Pex14 is the sole component of the peroxisomal translocon that is required for pexophagy (original) (raw)
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The Peroxisomal Matrix Import of Pex8p Requires Only PTS Receptors and Pex14p
Molecular Biology of the Cell, 2009
Pichia pastoris (Pp) Pex8p, the only known intraperoxisomal peroxin at steady state, is targeted to peroxisomes by either the peroxisomal targeting signal (PTS) type 1 or PTS2 pathway. Until recently, all cargoes entering the peroxisome matrix were believed to require the docking and really interesting new gene (RING) subcomplexes, proteins that bridge these two subcomplexes and the PTS receptor-recycling machinery. However, we reported recently that the import of PpPex8p into peroxisomes via the PTS2 pathway is Pex14p dependent but independent of the RING subcomplex ( Zhang et al., 2006 ). In further characterizing the peroxisome membrane-associated translocon, we show that two other components of the docking subcomplex, Pex13p and Pex17p, are dispensable for the import of Pex8p. Moreover, we demonstrate that the import of Pex8p via the PTS1 pathway also does not require the RING subcomplex or intraperoxisomal Pex8p. In receptor-recycling mutants (Δpex1, Δpex6, and Δpex4), Pex8p is...
Journal of Biological Chemistry, 2013
Background: PEX7 mediates the import of peroxisomal proteins from the cytosol. Results: The small GTPase RabE1c binds PEX7 on peroxisomes and mediates its degradation. Conclusion: RabE1c is responsible for PEX7 dislocation/degradation on the peroxisome membrane. Significance: By proteomic analysis, we identified a novel protein that regulates PEX7 function and revealed a crucial mechanism for PEX7 translocation from the peroxisome membrane. The biogenesis of peroxisomes is mediated by peroxins (PEXs). PEX7 is a cytosolic receptor that imports peroxisomal targeting signal type 2 (PTS2)-containing proteins. Although PEX7 is important for protein transport, the mechanisms that mediate its function are unknown. In this study, we performed proteomic analysis to identify PEX7-binding proteins using transgenic Arabidopsis expressing green fluorescent protein (GFP)-tagged PEX7. Our analysis identified RabE1c, a small GTPase, as a PEX7 binding partner. In vivo analysis revealed that GTP-bound RabE1c binds to PEX7 and that a subset of RabE1c localizes to peroxisomes and interacts with PEX7 on the peroxisome membrane. Unlike endogenous PEX7, which is predominantly localized to the cytosol, GFP-PEX7 accumulates abnormally on the peroxisomal membrane and induces degradation of endogenous PEX7, concomitant with a reduction in import of PTS2-containing proteins and decreased peroxisomal -oxidation activity. Thus, GFP-PEX7 on the peroxisomal membrane exerts a dominant negative effect. Mutation of RabE1c restored endogenous PEX7 protein expression and import of PTS2-containing proteins as well as peroxisomal -oxidation activity. Treatment with proteasome inhibitors also restored endogenous PEX7 protein levels in GFP-PEX7expressing seedlings. Based on these findings, we conclude that RabE1c binds PEX7 and facilitates PEX7 degradation in the presence of immobile GFP-PEX7 accumulated at the membrane. Because peroxisomes lack their own genome, all of their constituent proteins are encoded in the nuclear genome and translated on cytosolic ribosomes before being imported into peroxisomes. Two types of peroxisomal targeting signals, type 1 (PTS1) 2 and type 2 (PTS2), have been identified in peroxisomal matrix enzymes. PTS1 signals are found at C termini and consist of tripeptide variants (1), whereas PTS2 signals consist of nonapeptides found in cleavable N-terminal presequences (2). About one-fifth of peroxisomal enzymes contain PTS2, including several proteins involved in fatty acid metabolism (e.g. 3-ketoacyl-CoA thiolase, citrate synthase, malate dehydrogenase, and long-chain acyl-CoA oxidase) (2-5). These PTS2-containing proteins are synthesized as precursors in the cytosol before import into the peroxisomes. Upon import, the signal peptides are cleaved by the PTS2-processing enzyme, DEG15 endopeptidase (6). Studies in different organisms have revealed two soluble import receptors: PEX5 for PTS1 and PEX7 for PTS2 (7, 8). PEX5 and PEX7 interact with each other to facilitate peroxisomal protein import. Mammals possess two isoforms of PEX5, only the longer of which (PEX5L) binds to PEX7 (9, 10). In Arabidopsis, which has a single PEX5 isoform, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays have demonstrated that PEX7 directly binds to PEX5 in the cytosol. The middle region of PEX7, which contains the WD40 repeat domain, is necessary and sufficient for the interaction with PEX5 (11-13). An Arabidopsis pex5 mutant that lacks PEX7 binding activity is defective in PTS2-containing protein transport, demonstrating that the import of PTS2-containing proteins is dependent on PEX7 binding to PEX5 (8). In Saccharomyces cerevisiae, PTS2-containing protein transport requires both PEX7 and its auxiliary proteins, PEX18 and PEX21, instead of PEX5 (14). In Neurospora crassa, Yarrowia lipolytica, and Pichia pastoris, PEX18 and PEX21 are replaced by a single PEX7 docking protein, PEX20 (15, 16). These auxiliary proteins and mammalian PEX5L share common motif sequences involved in PEX7 interactions. The lack of these auxiliary pro
Pexophagy: The Selective Degradation of Peroxisomes
Peroxisomes are single-membrane-bounded organelles present in the majority of eukaryotic cells. Despite the existence of great diversity among different species, cell types, and under different environmental conditions, peroxisomes contain enzymes involved in β-oxidation of fatty acids and the generation, as well as detoxification, of hydrogen peroxide. The exigency of all eukaryotic cells to quickly adapt to different environmental factors requires the ability to precisely and efficiently control peroxisome number and functionality. Peroxisome homeostasis is achieved by the counterbalance between organelle biogenesis and degradation. The selective degradation of superfluous or damaged peroxisomes is facilitated by several tightly regulated pathways. The most prominent peroxisome degradation system uses components of the general autophagy core machinery and is therefore referred to as "pexophagy." In this paper we focus on recent developments in pexophagy and provide an overview of current knowledge and future challenges in the field. We compare different modes of pexophagy and mention shared and distinct features of pexophagy in yeast model systems, mammalian cells, and other organisms.
The Journal of biological chemistry, 2015
Pexophagy is a process that selectively degrades peroxisomes by autophagy. The Pichia pastoris pexophagy receptor Atg30 is recruited to peroxisomes under peroxisome proliferation conditions. During pexophagy, Atg30 undergoes phosphorylation, a prerequisite for its interactions with the autophagy scaffold protein Atg11 and the ubiquitin-like protein Atg8. Atg30 is subsequently shuttled to the vacuole along with the targeted peroxisome for degradation. Here, we defined the binding site for Atg30 on the peroxisomal membrane protein Pex3 and uncovered a role for Pex3 in the activation of Atg30 via phosphorylation and in the recruitment of Atg11 to the receptor protein complex. Pex3 is classically a docking protein for other proteins that affect peroxisome biogenesis, division, and segregation. We conclude that Pex3 has a role beyond simple docking of Atg30 and that its interaction with Atg30 regulates pexophagy in the yeast P. pastoris.
Journal of Biological Chemistry, 2000
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.
The Peroxin Pex19p Interacts with Multiple, Integral Membrane Proteins at the Peroxisomal Membrane
The Journal of Cell Biology, 2000
Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These d...
The FEBS Journal
PEX13 and PEX14 are two core components of the so-called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease-protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a N in-C out topology, and that PEX13 adopts a N out-C in topology, thus exposing its carboxyterminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery.