Non Miyata

Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export

ABSTRACT Peroxisomal matrix proteins are posttranslationally imported into peroxisomes with the peroxisome-targeting signal 1 receptor, Pex5. The longer isoform of Pex5, Pex5L, also transports Pex7-PTS2 protein complexes. After unloading the cargoes, Pex5 returns to the cytosol. To address molecular mechanisms underlying Pex5 functions, we constructed a cell-free Pex5 translocation system with a postnuclear supernatant fraction from CHO cell lines. In assays using the wild-type CHO-K1 cell fraction, 35S-labeled Pex5 was specifically imported into and exported from peroxisomes with multiple rounds. 35S-Pex5 import was also evident using peroxisomes isolated from rat liver. ATP was not required for 35S-Pex5 import but was indispensable for export. 35S-Pex5 was imported neither to peroxisome remnants from RING peroxin-deficient cell mutants nor to those from pex14 cells lacking a Pex5-docking site. In contrast, 35S-Pex5 was imported into the peroxisome remnants of PEX1-, PEX6-, and PEX26-defective cell mutants, including those from patients with peroxisome biogenesis disorders, from which, however, 35S-Pex5 was not exported, thereby indicating that Pex1 and Pex6 of the AAA ATPase family and their recruiter, Pex26, were essential for Pex5 export. Moreover, we analyzed the 35S-Pex5-associated complexes on peroxisomal membranes by blue-native polyacrylamide gel electrophoresis. 35S-Pex5 was in two distinct, 500- and 800-kDa complexes comprising different sets of peroxins, such as Pex14 and Pex2, implying that Pex5 transited between the subcomplexes. Together, results indicated that Pex5 most likely enters peroxisomes, changes its interacting partners, and then exits using ATP energy.

Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling

Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome‐targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine11 in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p‐C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild‐type cells. Pex5p‐C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N‐terminal 158‐amino‐acid region of Pex5p‐C11A, termed 158‐CA, is sufficient for such dominant‐negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p‐C11A or 158‐CA likewise inhibits the wild‐type Pex5p import into peroxisomes, strongly suggesting that Pex5p‐C11A exerts the dominant‐negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine11 of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p‐C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes.

Analysis of Nucleotide Binding to P97 Reveals the Properties of a Tandem AAA Hexameric ATPase

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5′-O-(thiotriphosphate) (ATPγS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPγS can saturate all 6 nucleotide binding sites in D1, only 3–4 of the 6 D2 sites can bind ATPγS simultaneously. ATPγS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

Mutations in novel peroxin gene PEX26 that cause peroxisome-biogenesis disorders of complementation group 8 provide a genotype-phenotype correlation.

The human disorders of peroxisome biogenesis (PBDs) are subdivided into 12 complementation groups (CGs). CG8 is one of the more common of these and is associated with varying phenotypes, ranging from the most severe, Zellweger syndrome (ZS), to the milder neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD). PEX26, encoding the 305-amino-acid membrane peroxin, has been shown to be deficient in CG8. We studied the PEX26 genotype in fibroblasts of eight CG8 patients--four with the ZS phenotype, two with NALD, and two with IRD. Catalase was mostly cytosolic in all these cell lines, but import of the proteins that contained PTS1, the SKL peroxisome targeting sequence, was normal. Expression of PEX26 reestablished peroxisomes in all eight cell lines, confirming that PEX26 defects are pathogenic in CG8 patients. When cells were cultured at 30 degrees C, catalase import was restored in the cell lines from patients with the NALD and IRD phenotypes, but to a much lesser extent in those with the ZS phenotype, indicating that temperature sensitivity varied inversely with the severity of the clinical phenotype. Several types of mutations were identified, including homozygous G89R mutations in two patients with ZS. Expression of these PEX26 mutations in pex26 Chinese hamster ovary cells resulted in cell phenotypes similar to those in the human cell lines. These findings confirm that the degree of temperature sensitivity in pex26 cell lines is predictive of the clinical phenotype in patients with PEX26 deficiency.

AWP1/ZFAND6 Functions in Pex5 Export by Interacting with Cys‐Monoubiquitinated Pex5 and Pex6 AAA ATPase

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal‐targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1‐containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin‐binding NF‐κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti‐AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1–Pex6 complexes. Preferential binding of AWP1 to the cysteine‐ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc‐finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1‐protein import into peroxisomes. Furthermore, in AWP1 knock‐down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.

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.

In vitro import of peroxisome-targeting signal type 2 (PTS2) receptor Pex7p into peroxisomes.

Pex7p, the peroxisome-targeting signal type 2 (PTS2) receptor, transports PTS2 proteins to peroxisomes from the cytosol. We here established a cell-free Pex7p translocation system. In assays using post-nuclear supernatant fractions each from wild-type CHO-K1 and pex7 ZPG207 cells, 35S-labeled Pex7p was imported into peroxisomes. 35S-Pex7p import was also evident using rat liver peroxisomes. 35S-Pex7p was not imported into peroxisomal remnants from a pex5 ZPG231 defective in PTS2 import and pex2 Z65. When the import of 35S-Pex5pL was inhibited with an excess amount of recombinant Pex5pS, 35S-Pex7p import was concomitantly abrogated, suggesting that Pex5pL was a transporter for Pex7p, unlike a yeast cochaperone, Pex18p. 35S-Pex7p as well as 35S-Pex5p was imported in an ATP-independent manner, whilst the import of PTS1 and PTS2 cargo-proteins was ATP-dependent. Thereby, ATP-independent import of Pex7p implicated that Pex5p export requiring ATP hydrolysis is not a limiting step for its cargo recruitment to peroxisomes. PTS1 protein import was indeed insensitive to N-ethylmaleimide, whereas Pex5p export was N-ethylmaleimide-sensitive. Taken together, the cargo-protein translocation through peroxisomal membrane more likely involves another ATP-requiring step in addition to the Pex5p export. Moreover, upon concurrent import into peroxisomes, 35S-Pex5pL and 35S-Pex7p were detected at mutually distinct ratios in the immunoprecipitates each of the import machinery peroxins including Pex14p, Pex13p, and Pex2p, hence suggesting that Pex7p as well as Pex5p translocated from the initial docking complex to RING complex on peroxisomes.

Phosphatidylserine transport by Ups2-Mdm35 in respiration-active mitochondria.

Phosphatidylethanolamine, an essential phospholipid for mitochondrial functions, is synthesized at the mitochondrial inner membrane. Miyata et al. demonstrate that Ups2–Mdm35, a protein complex in the mitochondrial intermembrane space, mediates phosphatidylserine transport for phosphatidylethanolamine synthesis in respiration-active mitochondria of Saccharomyces cerevisiae.

Dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p involved in shuttling of the PTS1 receptor Pex5p in peroxisome biogenesis.

The peroxisome is a single-membrane-bound organelle found in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient PBDs (peroxisome biogenesis disorders), such as Zellweger syndrome. Two AAA (ATPase associated with various cellular activities) peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for CG (complementation group) 1 and CG4 PBDs respectively. PEX26, which is responsible for CG8 PBDs, codes for Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. We recently assigned the binding regions between human Pex1p and Pex6p and elucidated the pivotal roles that the AAA cassettes, D1 and D2 domains, play in Pex1p-Pex6p interaction and in peroxisome biogenesis. ATP binding to both AAA cassettes of Pex1p and Pex6p was a prerequisite for the Pex1p-Pex6p interaction and peroxisomal localization, but ATP hydrolysis by the D2 domains was not required. Pex1p exists in two distinct oligomeric forms, a homo-oligomer in the cytosol and a hetero-oligomer on peroxisome membranes, with these possibly having distinct functions in peroxisome biogenesis. AAA peroxins are involved in the export from peroxisomes of Pex5p, the PTS1 (peroxisome-targeting signal type 1) receptor.

TMEM14C is required for erythroid mitochondrial heme metabolism

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.