A Cargo-centered Perspective on the PEX5 Receptor-mediated Peroxisomal Protein Import Pathway

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.     

Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility

Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.     

Mechanistic Insights into PTS2-mediated Peroxisomal Protein Import: THE CO-RECEPTOR PEX5L DRASTICALLY INCREASES THE INTERACTION STRENGTH BETWEEN THE CARGO PROTEIN AND THE RECEPTOR PEX7*

The destination of peroxisomal matrix proteins is encoded by short peptide sequences, which have been characterized as peroxisomal targeting signals (PTS) residing either at the C terminus (PTS1) or close to the N terminus (PTS2). PTS2-carrying proteins interact with their cognate receptor protein PEX7 that mediates their transport to peroxisomes by a concerted action with a co-receptor protein, which in mammals is the PTS1 receptor PEX5L. Using a modified version of the mammalian two-hybrid assay, we demonstrate that the interaction strength between cargo and PEX7 is drastically increased in the presence of the co-receptor PEX5L. In addition, cargo binding is a prerequisite for the interaction between PEX7 and PEX5L and ectopic overexpression of PTS2-carrying cargo protein drastically increases the formation of PEX7-PEX5L complexes in this assay. Consistently, we find that the peroxisomal transfer of PEX7 depends on cargo binding and that ectopic overexpression of cargo protein stimulates this process. Thus, the sequential formation of a highly stable trimeric complex involving cargo protein, PEX7 and PEX5L stabilizes cargo binding and is a prerequisite for PTS2-mediated peroxisomal import.     

A Mobile PTS2 Receptor for Peroxisomal Protein Import in Pichia pastoris

Abstract. Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Δ mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Δ cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Δ mutant and were impaired in binding to the PTS2 sequence.     

PEX5, the Shuttling Import Receptor for Peroxisomal Matrix Proteins,Is a Redox‐Sensitive Protein

Peroxisome maintenance depends on the import of nuclear‐encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C‐terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N‐terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox‐sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination‐deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.      

Distinct Ubiquitination Cascades Act on the Peroxisomal Targeting Signal Type 2 Co‐receptor Pex18p

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 .     

Pex2 and Pex12 Function as Protein-Ubiquitin Ligases in Peroxisomal Protein Import

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.     

Rhizomelic Chondrodysplasia Punctata,a Peroxisomal Biogenesis Disorder Caused by Defects in Pex7p,a Peroxisomal Protein Import Receptor: A Minireview

Rhizomelic chondrodysplasia punctata (RCDP) is a lethal autosomal recessive disease correspondingto complementation group 11 (CG 11), the second most common of the thirteen CGs of peroxisomalbiogenesis disorders (PBDs). RCDP is characterized by proximal limb shortening, severely disturbedendochondrial bone formation, and mental retardation, but there is an absence of the neuronal migrationdefect found in the other PBDs. Plasmalogen biosynthesis and phytanic acid oxidation are deficient, butvery long chain fatty acid (VLCFA) oxidation is normal. At the cellular level, RCDP is unique in thatthe biogenesis of most peroxisomal proteins is normal, but a specific subset of at least four, and maybemore, peroxisomal matrix proteins fail to be imported from the cytosol. In this review, we discuss recentadvances in understanding RCDP, most prominently the cloning of the affected gene, PEX7,and identification of PEX7 mutations in RCDP patients. Human PEX7 wasidentified by virtue of its sequence similarity to its Saccharomyces cerevisiae ortholog, whichhad previously been shown to encode Pex7p, an import receptor for type 2 peroxisomal targetingsequences (PTS2). Normal human PEX7 expression rescues the cellular defects in culturedRCDP cells, and cDNA sequence analysis has identified a variety of PEX7 mutations in RCDP patients,including a deletion of 100 nucleotides, probably due to a splice site mutation, and a prevalent nonsensemutation which results in loss of the carboxyterminal 32 amino acids. Identification of RCDP as a PTS2import disorder explains the observation that several, but not all, peroxisomal matrix proteins aremistargeted in this disease; three of the four proteins deficient in RCDP have now been shown to bePTS2-targeted.     

Arabidopsis LON2 Is Necessary for Peroxisomal Function and Sustained Matrix Protein Import

Relatively little is known about the small subset of peroxisomal proteins with predicted protease activity. Here, we report that the peroxisomal LON2 (At5g47040) protease facilitates matrix protein import into Arabidopsis (Arabidopsis thaliana) peroxisomes. We identified T-DNA insertion alleles disrupted in five of the nine confirmed or predicted peroxisomal proteases and found only two—lon2 and deg15, a mutant defective in the previously described PTS2-processing protease (DEG15/At1g28320)—with phenotypes suggestive of peroxisome metabolism defects. Both lon2 and deg15 mutants were mildly resistant to the inhibitory effects of indole-3-butyric acid (IBA) on root elongation, but only lon2 mutants were resistant to the stimulatory effects of IBA on lateral root production or displayed Suc dependence during seedling growth. lon2 mutants displayed defects in removing the type 2 peroxisome targeting signal (PTS2) from peroxisomal malate dehydrogenase and reduced accumulation of 3-ketoacyl-CoA thiolase, another PTS2-containing protein; both defects were not apparent upon germination but appeared in 5- to 8-d-old seedlings. In lon2 cotyledon cells, matrix proteins were localized to peroxisomes in 4-d-old seedlings but mislocalized to the cytosol in 8-d-old seedlings. Moreover, a PTS2-GFP reporter sorted to peroxisomes in lon2 root tip cells but was largely cytosolic in more mature root cells. Our results indicate that LON2 is needed for sustained matrix protein import into peroxisomes. The delayed onset of matrix protein sorting defects may account for the relatively weak Suc dependence following germination, moderate IBA-resistant primary root elongation, and severe defects in IBA-induced lateral root formation observed in lon2 mutants.Peroxisomes are single-membrane-bound organelles found in most eukaryotes. Peroxin (PEX) proteins are necessary for various aspects of peroxisome biogenesis, including matrix protein import (for review, see Distel et al., 1996; Schrader and Fahimi, 2008). Most matrix proteins are imported into peroxisomes from the cytosol using one of two targeting signals, a C-terminal type 1 peroxisome-targeting signal (PTS1) or a cleavable N-terminal type 2 peroxisome-targeting signal (PTS2) (Reumann, 2004). PTS1- and PTS2-containing proteins are bound in the cytosol by soluble matrix protein receptors, escorted to the peroxisome membrane docking complex, and translocated into the peroxisome matrix (for review, see Platta and Erdmann, 2007). Once in the peroxisome, many matrix proteins participate in metabolic pathways, such as β-oxidation, hydrogen peroxide decomposition, and photorespiration (for review, see Gabaldon et al., 2006; Poirier et al., 2006).In addition to metabolic enzymes, several proteases are found in the peroxisome matrix. Only one protease, DEG15/Tysnd1, has a well-defined role in peroxisome biology. The rat Tysnd1 protease removes the targeting signal after PTS2-containing proteins enter the peroxisome and also processes certain PTS1-containing β-oxidation enzymes (Kurochkin et al., 2007). Similarly, the Arabidopsis (Arabidopsis thaliana) Tysnd1 homolog DEG15 (At1g28320) is a peroxisomal Ser protease that removes PTS2 targeting signals (Helm et al., 2007; Schuhmann et al., 2008).In contrast with DEG15, little is known about the other eight Arabidopsis proteins that are annotated as proteases in the AraPerox database of putative peroxisomal proteins (Reumann et al., 2004; Carter et al., 2004; Shimaoka et al., 2004), which, in combination with the minor PTS found in both of these predicted proteases (Reumann, 2004), suggests that these enzymes may not be peroxisomal. Along with DEG15, only two of the predicted peroxisomal proteases, an M16 metalloprotease (At2g41790), which we have named PXM16 for peroxisomal M16 protease, and a Lon-related protease (At5g47040/LON2; Ostersetzer et al., 2007), are found in the proteome of peroxisomes purified from Arabidopsis suspension cells (Eubel et al., 2008). DEG15 and LON2 also have been validated as peroxisomally targeted using GFP fusions (Ostersetzer et al., 2007; Schuhmann et al., 2008).

Table I.

Putative Arabidopsis proteases predicted or demonstrated to be peroxisomal

Tryptanthrin Inhibits Angiogenesis by Targeting the VEGFR2-Mediated ERK1/2 Signalling Pathway

Angiogenesis is a key step for tumour growth and metastasis, and anti-angiogenesis has been proposed as an important strategy for cancer therapy. Tryptanthrin is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants and has been shown to possess anti-tumour activities on various cancer cell types. This study aims to investigate the in vitro and in vivo anti-angiogenic activities of tryptanthrin and to unravel its underlying molecular action mechanisms. Our results show that tryptanthrin inhibited the in vitro proliferation, migration, and tube formation of the human microvascular endothelial cells (HMEC-1) in a concentration-dependent manner and significantly suppressed angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that tryptanthrin reduced the expression of several pro-angiogenic factors (Ang-1, PDGFB and MMP2). Tryptanthrin was also found to suppress the VEGFR2-mediated ERK1/2 signalling pathway in HMEC-1 cells and molecular docking simulation indicated that tryptanthrin could bound to the ATP-binding site of VEGFR2. Collectively, the present study demonstrated that tryptanthrin exhibited both in vitro and in vivo anti-angiogenic activities by targeting the VEGFR2-mediated ERK1/2 signalling pathway and might have therapeutic potential for the treatment of angiogenesis-related diseases.     

Proteomic Analysis Reveals That the Rab GTPase RabE1c Is Involved in the Degradation of the Peroxisomal Protein Receptor PEX7 (Peroxin 7)

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 β-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 β-oxidation activity. Treatment with proteasome inhibitors also restored endogenous PEX7 protein levels in GFP-PEX7-expressing 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.     

A Nuclear Import Pathway for a Protein Involved in tRNA Maturation

A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing.     

Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2

The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.     

Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.     

A Distinct and Parallel Pathway for the Nuclear Import of an mRNA-binding Protein

Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. Here we have characterized a new pathway to the nucleus, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p bound peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways.     

Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor,Pex5

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.     

Systematic Identification of Regulators of Oxidative Stress Reveals Non-canonical Roles for Peroxisomal Import and the Pentose Phosphate Pathway

1. Download : Download high-res image (247KB)
2. Download : Download full-size image