Arabidopsis PEX19 is a dimeric protein that binds the peroxin PEX10

Peroxisomes are organelles found in all eukaryotic cells. Peroxisomes import integral membrane proteins post-translationally, and PEX19 is a predominantly cytosolic, farnesylated protein of mammalian and yeast cells that binds multiple peroxisome membrane proteins and is required for their correct targeting/insertion to the peroxisome membrane. We report the characterisation of the Arabidopsis thaliana homologue of PEX19 which is a predominantly cytosolic protein. AtPEX19 is encoded by two genes (designated AtPEX19-1 and AtPEX19-2) that are expressed in all tissues and at all developmental stages of the plant. Quantitative real time PCR shows that AtPEX19-1 and AtPEX19-2 have distinct expression profiles. Using in vitro translation and co-immunoprecipitation AtPEX19-1 was shown to bind to the Arabidopsis peroxisomal membrane protein PEX10. Additionally, bacterially expressed recombinant AtPEX19-1 was able to bind a fusion protein consisting of the C-terminus of PEX10 and glutathione S-transferase in pull-down assays, thereby demonstrating that non-farnesylated AtPEX19 can interact with the C-terminus of AtPEX10. Purified recombinant AtPEX19-1 was analysed by gel filtration chromatography and was found to have a molecular weight consistent with it forming a dimer and a dimer was detected in Arabidopsis cell extracts that was slightly destabilised in the presence of DTT. Moreover, cross-linking studies of native AtPEX19 suggest that in vivo it is the dimeric species of the protein that preferentially forms complexes with other proteins.     

Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) is a group of diseases caused by mutations in one of the peroxins, proteins responsible for biogenesis of the peroxisomes. In recent years, it became clear that many peroxins (e.g., PEX3 and PEX14) play additional roles in peroxisome homeostasis (such as promoting autophagic degradation of peroxisomes or pexophagy), which are often opposite to their originally established functions in peroxisome formation and maintenance. Even more interesting, the peroxins that make up the peroxisomal AAA ATPase complex (AAA-complex) in yeast (Pex1, Pex6 and Pex15) or mammals (PEX1, PEX6, PEX26) are responsible for the downregulation of pexophagy. Moreover, this might be even their primary role in human: to prevent pexophagy by removing from the peroxisomal membrane the ubiquitinated peroxisomal matrix protein import receptor, Ub-PEX5, which is also a signal for the Ub-binding pexophagy receptor, NBR1. Remarkably, the peroxisomes rescued from pexophagy by autophagic inhibitors in PEX1^G843D (the most common PBD mutation) cells are able to import matrix proteins and improve their biochemical function suggesting that the AAA-complex per se is not essential for the protein import function in human. This paradigm-shifting discovery published in the current issue of Autophagy has raised hope for up to 65% of all PBD patients with various deficiencies in the AAA-complex. Recognizing PEX1, PEX6 and PEX26 as pexophagy suppressors will allow treating these patients with a new range of tools designed to target mammalian pexophagy.     

Novel proteins interacting with peroxisomal protein receptor PEX7 in Arabidopsis thaliana

Peroxisomal matrix protein transport relies on 2 cytosolic receptors, PEX5 and PEX7, which import peroxisomal targeting signal type 1 (PTS1) and PTS2-containing proteins, respectively. To better understand the transport mechanism of PEX7, we isolated PEX7 complexes using proteomics. We identified PEX5 as well as PTS1- and PTS2-containing proteins within the complex, thereby confirming the interaction between PEX5 and PEX7 during cargo transport that had been previously characterized by biochemical approaches. In addition, a chaperone T-complex and 2 small Rab GTPases were identified. We recently reported that the RabE1c is involved in the degradation of the PEX7 when abnormal PEX7 is accumulated on the peroxisomal membrane. This study expands our knowledge on the transport machinery via PEX7 by identifying both known and novel PEX7-interacting proteins and thus is helpful for further investigation of the regulation of the peroxisomal protein receptor during its translocation.     

Zellweger spectrum disorder patient–derived fibroblasts with the PEX1-Gly843Asp allele recover peroxisome functions in response to flavonoids

Zellweger spectrum disorder (ZSD) results from biallelic mutations in PEX genes required for peroxisome biogenesis. PEX1-G843D is a common hypomorphic allele in the patient population that is associated with milder disease. In prior work using a PEX1-G843D/null patient fibroblast line expressing a green fluorescent protein (GFP) reporter with a peroxisome-targeting signal (GFP-PTS1), we demonstrated that treatments with the chemical chaperone betaine and flavonoid acacetin diacetate recovered peroxisome functions. To identify more effective compounds for preclinical investigation, we evaluated 54 flavonoids using this cell-based phenotype assay. Diosmetin showed the most promising combination of potency and efficacy (EC50 2.5 µM). All active 5′,7′-dihydroxyflavones showed greater average efficacy than their corresponding flavonols, whereas the corresponding flavanones, isoflavones, and chalcones tested were inactive. Additional treatment with the proteostasis regulator bortezomib increased the percentage of import-rescued cells over treatment with flavonoids alone. Cotreatments of diosmetin and betaine showed the most robust additive effects, as confirmed by three independent functional assays in primary PEX1-G843D patient cells, but neither agent was active alone or in combination in patient cells homozygous for the PEX1 c.2097_2098insT null allele. Moreover, diosmetin treatment increased PEX1, PEX6, and PEX5 protein levels in PEX1-G843D patient cells, but none of these proteins increased in PEX1 null cells. We propose that diosmetin acts as a pharmacological chaperone that improves the stability, conformation, and functions of PEX1/PEX6 exportomer complexes required for peroxisome assembly. We suggest that diosmetin, in clinical use for chronic venous disease, and related flavonoids warrant further preclinical investigation for the treatment of PEX1-G843D–associated ZSD.     

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.     

Recruiting mechanism of the AAA peroxins, Pex1p and Pex6p, to Pex26p on the peroxisomal membrane

A peroxisomal C-tail-anchored type-II membrane protein, Pex26p, recruits AAA ATPase Pex1p-Pex6p complexes to peroxisomes. We herein attempted to gain mechanistic insight into Pex26p function. Pex26pΔ33-40 truncated in amino-acid residues at 33-40 abolishes the recruiting of Pex1p-Pex6p complex to peroxisomes and fails to complement the impaired phenotype of pex26 CHO cell mutant ZP167, thereby suggesting that peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins. In in vitro transport assay using semipermeabilized CHO cells, Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. This finding is confirmed by the assay using Walker-motif mutants. Transport of Pex1p and Pex6p is temperature-dependent. In vitro binding assays with glutathione-S-transferase-fused Pex26p, Pex1p and Pex6p bind to Pex26p in a manner dependent on ATP binding but not ATP hydrolysis. These results suggest that ATP hydrolysis is required for stable localization of Pex1p to peroxisomes, but not for binding to Pex26p. Moreover, Pex1p and Pex6p are altered to a more compact conformation upon binding to ATP, as verified by limited proteolysis. Taken together, Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by the ATPase cycle.     

Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains

A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome. Here, we have studied the mechanism by which the p97-Ufd1-Npl4 complex functions in this retrotranslocation pathway. Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain. The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol. The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane. Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1. We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.     

The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) are metabolic disorders caused by the loss of peroxisomes. The majority of PBDs result from mutation in one of 3 genes that encode for the peroxisomal AAA ATPase complex (AAA-complex) required for cycling PEX5 for peroxisomal matrix protein import. Mutations in these genes are thought to result in a defect in peroxisome assembly by preventing the import of matrix proteins. However, we show here that loss of the AAA-complex does not prevent matrix protein import, but instead causes an upregulation of peroxisome degradation by macroautophagy, or pexophagy. The loss of AAA-complex function in cells results in the accumulation of ubiquitinated PEX5 on the peroxisomal membrane that signals pexophagy. Inhibiting autophagy by genetic or pharmacological approaches rescues peroxisome number, protein import and function. Our findings suggest that the peroxisomal AAA-complex is required for peroxisome quality control, whereas its absence results in the selective degradation of the peroxisome. Thus the loss of peroxisomes in PBD patients with mutations in their peroxisomal AAA-complex is a result of increased pexophagy. Our study also provides a framework for the development of novel therapeutic treatments for PBDs.     

cDNA isolation, characterization, and protein intracellular localization of a katanin-like p60 subunit from Arabidopsis thaliana

Summary Katanin, a heterodimeric protein with ATP-dependent microtubule-severing activity, localizes to the centrosome in animal cells. Widespread occurrence is suspected as several species contain homologs to the katanin p60 subunit. Recently we isolated anArabidopsis thaliana cDNA with significant identity to the p60 subunit of sea urchin katanin. Like p60, the encoded protein is a member of the AAA superfamily of ATPases, containing the Walker ATP binding consensus and the signature AAA minimal consensus sequences within a single larger AAA/CAD amino acid motif. Phylogenetic analysis placed the encoded protein in the AAA subfamily of cytoskeleton-interactive proteins, where it formed a strongly supported clade with 4 other members identified as katanin p60 subunits. The clone was named AtKSSArabidopsis thaliana kataninlike protein small subunit). Western blots, performed using a polyclonal antibody raised against recombinant AtKSS, revealed AtKSS is present in protein extracts of all Arabidopsis organs examined. To evaluate potential interactions between AtKSS and the cytoskeleton, the intracellular localization of AtKSS was correlated with that of tubulin. AtKSS was found in perinuclear regions during interphase, surrounding the spindle poles during mitosis, but was absent from the preprophase band and phragmoplast microtubule arrays. These data support the thesis that AtKSS is an Arabidopsis homolog of the p60 subunit of katanin. Its cell cycle-dependent distribution is consistent with microtubule-severing activity, but additional studies will better define its role.     

膜蛋白ATAD3A在线粒体质量控制中的作用

线粒体质量控制对于线粒体网络的稳态和线粒体功能的正常发挥具有重要意义。三磷酸腺苷酶家族蛋白3A(ATAD3A)是同时参与调节线粒体结构功能、线粒体动力学和线粒体自噬等重要生物学过程的线粒体膜蛋白之一。近期研究表明,ATAD3A既可与Mic60/Mitofilin和线粒体转录因子A (TFAM)等因子相互作用以维持线粒体嵴的形态和氧化磷酸化功能,又能与发动蛋白相关蛋白1 (Drp1)结合而正性/负性调节线粒体分裂,还可作为线粒体外膜转位酶（TOM）复合物和线粒体内膜转位酶（TIM）复合物之间的桥接因子而介导PTEN诱导激酶（PINK1）输入线粒体进行加工,显示出促自噬或抗自噬活性。本文对ATAD3A在调控线粒体质量控制中的作用及其机制进行了综述。     

Search and Destroy: ER Quality Control and ER-Associated Protein Degradation

Abstract

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms. The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.     

蛋白质的选择性降解机制——2004年诺贝尔化学奖部分工作介绍

如何识别和选择性降解蛋白质是细胞生命过程中的重要环节.泛素-蛋白酶体需能降解途径的发现,揭示了蛋白质在细胞内选择性降解的普遍方式.对于需要清除的蛋白质,通过其赖氨酸残基侧链ε-氨基连接多聚泛素链（降解标签）,继而在蛋白酶体中被降解.这种选择性降解机制对于维持蛋白质在细胞内含量的动态平衡起到了关键性作用.     

拟南芥中一个未知功能蛋白的叶绿体亚细胞定位研究

生物信息学分析表明,模式植物拟南芥叶绿体中含有大约4000多种蛋白质,目前只分离得到1000多种,其他预测的叶绿体蛋白的实验验证对叶绿体功能研究有重要意义。本文对一个预测的叶绿体未知功能蛋白AT5G48790进行了亚细胞定位研究。我们克隆了该基因5端长178bp的DNA片段,与绿色荧光蛋白（GFP）基因构建重组载体pMON530-cTP-GFP。转基因植株通过激光共聚焦显微镜观察,GFP只在叶绿体中特异表达。实验结果表明,AT5G48790的确为叶绿体蛋白。本实验方法也可用于其他预测的蛋白质的实验验证。     

Two splice variants of human PEX19 exhibit distinct functions in peroxisomal assembly

PEX19 has been shown to play a central role in the early steps of peroxisomal membrane synthesis. Computational database analysis of the PEX19 sequence revealed three different conserved domains: D1 (aa 1--87), D2 (aa 88--272), and D3 (aa 273--299). However, these domains have not yet been linked to specific biological functions. We elected to functionally characterize the proteins derived from two naturally occurring PEX19 splice variants: PEX19DeltaE2 lacking the N-terminal domain D1 and PEX19DeltaE8 lacking the domain D3. Both interact with peroxisomal ABC transporters (ALDP, ALDRP, PMP70) and with full-length PEX3 as shown by in vitro protein interaction studies. PEX19DeltaE8 also interacts with a PEX3 protein lacking the peroxisomal targeting region located at the N-terminus (Delta66aaPEX3), whereas PEX19DeltaE2 does not. Functional complementation studies in PEX19-deficient human fibroblasts revealed that transfection of PEX19DeltaE8-cDNA leads to restoration of both peroxisomal membranes and of functional peroxisomes, whereas transfection of PEX19DeltaE2-cDNA does not restore peroxisomal biogenesis. Human PEX19 is partly farnesylated in vitro and in vivo. The farnesylation consensus motif CLIM is located in the PEX19 domain D3. The finding that the protein derived from the splice variant lacking D3 is able to interact with several peroxisomal membrane proteins and to restore peroxisomal biogenesis challenges the previous assumption that farnesylation of PEX19 is essential for its biological functionality. The data presented demonstrate a considerable functional diversity of the proteins encoded by two PEX19 splice variants and thereby provide first experimental evidence for specific biological functions of the different predicted domains of the PEX19 protein.     

Visualization of plastid movement in the pollen tube of Arabidopsis thaliana

Organelle dynamics in the plant male gametophyte has received attention for its importance in pollen tube growth and cytoplasmic inheritance. We recently revealed the dynamic behaviors of plastids in living Arabidopsis pollen grains and tubes, using an inherent promoter-driven FtsZ1–green fluorescent protein (GFP) fusion. Here, we further monitored the movement of pollen tube plastids with an actin1 promoter-driven, stroma-targeted yellow fluorescent protein (YFP). In elongating pollen tubes, most plastids localized to the tube shank, where they displayed either retarded and unsteady motion, or fast, directional, and long-distance movement along the tube polarity. Efficient plastid tracking further revealed a population of tip-forwarding plastids that undergo a fluctuating motion(s) before traveling backward. The behavior of YFP-labeled plastids in pollen basically resembled that of FtsZ1–GFP-labeled plastids, thus validating the use of FtsZ1–GFP for simultaneous visualization of the stroma and the plastid-dividing FtsZ ring.     

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.     

Cryo-EM Structures of the Hsp104 Protein Disaggregase Captured in the ATP Conformation

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靶向蛋白质降解技术研究进展

靶向蛋白质降解技术可有效克服DNA敲除、RNA干扰等传统药物靶点确认及干扰策略的局限性。近年来,一系列新型靶向蛋白质降解技术不断涌现,在药物研发领域展现出极好的应用前景。本文综述了靶向蛋白质降解技术的最新研究进展,重点介绍各种技术的作用机制、应用情况、技术优势及目前存在问题,以期为药物靶点确认及新药开发提供有力理论及技术支持。     

Pex20p functions as the receptor for non‐PTS1/non‐PTS2 acyl‐CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica

Most soluble proteins targeted to the peroxisomal matrix contain a C‐terminal peroxisome targeting signal type 1 (PTS1) or an N‐terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl‐CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co‐receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2‐containing protein 3‐ketoacyl‐CoA thiolase (Pot1p) but also the non‐PTS1/non‐PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C‐terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C‐terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non‐PTS1/non‐PTS2 Aox isozymes into peroxisomes.