-existing, morphologically recognizable peroxisomes. Impaired peroxisome assembly causes peroxisome biogenesis disorders such as Zellweger syndrome.     

New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p.     

p24 proteins play a role in peroxisome proliferation in yeast

Emp24 is a member of the p24 protein family, which was initially localized to the endoplasmic reticulum, Golgi and COP vesicles, but has recently shown to be associated with Saccharomyces cerevisiae peroxisomes as well. Using cell fractionation and electron- and fluorescence microscopy, we show that in the yeast Hansenula polymorpha, Emp24 also associates with peroxisomes. In addition, we show that peroxisome numbers are strongly decreased in H. polymorpha cells lacking two proteins of the p24 complex, Emp24 and Erp3. Detailed fluorescence microscopy analyses suggest that emp24.erp3 cells are disturbed in peroxisome fission and inheritance.     

Pex18p and Pex21p,a Novel Pair of Related Peroxins Essential for Peroxisomal Targeting by the PTS2 Pathway

We have identified ScPex18p and ScPex21p, two novel S. cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis. Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor. Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor. Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes. They each interact specifically with Pex7p both in two-hybrid analyses and in vitro. In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished. Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting.