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.     

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.     

PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins

Integral peroxisomal membrane proteins (PMPs) are synthesized in the cytoplasm and imported posttranslationally. Here, we demonstrate that PEX19 binds and stabilizes newly synthesized PMPs in the cytosol, binds to multiple PMP targeting signals (mPTSs), interacts with the hydrophobic domains of PMP targeting signals, and is essential for PMP targeting and import. These results show that PEX19 functions as both a chaperone and an import receptor for newly synthesized PMPs. We also demonstrate the existence of two PMP import mechanisms and two classes of mPTSs: class 1 mPTSs, which are bound by PEX19 and imported in a PEX19-dependent manner, and class 2 mPTSs, which are not bound by PEX19 and mediate protein import independently of PEX19.     

Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7

The import of a subset of peroxisomal matrix proteins is mediated by the peroxisomal targeting signal 2 (PTS2). The results of our sequence and physical property analysis of known PTS2 signals and of a mutational study of the least characterized amino acids of a canonical PTS2 motif indicate that PTS2 forms an amphipathic helix accumulating all conserved residues on one side. Three-dimensional structural modeling of the PTS2 receptor PEX7 reveals a groove with an evolutionarily conserved charge distribution complementary to PTS2 signals. Mammalian two-hybrid assays and cross-complementation of a mutation in PTS2 by a compensatory mutation in PEX7 confirm the interaction site. An unstructured linker region separates the PTS2 signal from the core protein. This additional information on PTS2 signals was used to generate a PTS2 prediction algorithm that enabled us to identify novel PTS2 signals within human proteins and to describe KChIP4 as a novel peroxisomal protein.     

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.     

The peroxisomal membrane targeting elements of human peroxin 2 (PEX2)

Peroxin 2 (PEX2) is a 35-kDa integral peroxisomal membrane protein with two transmembrane regions and a zinc RING domain within its cytoplasmically exposed C-terminus. Although its role in peroxisome biogenesis and function is poorly understood, it seems to be involved in peroxisomal matrix protein import. PEX2 is synthesized on free cytosolic ribosomes and is posttranslationally imported into the peroxisome membrane by specific targeting information. While a clear picture of the basic targeting mechanisms for peroxisomal matrix proteins has emerged over the past years, the targeting processes for peroxisomal membrane proteins are less well understood. We expressed various deletion constructs of PEX2 in fusion with the green fluorescent protein in COS-7 cells and determined their intracellular localization. We found that the minimum peroxisomal targeting signal of human PEX2 consists of an internal protein region of 30 amino acids (AA130 to AA159) and the first transmembrane domain, and that adding the second transmembrane domain increases targeting efficiency. Within the minimum targeting region we identified the motif "KX6(I/L)X(L/F/I)LK(L/F/I)" that includes important targeting information and is also present in the targeting regions of the 22-kDa peroxisomal membrane protein (PMP22) and the 70-kDa peroxisomal membrane protein (PMP70). Mutations in this targeting motif mislocalize PEX2 to the cytosol. In contrast, the second transmembrane domain does not seem to have specific peroxisomal membrane targeting information. Replacing the second transmembrane domain of human PEX2 with the transmembrane domain of human cytochrome c oxidase subunit IV does not alter PEX2 peroxisome targeting function and efficiency.     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.     

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.     

PEX5参与调控拟南芥根系抗逆性的初步研究

以拟南芥过氧化物酶体生成蛋白突变体pex5和野生型为试验材料,初步研究PEX5对拟南芥根系抗逆性的影响。结果表明,在MS基础培养基中,突变体pex5的根长相较于野生型根长显著变短;同时,拟南芥pex5突变体株高也低于野生型,说明PEX5基因功能缺失影响了拟南芥根系和茎的生长。pex5突变体在添加氯化钠或甘露醇的MS培养基上培养时,萌发和根伸长受到的抑制显著高于野生型,突变体幼苗的SOD和POD活性也显著低于野生型,说明PEX5参与调控拟南芥根系的抗逆性。本研究为解析PEX5在植物抗逆中的调控机制提供了重要参考。     

PEX5 translocation into and out of peroxisomes drives matrix protein import

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An Arabidopsis dynamin-related protein, DRP3A, controls both peroxisomal and mitochondrial division

Peroxisomes undergo dramatic changes in size, shape, number, and position within the cell, but the division process of peroxisomes has not been characterized. We screened a number of Arabidopsis mutants with aberrant peroxisome morphology (apm mutants). In one of these mutants, apm1, the peroxisomes are long and reduced in number, apparently as a result of inhibition of division. We showed that APM1 encodes dynamin-related protein 3A (DRP3A), and that mutations in APM1/DRP3A also caused aberrant morphology of mitochondria. The transient expression analysis showed that DRP3A is associated with the cytosolic side of peroxisomes. These findings indicate that the same dynamin molecule is involved in peroxisomal and mitochondrial division in higher plants. We also report that the growth of Arabidopsis, which requires the cooperation of various organelles, including peroxisomes and mitochondria, is repressed in apm1, indicating that the changes of morphology of peroxisomes and mitochondria reduce the efficiency of metabolism in these organelles.     

Identification of a novel family of 70 kDa microtubule-associated proteins in Arabidopsis cells

Most plant microtubule-associated proteins (MAPs) have homologues across the phylogenetic spectrum. To find potential plant-specific MAPs that will have evaded bioinformatic searches we devised a low stringency method for isolating proteins from an Arabidopsis cell suspension on endogenous taxol-microtubules. By tryptic peptide mass fingerprinting we identified 55 proteins that were enriched on taxol-microtubules. Amongst a range of known MAPs, such as kinesins, MAP65 isoforms and MOR1, we detected 'unknown' 70 kDa proteins that belong to a family of five closely related Arabidopsis proteins having no known homologues amongst non-plant organisms. To verify that AtMAP70-1 associates with microtubules in vivo, it was expressed as a GFP fusion. This confirmed that the protein decorates all four microtubule arrays in both transiently infected Arabidopsis and stably transformed tobacco BY-2 suspension cells. Microtubule-directed drugs perturbed the localization of AtMAP70-1 but cytochalasin D did not. AtMAP70-1 contains four predicted coiled-coil domains and truncation studies identified a central domain that targets the fusion protein to microtubules in vivo. This study therefore introduces a novel family of plant-specific proteins that interact with microtubules.     

An Arabidopsis thaliana protein homologous to cyanobacterial high-light-inducible proteins

An Arabidopsis thaliana cDNA clone encoding a novel 110 amino acid thylakoid protein has been sequenced. The in vitro synthesized protein is taken up by intact chloroplasts, inserted into the thylakoid membrane and the transit peptide is cleaved off during this process. The mature protein is predicted to contain 69 amino acids, to form one membrane-spanning -helix and to have its N-terminus at the stromal side of the thylakoid membrane. The protein showed similarity to the LHC, ELIP and PsbS proteins of higher plants, but more pronounced to the high-light-inducible proteins (HLIPs) of cyanobacteria and red algae, to which no homologue previously has been detected in higher plants. As for HLIP and ELIP, high light increases the mRNA levels of the corresponding gene. Sequence comparisons indicate that the protein may bind chlorophyll and form dimers in the thylakoid membrane. The level of expression of the protein seems to be far lower than that of normal PSI and PSII subunits.     

The mitochondrial complexome of Arabidopsis thaliana

Mitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein–protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot‐gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re‐annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource ( https://complexomemap.de/at_mito_leaves ) to deposit the data and to allow straightforward access and custom data analyses.     

Tetratricopeptide repeat protein Pyg7 is essential for photosystem I assembly by interacting with PsaC in Arabidopsis

Although progress has been made in determining the structure and understanding the function of photosystem I (PSI), the PSI assembly process remains poorly understood. PsaC is an essential subunit of PSI and participates in the transfer of electrons to ferredoxin. However, how PsaC is assembled during accumulation of the PSI complex is unknown. In the present study, we showed that Pyg7 localized to the stromal thylakoid and associated with the PSI complex. We also showed that Pyg7 interacted with PsaC. Furthermore, we found that the PSI assembly process was blocked following formation of the PsaAB heterodimer in the pyg7 mutant. In addition, the analyses of PSI stability in Pyg7RNAi plants showed that Pyg7 is involved in maintaining the assembled PSI complex under excess‐light conditions. Moreover, we demonstrated that decreased Pyg7 content resulted in decreased efficiency of PSI assembly in Pyg7RNAi plants. These findings suggest that the role of Pyg7 in PSI biogenesis has evolved as an essential assembly factor by interacting with PsaC in Arabidopsis, in addition to being a stability factor for PSI as seen in Synechocystis.     

Construction of a chloroplast protein interaction network and functional mining of photosynthetic proteins in Arabidopsis thaliana

Chloroplast is a typical plant cell organelle where photosynthesis takes place. In this study, a total of 1 808 chloroplast core proteins in Arabidopsis thaliana were reliably identified by combining the results of previously published studies and our own predictions. We then constructed a chloroplast protein interaction network primarily based on these core protein interactions. The network had 22 925 protein interaction pairs which involved 2 214 proteins. A total of 160 previously uncharacterized proteins were annotated in this network. The subunits of the photosynthetic complexes were modularized, and the functional relationships among photosystem Ⅰ （PSI）, photosystem Ⅱ （PSII）, light harvesting complex of photosystem Ⅰ （LHC Ⅰ） and light harvesting complex of photosystem Ⅰ （LHC Ⅱ） could be deduced from the predicted protein interactions in this network. We further confirmed an interaction between an unknown protein AT1G52220 and a photosynthetic subunit PSI-D2 by yeast two-hybrid analysis. Our chloroplast protein interaction network should be useful for functional mining of photosynthetic proteins and investigation of chloroplast-related functions at the systems biology level in Arabidopsis.