PEX12 interacts with PEX5 and PEX10 and acts downstream of receptor docking in peroxisomal matrix protein import

Peroxisomal matrix protein import requires PEX12, an integral peroxisomal membrane protein with a zinc ring domain at its carboxy terminus. Mutations in human PEX12 result in Zellweger syndrome, a lethal neurological disorder, and implicate the zinc ring domain in PEX12 function. Using two-hybrid studies, blot overlay assays, and coimmunoprecipitation experiments, we observed that the zinc-binding domain of PEX12 binds both PEX5, the PTS1 receptor, and PEX10, another integral peroxisomal membrane protein required for peroxisomal matrix protein import. Furthermore, we identified a patient with a missense mutation in the PEX12 zinc-binding domain, S320F, and observed that this mutation reduces the binding of PEX12 to PEX5 and PEX10. Overexpression of either PEX5 or PEX10 can suppress this PEX12 mutation, providing genetic evidence that these interactions are biologically relevant. PEX5 is a predominantly cytoplasmic protein and previous PEX5-binding proteins have been implicated in docking PEX5 to the peroxisome surface. However, we find that loss of PEX12 or PEX10 does not reduce the association of PEX5 with peroxisomes, demonstrating that these peroxins are not required for receptor docking. These and other results lead us to propose that PEX12 and PEX10 play direct roles in peroxisomal matrix protein import downstream of the receptor docking event.     

High level expression and purification of recombinant PEX protein in cultured skeletal muscle cell expression system

Large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications. To achieve high-level expression in eukaryotic cells, the choice of cell line as well as the expression vector is critical. In this report, we demonstrate that a combination of the skeletal muscle cell line, QM7 and a cytomegalovirus promoter-based expression vector can achieve high-level expression of secretory recombinant proteins in eukaryotic cells. We also screened a serum-free medium containing 3 microg/ml insulin suitable for QM7 differentiation and identified a very potent signal peptide from MMP9, which effectively directs secretion of heterologous proteins. The C-terminal hemopexin-like domain of MMP-2, PEX, a powerful candidate for the treatment of diseases associated with neovascularization was expressed in QM7 cells with bioactivity. This skeletal muscle cell-based system may be employed for the production of human proteins of special interests, such as those for structural determination or therapeutical development.     

Disparate peroxisome‐related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization

Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail‐anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β‐oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome‐associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.     

真菌类过氧化物酶体增殖因子PEX11基因家族生物信息学研究

PEX11基因家族成员是参与过氧化物酶体增殖调控的关键因子, 文章利用生物信息学方法对26种代表性真菌的PEX11基因家族成员进行了检索和进化分析。研究发现：(1)26种真菌中共有66个可能的PEX11p。酵母类真菌有1个或2个PEX11p, 而大多数丝状真菌中包含2到3个, 其中子囊菌中PEX11p的个数偏多, 个别种类达到5个; (2)真菌PEX11p可分为3类, 大多数真菌含有类型Ⅰ和类型Ⅲ的PEX11p, 类型Ⅱ是盘菌亚门真菌所特有的, 可能与类型Ⅰ和类型Ⅲ在功能上有冗余; (3)通过MEME分析, 发现PEX11p含有多个保守区域, 其中C末端的Motif8具有很高的保守性, 推测可能对PEX11p发挥功能具有重要作用。文章对进一步研究真菌PEX11p的进化与功能以及过氧化物酶体的增殖具有重要意义。     

Role of PEX5 ubiquitination in maintaining peroxisome dynamics and homeostasis

Peroxisomes are essential and dynamic organelles that allow cells to rapidly adapt and cope with changing environments and/or physiological conditions by modulation of both peroxisome biogenesis and turnover. Peroxisome biogenesis involves the assembly of peroxisome membranes and the import of peroxisomal matrix proteins. The latter depends on the receptor, PEX5, which recognizes peroxisomal matrix proteins in the cytosol directly or indirectly, and transports them to the peroxisomal lumen. In this review, we discuss the role of PEX5 ubiquitination in both peroxisome biogenesis and turnover, specifically in PEX5 receptor recycling, stability and abundance, as well as its role in pexophagy (autophagic degradation of peroxisomes).     

Genetic Dissection of Peroxisome-Associated Matrix Protein Degradation in Arabidopsis thaliana

Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H₂O₂, which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP–ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICL fluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.     

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.