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1.
Kanji Okumoto Hiromi Noda Yukio Fujiki 《The Journal of biological chemistry》2014,289(20):14089-14108
Peroxisome targeting signal type-1 (PTS1) receptor, Pex5p, is a key player in peroxisomal matrix protein import. Pex5p recognizes PTS1 cargoes in the cytosol, targets peroxisomes, translocates across the membrane, unloads the cargoes, and shuttles back to the cytosol. Ubiquitination of Pex5p at a conserved cysteine is required for the exit from peroxisomes. However, any potential ubiquitin ligase (E3) remains unidentified in mammals. Here, we establish an in vitro ubiquitination assay system and demonstrate that RING finger Pex10p functions as an E3 with an E2, UbcH5C. The E3 activity of Pex10p is essential for its peroxisome-restoring activity, being enhanced by another RING peroxin, Pex12p. The Pex10p·Pex12p complex catalyzes monoubiquitination of Pex5p at one of multiple lysine residues in vitro, following the dissociation of Pex5p from Pex14p and the PTS1 cargo. Several lines of evidence with lysine-to-arginine mutants of Pex5p demonstrate that Pex10p RING E3-mediated ubiquitination of Pex5p is required for its efficient export from peroxisomes to the cytosol and peroxisomal matrix protein import. RING peroxins are required for both modes of Pex5p ubiquitination, thus playing a pivotal role in Pex5p shuttling. 相似文献
2.
Fouzi El Magraoui Rebecca Brinkmeier Andreas Schrötter Wolfgang Girzalsky Thorsten Müller Katrin Marcus Helmut E. Meyer Ralf Erdmann Harald W. Platta 《Traffic (Copenhagen, Denmark)》2013,14(12):1290-1301
Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1‐receptor. While polyubiquitination of PTS‐receptors results in their proteasomal degradation, the monoubiquitinated PTS‐receptors are exported to the cytosol and recycled for further rounds of protein import. Here, we describe the identification of two ubiquitination cascades acting on the PTS2 co‐receptor Pex18p. Using in vivo and in vitro approaches, we demonstrate that the polyubiquitination of Pex18p requires the ubiquitin‐conjugating enzyme (E2) Ubc4p, which cooperates with the RING (really interesting new gene)‐type ubiquitin‐protein ligases (E3) Pex2p as well as Pex10p. Monoubiquitination of Pex18p depends on the E2 enzyme Pex4p (Ubc10p), which functions in concert with the E3 enzymes Pex12p and Pex10p. Our findings for the PTS2‐pathway complement the data on PTS1‐receptor ubiquitination and add up to a unified concept of the ubiquitin‐based regulation of peroxisomal import . 相似文献
3.
Two independent pathways traffic the intraperoxisomal peroxin PpPex8p into peroxisomes: mechanism and evolutionary implications 总被引:1,自引:0,他引:1
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Among peroxins involved in peroxisome biogenesis, only Pex8p is predominantly intraperoxisomal at steady state. Pex8p is necessary for peroxisomal matrix protein import via the PTS1 and PTS2 pathways. It is proposed to bridge two peroxisomal membrane subcomplexes comprised of the docking (Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) peroxins and is also implicated in cargo release of PTS1 proteins in the matrix. We show that Pichia pastoris Pex8p (PpPex8p) enters the peroxisome matrix using two redundant pathways in a Pex14p-dependent, but Pex2p-independent, manner, showing that the intact importomer and RING subcomplex are not required for its import. One pathway depends on the TPR motifs in Pex5p, the C-terminal PTS1 sequence (AKL) in PpPex8p, and the intraperoxisomal presence of this peroxin. The alternative pathway uses the PTS2 receptor, Pex7p, its accessory protein, Pex20p, and a putative PTS2 motif in PpPex8p, but does not require intraperoxisomal PpPex8p. Pex20p interaction with PpPex8p is independent of Pex7p, but the interaction of PpPex8p with Pex7p requires Pex20p. These data suggest a direct interaction between PpPex8p and Pex20p. Our studies shed light on the mechanism and evolution of the dual import pathways for PpPex8p. 相似文献
4.
The peroxisomal targeting signal type1 (PTS1) receptor Pex5 is required for the peroxisomal targeting of most matrix proteins. Pex5 recognises target proteins in the cytosol and directs them to the peroxisomal membrane where cargo is released into the matrix, and the receptor shuttles back to the cytosol. Recently, it has become evident that the membrane-bound Pex5 can be modified by mono- and polyubiquitination. This review summarises recent results on Pex5 ubiquitination and on the role of the AAA peroxins Pex1 and Pex6 as dislocases required for the release of Pex5 from the membrane to the cytosol where the receptor is either degraded by proteasomes or made available for another round of protein import into peroxisomes. 相似文献
5.
PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis. 相似文献
6.
Benjamin Schwartzkopff Harald?W. Platta Sohel Hasan Wolfgang Girzalsky Ralf Erdmann 《Bioscience reports》2015,35(3)
Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p. 相似文献
7.
Hensel A Beck S El Magraoui F Platta HW Girzalsky W Erdmann R 《The Journal of biological chemistry》2011,286(50):43495-43505
The peroxisomal matrix protein import is facilitated by cycling receptor molecules that shuttle between the cytosol and the peroxisomal membrane. In the yeast Saccharomyces cerevisiae, the import of proteins harboring a peroxisomal targeting signal of type II (PTS2) is mediated by the receptor Pex7p and its co-receptor Pex18p. Here we demonstrate that Pex18p undergoes two kinds of ubiquitin modifications. One of these ubiquitination events depends on lysines 13 and 20 and forces rapid Pex18p turnover by proteasomal degradation. A cysteine residue near the extreme Pex18p amino-terminus is required for the second type of ubiquitination. It turned out that this cysteine residue at position 6 is essential for the function of Pex18p in peroxisomal protein import but does not contribute to receptor-cargo association and binding to the peroxisomal import apparatus. However, in contrast to the wild-type protein, cysteine 6-mutated Pex18p is arrested in a membrane-protected state, whereas Pex7p is accessible in a protease protection assay. This finding indicates that Pex18p export is linked to cargo translocation, which supports the idea of an export-driven import of proteins into peroxisomes. 相似文献
8.
Harald W. Platta Fouzi El Magraoui Bastian E. B?umer Daniel Schlee Wolfgang Girzalsky Ralf Erdmann 《Molecular and cellular biology》2009,29(20):5505-5516
The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane docking of the cargo-receptor complex, cargo release, and recycling of the receptor. The final step is controlled by the ubiquitination status of Pex5. While polyubiquitinated Pex5 is degraded by the proteasome, monoubiquitinated Pex5 is destined for a new round of the receptor cycle. Recently, the ubiquitin-conjugating enzymes involved in Pex5 ubiquitination were identified as Ubc4 and Pex4 (Ubc10), whereas the identity of the corresponding protein-ubiquitin ligases remained unknown. Here we report on the identification of the protein-ubiquitin ligases that are responsible for the ubiquitination of the peroxisomal protein import receptor Pex5. It is demonstrated that each of the three RING peroxins Pex2, Pex10, and Pex12 exhibits ubiquitin-protein isopeptide ligase activity. Our results show that Pex2 mediates the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.The maintenance of peroxisome function depends on the formation of the peroxisomal membrane and the subsequent import of both membrane and matrix proteins. Without exception, peroxisomal matrix proteins are nucleus encoded, synthesized on free ribosomes, and subsequently imported in a posttranslational manner (40). The peroxisomal import apparatus can facilitate the transport of folded and oligomeric proteins over the peroxisomal membrane, with the basic principle of this translocation event still being unknown. Based on the concept of cycling receptors (9, 31), the receptor cycle is divided into four steps. In the first step, the cargo proteins are recognized in the cytosol by their cognate receptor protein Pex5 or Pex7. In general, this initial step depends on either one of the two well-characterized PTSs (peroxisomal targeting signals), PTS1 and PTS2, which are recognized and bound by the corresponding receptor proteins Pex5 and Pex7, respectively. In the second step, the cargo-loaded receptors dock with distinct proteins accessible at the surface of the peroxisomal membrane, namely, Pex13 and Pex14. These two proteins together with Pex17 are established components of the docking complex. A second complex of the peroxisomal protein import machinery acts downstream of the docking event and consists of the three peroxins Pex2, Pex10, and Pex12. A common feature of these proteins is a C-terminal RING (really interesting new gene) finger domain. The RING finger subcomplex and the docking subcomplex are both linked in a Pex8-dependent manner to form a larger assembly, the importomer (1). In the third step of the receptor cycle, the cargo is delivered to the peroxisomal matrix, and finally, the receptor is released from the peroxisomal membrane in an ATP-dependent manner and thus made available for proteasomal degradation or another round of import (for a review, see reference 27).With respect to the PTS1 receptor Pex5, recent reports demonstrated that this final ATP-dependent step in the receptor cycle is catalyzed by the AAA (ATPases associated with various cellular activities) peroxins Pex1 and Pex6 (33, 37). The signal for the export process is the attachment of a monoubiquitin moiety or, alternatively, the anchoring of a polyubiquitin chain (5, 35). This protein modification is in general facilitated by a three-step enzyme cascade (20). The ubiquitin (Ub)-activating enzyme (E1) activates the Ub and transfers it to the Ub conjugation enzyme (E2). In a final step, a protein-Ub ligase (E3) binds both E2 and substrate and thereby facilitates the conjugation of the Ub moiety onto the substrate protein. Saccharomyces cerevisiae harbors genes coding for one E1 enzyme, 11 E2 enzymes, and approximately 80 to 100 putative E3 enzymes (18, 29). It was demonstrated that the polyubiquitination of Pex5 primarily depends on the E2 protein Ubc4, which upon deletion can be partly replaced by Ubc5 or Ubc1 (22, 25, 36). Polyubiquitination of Pex5 is not a prerequisite for its function in peroxisomal protein import but might be a crucial step of a quality control system for the disposal of dysfunctional Pex5 (10, 22, 25, 36). Pex5 monoubiquitination is facilitated by the E2 protein Pex4 (Ubc10) in yeast or the Pex4-like UbcH5a/b/c in humans (14, 35, 47). The modification of Pex5 by a single Ub primes the receptor for its export back to the cytosol, where the Ub supposedly is removed prior to the initiation of a new receptor cycle (5, 14, 35). Although the functional relevance and the cognate E2 protein required for the different Ub modifications of Pex5 were identified, the factor(s) determining the substrate specificity, the protein-Ub ligase(s), remained unknown.Here we report on the discovery of the function of Pex2 and Pex12 as E3 proteins required for ubiquitination of the import receptor Pex5. These RING peroxins, defects of which cause the lethal peroxisome biogenesis disorders in humans, exhibit Ub-protein isopeptide ligase activity with Pex5 as the molecular target. Pex2 is shown to mediate the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5. 相似文献
9.
Peroxisomal import receptors bind their cargo proteins in the cytosol and target them to docking and translocation machinery at the peroxisomal membrane (reviewed in ref. 1). The receptors release the cargo proteins into the peroxisomal lumen and, according to the model of cycling receptors, they are supposed to shuttle back to the cytosol. This shuttling of the receptors has been assigned to peroxins including the AAA peroxins Pex1p and Pex6p, as well as the ubiquitin-conjugating enzyme Pex4p (reviewed in ref. 2). One possible target for Pex4p is the PTS1 receptor Pex5p, which has recently been shown to be ubiquitinated. Pex1p and Pex6p are both cytosolic and membrane-associated AAA ATPases of the peroxisomal protein import machinery, the exact function of which is still unknown. Here we demonstrate that the AAA peroxins mediate the ATP-dependent dislocation of the peroxisomal targeting signal-1 (PTS1) receptor from the peroxisomal membrane to the cytosol. 相似文献
10.
Otera H Harano T Honsho M Ghaedi K Mukai S Tanaka A Kawai A Shimizu N Fujiki Y 《The Journal of biological chemistry》2000,275(28):21703-21714
In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p. 相似文献
11.
Pex7p and Pex20p of Neurospora crassa function together in PTS2-dependent protein import into peroxisomes 总被引:4,自引:0,他引:4
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Sichting M Schell-Steven A Prokisch H Erdmann R Rottensteiner H 《Molecular biology of the cell》2003,14(2):810-821
Recruiting matrix proteins with a peroxisomal targeting signal type 2 (PTS2) to the peroxisomal membrane requires species-specific factors. In Saccharomyces cerevisiae, the PTS2 receptor Pex7p acts in concert with the redundant Pex18p/Pex21p, whereas in Yarrowia lipolytica, Pex20p might unite the function of both S. cerevisiae peroxins. Herein, the genome of the filamentous fungus Neurospora crassa was analyzed for peroxin-encoding genes. We identified a set of 18 peroxins that resembles that of Y. lipolytica rather than that of S. cerevisiae. Interestingly, proteins homologous to both S. cerevisiae Pex7p and Y. lipolytica Pex20p exist in N. crassa. We report on the isolation of these PTS2-specific peroxins and demonstrate that NcPex20p can substitute for S. cerevisiae Pex18p/Pex21p, but not for ScPex7p. Like Pex18p, NcPex20p did not bind PTS2 protein or the docking proteins in the absence of ScPex7p. Rather, NcPex20p was required before docking to form an import-competent complex of cargo-loaded PTS2 receptors. NcPex7p did not functionally replace yeast Pex7p, probably because the N. crassa PTS2 receptor failed to associate with Pex18p/Pex21p. However, once NcPex7p and NcPex20p had been coexpressed, it proved possible to replace yeast Pex7p. Pex20p and Pex18p/Pex21p are therefore true orthologues, both of which are in need of Pex7p for PTS2 protein import. 相似文献
12.
Pex14 is the sole component of the peroxisomal translocon that is required for pexophagy 总被引:1,自引:0,他引:1
Pex14 was initially identified as a peroxisomal membrane protein that is involved in docking of the soluble receptor proteins Pex5 and Pex7, which are required for import of PTS1- or PTS2-containing peroxisomal matrix proteins. However, Hansenula polymorpha Pex14 is also required for selective degradation of peroxisomes (pexophagy). Previously we showed that Pex1, Pex4, Pex6 and Pex8 are not required for this process. Here we show that also in the absence of various other peroxins, namely Pex2, Pex10, Pex12, Pex13 and Pex17, pexophagy can normally occur. These peroxins are, like Pex14, components of the peroxisomal translocon. Our data confirm that Pex14 is the sole peroxin that has a unique dual function in two apparent opposite processes, namely peroxisome formation and selective degradation. 相似文献
13.
Changle Ma Danielle Hagstrom Soumi Guha Polley Suresh Subramani 《The Journal of biological chemistry》2013,288(38):27220-27231
In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1–110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle. 相似文献
14.
Daniel Effelsberg Luis Daniel Cruz-Zaragoza Jason Tonillo Wolfgang Schliebs Ralf Erdmann 《The Journal of biological chemistry》2015,290(42):25333-25342
Proteins designated for peroxisomal protein import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here, we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, whereas Pex18p is especially important for oleate-induced import of PTS2 proteins. The glycerol-producing PTS2 protein glycerol-3-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol, and in the nucleus under osmotic stress conditions. We show the following: (i) Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD+ salvage pathway, Pnc1p; (ii) Pnc1p, a nicotinamidase without functional PTS2, is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions. 相似文献
15.
Kragt A Voorn-Brouwer T van den Berg M Distel B 《The Journal of biological chemistry》2005,280(9):7867-7874
Pex5p is a mobile receptor for peroxisomal targeting signal type I-containing proteins that cycles between the cytoplasm and the peroxisome. Here we show that Pex5p is a stable protein that is monoubiquitinated in wild type cells. By making use of mutants defective in vacuolar or proteasomal degradation we demonstrate that monoubiquitinated Pex5p is not a breakdown intermediate of either system. Monoubiquitinated Pex5p is localized to peroxisomes, and ubiquitination requires the presence of functional docking and RING finger complexes, which suggests that it is a late event in peroxisomal matrix protein import. In pex1, pex4, pex6, pex15, and pex22 mutants, all of which are blocked in the terminal steps of peroxisomal matrix protein import, polyubiquitinated forms of Pex5p accumulate, ubiquitination being dependent on the ubiquitin-conjugating enzyme Ubc4p. However, Ubc4p is not required for Pex5p ubiquitination in wild type cells, and cells lacking Ubc4p are not affected in peroxisome biogenesis. These results indicate that Pex5p monoubiquitination in wild type cells serves to regulate rather than to degrade Pex5p, which is supported by the observed stability of Pex5p. We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, whereas Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway. 相似文献
16.
Danielle Hagstrom Changle Ma Soumi Guha-Polley Suresh Subramani 《Molecular biology of the cell》2014,25(17):2634-2643
Peroxisomal matrix protein import uses two peroxisomal targeting signals (PTSs). Most matrix proteins use the PTS1 pathway and its cargo receptor, Pex5. The PTS2 pathway is dependent on another receptor, Pex7, and its coreceptor, Pex20. We found that during the matrix protein import cycle, the stability and dynamics of Pex7 differ from those of Pex5 and Pex20. In Pichia pastoris, unlike Pex5 and Pex20, Pex7 is constitutively degraded in wild-type cells but is stabilized in pex mutants affecting matrix protein import. Degradation of Pex7 is more prevalent in cells grown in methanol, in which the PTS2 pathway is nonessential, in comparison with oleate, suggesting regulation of Pex7 turnover. Pex7 must shuttle into and out of peroxisomes before it is polyubiquitinated and degraded by the proteasome. The shuttling of Pex7, and consequently its degradation, is dependent on the receptor recycling pathways of Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. We also found that blocking the export of Pex20 from peroxisomes inhibits PTS1-mediated import, suggesting sharing of limited components in the export of PTS receptors/coreceptors. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20, exemplifying a novel interdependence of the PTS1 and PTS2 pathways. 相似文献
17.
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. 相似文献
18.
PTS2 co-receptors: diverse proteins with common features 总被引:1,自引:0,他引:1
One feature of the PTS2 import pathway is the separation of the roles of the PTS receptor between two proteins. Pex7p alone is insufficient to act as the receptor for the import cycle for peroxisomal matrix proteins. In all cases, Pex7p needs a PTS2 co-receptor to form an import-competent PTS2 receptor complex together with the PTS2 cargo. We provide an overview of the proteins that have been identified as PTS2 co-receptors and discuss their proposed functions. 相似文献
19.
Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation 总被引:1,自引:0,他引:1
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Léon S Zhang L McDonald WH Yates J Cregg JM Subramani S 《The Journal of cell biology》2006,172(1):67-78
We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes. PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p. Similarities between Pex5p and Pex20p were noted in their protein interactions and dynamics during import, and both contain a conserved NH2-terminal domain. In the absence of the E2-like Pex4p or the AAA proteins Pex1p and Pex6p, Pex20p is degraded via polyubiquitylation of residue K19, and the K19R mutation causes accumulation of Pex20p in peroxisome remnants. Finally, either interference with K48-branched polyubiquitylation or removal of the conserved NH2-terminal domain causes accumulation of Pex20p in peroxisomes, mimicking a defect in its recycling to the cytosol. Our data are consistent with a model in which Pex20p enters peroxisomes and recycles back to the cytosol in an ubiquitin-dependent manner. 相似文献
20.
Yun-Ting Kao Wendell A. Fleming Meredith J. Ventura Bonnie Bartel 《Plant physiology》2016,172(3):1643-1656
Most eukaryotic cells require peroxisomes, organelles housing fatty acid β-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired β-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.Oilseed plants obtain energy for germination and early development by utilizing stored fatty acids (Graham, 2008). This β-oxidation of fatty acids to acetyl-CoA occurs in peroxisomes, organelles that also house other important metabolic reactions, including the glyoxylate cycle, several steps in photorespiration, and phytohormone production (Hu et al., 2012). For example, indole-3-butyric acid (IBA) is β-oxidized into the active auxin indole-3-acetic acid (IAA) in peroxisomes (Zolman et al., 2000, 2007, 2008; Strader et al., 2010; Strader and Bartel, 2011). Many peroxisomal metabolic pathways generate reactive oxygen species (Inestrosa et al., 1979; Hu et al., 2012), and peroxisomes also house antioxidative enzymes, like catalase and ascorbate peroxidase, to detoxify hydrogen peroxide (Wang et al., 1999; Mhamdi et al., 2012).Peroxisomes can divide by fission or be synthesized de novo from the endoplasmic reticulum (ER). Preperoxisomes with peroxisomal membrane proteins bud from the ER and fuse, allowing matrix proteins to be imported to form mature peroxisomes (van der Zand et al., 2012; Mayerhofer, 2016). Peroxin (PEX) proteins facilitate peroxisome biogenesis and matrix protein import. Most peroxins are involved in importing proteins destined for the peroxisome matrix, which are imported after recognition of a type 1 or type 2 peroxisome-targeting signal (PTS). The PTS1 is a tripeptide located at the C terminus of most peroxisome-bound proteins (Gould et al., 1989; Chowdhary et al., 2012). The less common PTS2 is a nonapeptide usually located near the N terminus (Swinkels et al., 1991; Reumann, 2004). PTS1 proteins are recognized by PEX5 (van der Leij et al., 1993; Zolman et al., 2000), PTS2 proteins are recognized by PEX7 (Marzioch et al., 1994; Braverman et al., 1997; Woodward and Bartel, 2005), and PEX7 binds to PEX5 to allow matrix protein delivery in plants and mammals (Otera et al., 1998; Hayashi et al., 2005; Woodward and Bartel, 2005). The cargo-receptor complex docks with the membrane peroxins PEX13 and PEX14 (Urquhart et al., 2000; Otera et al., 2002; Woodward et al., 2014), and PEX5 assists cargo translocation into the peroxisomal matrix (Meinecke et al., 2010) before dissociating from its cargo (Freitas et al., 2011).After cargo delivery, PEX5 is recycled to enable further rounds of cargo recruitment (Thoms and Erdmann, 2006). This process requires a set of peroxins that is implicated in ubiquitinating PEX5 so that it can be retrotranslocated back to the cytosol. PEX5 ubiquitination is best understood in yeast. In Saccharomyces cerevisiae, Pex5 is monoubiquitinated through the action of the peroxisome-tethered ubiquitin-conjugating enzyme Pex4 and the peroxisomal ubiquitin-protein ligase Pex12 (Platta et al., 2009) and returned to the cytosol with the assistance of a peroxisome-tethered ATPase complex containing Pex1 and Pex6 (Grimm et al., 2012). S. cerevisiae Pex5 also can be polyubiquitinated and targeted for proteasomal degradation (Kiel et al., 2005). The cytosolic ubiquitin-conjugating enzyme Ubc4 cooperates with the peroxisomal ubiquitin-protein ligase Pex2 to polyubiquitinate Pex5 (Platta et al., 2009). Pex10 has ubiquitin-protein ligase activity (Williams et al., 2008; Platta et al., 2009; El Magraoui et al., 2012), but whether Pex10 directly ubiquitinates Pex5 is controversial. Pex10 promotes Ubc4-dependent Pex5 polyubiquitination when Pex4 is absent (Williams et al., 2008); however, Pex10 is not essential for Pex5 mono- or polyubiquitination (Platta et al., 2009), but rather enhances both Pex4/Pex12- and Ubc4/Pex2-mediated ubiquitination (El Magraoui et al., 2012). Recycling of the PTS2 receptor PEX7 is less understood, although the Pex5 recycling pathways are implicated in shuttling and degrading Pex7 in Pichia pastoris (Hagstrom et al., 2014).Although PEX5 ubiquitination has not been directly demonstrated in plants, the implicated peroxins are conserved in Arabidopsis, and several have been connected to PEX5 retrotranslocation. The PEX4 ubiquitin-conjugating enzyme binds to PEX22, which is predicted to be a peroxisomal membrane protein based on ability to restore peroxisome function to yeast mutants (Zolman et al., 2005). The pex4-1 mutant displays increased membrane-associated PEX5 (Ratzel et al., 2011; Kao and Bartel, 2015), suggesting that ubiquitin supplied by PEX4 promotes PEX5 retrotranslocation. PEX1 and PEX6 are members of the ATPases associated with diverse cellular activities (AAA) family and are tethered to peroxisomes by the peroxisomal membrane protein PEX26 (Goto et al., 2011; Li et al., 2014). The pex6-1 mutant displays PTS1 import defects and decreased PEX5 levels (Zolman and Bartel, 2004), suggesting that impaired PEX5 recycling can lead to increased PEX5 degradation. Indeed, pex4-1 restores PEX5 levels in the pex6-1 mutant (Ratzel et al., 2011), suggesting that Arabidopsis PEX4 also is involved in PEX5 ubiquitination and degradation when retrotranslocation is impeded.In addition to allowing for further rounds of PTS1 cargo import, several lines of evidence suggest that in the absence of efficient retrotranslocation, PEX5 retention in the peroxisomal membrane impairs peroxisome function. Slightly reducing levels of the PEX13 docking peroxin ameliorates the physiological defects of pex4-1 without restoring matrix protein import (Ratzel et al., 2011), presumably because decreasing PEX5 docking reduces its accumulation in the peroxisomal membrane. In addition, overexpressing PEX5 exacerbates rather than ameliorates the peroxisomal defects of pex4-1 (Kao and Bartel, 2015), suggesting that pex4-1 defects are linked to excessive PEX5 lingering in the peroxisome membrane rather than a lack of PEX5 available for import.The three Really Interesting New Gene (RING) peroxins (PEX2, PEX10, and PEX12) from Arabidopsis each possesses in vitro ubiquitin-protein ligase activity (Kaur et al., 2013). Null mutations in the RING peroxin genes confer embryo lethality in Arabidopsis (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Prestele et al., 2010), necessitating other approaches to study the in vivo functions of these peroxins. Expressing RING peroxins with mutations in the C-terminal zinc-binding RING domains (ΔZn) confers matrix protein import defects for PEX2-ΔZn and photorespiration defects for PEX10-ΔZn but no apparent defects for PEX12-ΔZn (Prestele et al., 2010). Targeting individual RING peroxins using RNAi confers β-oxidation deficiencies and impairs PTS1 cargo import (Fan et al., 2005; Nito et al., 2007). A screen for delayed matrix protein degradation (Burkhart et al., 2013) uncovered a missense pex2-1 mutant and a splicing pex10-2 mutant that both display PTS1 import defects (Burkhart et al., 2014), suggesting roles in regulating the PTS1 receptor, PEX5. A missense pex12 mutant (aberrant peroxisome morphology 4, apm4) has defects in β-oxidation and PTS1 import and increased membrane-associated PEX5 (Mano et al., 2006). These findings highlight the essential roles of the RING peroxins in Arabidopsis development and peroxisomal functions, but the RING peroxin interactions and the individual roles of the RING peroxins in PEX5 retrotranslocation remain incompletely understood.In this study, we describe a missense pex12-1 mutant recovered from a forward genetic screen for β-oxidation deficient mutants. The pex12-1 mutant displayed severe peroxisomal defects, including reduced growth, β-oxidation deficiencies, matrix protein import defects, and inefficient processing of PTS2 proteins. Comparing single and double mutants with impaired RING peroxins revealed that each RING peroxin contributes to complex stability and influences PEX5 accumulation. Furthermore, decreasing PEX4 function ameliorated pex12-1 defects, suggesting that the Glu-to-Lys substitution in pex12-1 lures ubiquitination, perhaps by pex12-1 itself, leading to PEX4-dependent degradation of the mutant protein. 相似文献