Peroxisomal Localization Research Articles

Peroxisomal Localization of Benzyl Alcohol O-Benzoyltransferase HSR201 Is Mediated by a Non-canonical Peroxisomal Targeting Signal and Required for Salicylic Acid Biosynthesis.

The phytohormone salicylic acid (SA) regulates plant responses to various types of environmental stress, particularly pathogen infections. We previously revealed that the benzyl alcohol O-benzoyltransferase HSR201 was required for pathogen signal-induced SA synthesis, and its overexpression together with NtCNL, encoding a cinnamate-coenzyme A ligase, was sufficient for the production of significant amounts of SA in tobacco. We herein examined the subcellular localization of HSR201 and found that it fused to a yellow fluorescent protein localized in peroxisomes. Most peroxisomal matrix proteins possess peroxisomal targeting signal type-1 (PTS1) located at the extreme C terminus or PTS2 located at the N terminus; however, a bioinformatics analysis failed to identify similar signals for HSR201. Deletion and mutation analyses of HSR201 identified one essential (extreme C-terminal Leu46°) and three important (Ile455, Ile456 and Ala459) amino acid residues for its peroxisomal localization. The virus-induced gene silencing (VIGS) of PEX5, a PTS1 receptor, but not PEX7, a PTS2 receptor, compromised the peroxisomal targeting of HSR201 in Nicotiana benthamiana. When overexpressed with NtCNL, HSR201 mutants with reduced or non-peroxisomal targeting induced lower SA levels than the wild type; however, these mutations did not affect the protein stability or activity of HSR201. VIGS of the HSR201 homolog compromised pathogen signal-induced SA accumulation in N. benthamiana, which was complemented by the HSR201 wild type, but not the mutant with non-peroxisomal targeting. These results suggest that the peroxisomal localization of HSR201 is mediated by a non-canonical PTS1 and required for SA biosynthesis.

The lipid-metabolism enzyme ECI2 reduces neutrophil extracellular traps formation for colorectal cancer suppression

Abnormalities in ether lipid metabolism as well as the formation of neutrophil extracellular traps have recently been recognized as detrimental factors affecting tumorigenesis and progression. However, the role of abnormal ether lipid metabolism in colorectal cancer (CRC) evolution has not been reported. Here we show that the lipid metabolism-related gene enoyl-CoA δ-isomerase 2 (ECI2) plays a tumor-suppressor role in CRC and is negatively associated with poor prognosis in CRC patients. We mechanistically demonstrate that ECI2 reduces ether lipid-mediated Interleukin 8 (IL-8) expression leading to decreased neutrophil recruitment and neutrophil extracellular traps formation for colorectal cancer suppression. In particular, ECI2 inhibits ether lipid production in CRC cells by inhibiting the peroxisomal localization of alkylglycerone phosphate synthase (AGPS), the rate-limiting enzyme for ether lipid synthesis. These findings not only deepen our understanding of the role of metabolic reprogramming and neutrophil interactions in the progression of CRC, but also provide ideas for identifying potential diagnostic markers and therapeutic targets for CRC.

The Hansenula polymorpha mitochondrial carrier family protein Mir1 is dually localized at peroxisomes and mitochondria

Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3–6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full-length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full-length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information. Data are available via ProteomeXchange with identifier PXD050324.

The Bbotf1 Zn(Ⅱ)2Cys6 transcription factor contributes to antioxidant response, fatty acid assimilation, peroxisome proliferation and infection cycles in insect pathogenic fungus Beauveria bassiana

The abilities to withstand oxidation and assimilate fatty acids are critical for successful infection by many pathogenic fungi. Here, we characterized a Zn(II)2Cys6 transcription factor Bbotf1 in the insect pathogenic fungus Beauveria bassiana, which links oxidative response and fatty acid assimilation via regulating peroxisome proliferation. The null mutant ΔBbotf1 showed impaired resistance to oxidants, accompanied by decreased activities of antioxidant enzymes including CATs, PODs and SODs, and down-regulated expression of many antioxidation-associated genes under oxidative stress condition. Meanwhile, Bbotf1 acts as an activator to regulate fatty acid assimilation, lipid and iron homeostasis as well as peroxisome proliferation and localization, and the expressions of some critical genes related to glyoxylate cycle and peroxins were down-regulated in ΔBbotf1 in presence of oleic acid. In addition, ΔBbotf1 was more sensitive to osmotic stressors, CFW, SDS and LDS. Insect bioassays revealed that insignificant changes in virulence were seen between the null mutant and parent strain when conidia produced on CZP plates were used for topical application. However, propagules recovered from cadavers killed by ΔBbotf1 exhibited impaired virulence as compared with counterparts of the parent strain. These data offer a novel insight into fine-tuned aspects of Bbotf1 concerning multi-stress responses, lipid catabolism and infection cycles.

Peroxisomal Localization of a Truncated HMG-CoA Reductase under Low Cholesterol Conditions.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGCR) is one of the rate-limiting enzymes in the mevalonate pathway required for cholesterol biosynthesis. It is an integral membrane protein of the endoplasmic reticulum (ER) but has occasionally been described in peroxisomes. By co-immunofluorescence microscopy using different HMGCR antibodies, we present evidence for a dual localization of HMGCR in the ER and peroxisomes in differentiated human monocytic THP-1 cells, primary human monocyte-derived macrophages and human primary skin fibroblasts under conditions of low cholesterol and statin treatment. Using density gradient centrifugation and Western blot analysis, we observed a truncated HMGCR variant of 76 kDa in the peroxisomal fractions, while a full-length HMGCR of 96 kDa was contained in fractions of the ER. In contrast to primary human control fibroblasts, peroxisomal HMGCR was not found in fibroblasts from patients suffering from type-1 rhizomelic chondrodysplasia punctata, who lack functional PEX7 and, thus, cannot import peroxisomal matrix proteins harboring a type-2 peroxisomal targeting signal (PTS2). Moreover, in the N-terminal region of the soluble 76 kDa C-terminal catalytic domain, we identified a PTS2-like motif, which was functional in a reporter context. We propose that under sterol-depleted conditions, part of the soluble HMGCR domain, which is released from the ER by proteolytic processing for further turnover, remains sufficiently long in the cytosol for peroxisomal import via a PTS2/PEX7-dependent mechanism. Altogether, our findings describe a dual localization of HMGCR under combined lipid depletion and statin treatment, adding another puzzle piece to the complex regulation of HMGCR.

Analysis of the Mouse Hepatic Peroxisome Proteome-Identification of Novel Protein Constituents Using a Semi-Quantitative SWATH-MS Approach.

Ongoing technical and bioinformatics improvements in mass spectrometry (MS) allow for the identifying and quantifying of the enrichment of increasingly less-abundant proteins in individual fractions. Accordingly, this study reassessed the proteome of mouse liver peroxisomes by the parallel isolation of peroxisomes from a mitochondria- and a microsome-enriched prefraction, combining density-gradient centrifugation with a semi-quantitative SWATH-MS proteomics approach to unveil novel peroxisomal or peroxisome-associated proteins. In total, 1071 proteins were identified using MS and assessed in terms of their distribution in either high-density peroxisomal or low-density gradient fractions, containing the bulk of organelle material. Combining the data from both fractionation approaches allowed for the identification of specific protein profiles characteristic of mitochondria, the ER and peroxisomes. Among the proteins significantly enriched in the peroxisomal cluster were several novel peroxisomal candidates. Five of those were validated by colocalization in peroxisomes, using confocal microscopy. The peroxisomal import of HTATIP2 and PAFAH2, which contain a peroxisome-targeting sequence 1 (PTS1), could be confirmed by overexpression in HepG2 cells. The candidates SAR1B and PDCD6, which are known ER-exit-site proteins, did not directly colocalize with peroxisomes, but resided at ER sites, which frequently surrounded peroxisomes. Hence, both proteins might concentrate at presumably co-purified peroxisome-ER membrane contacts. Intriguingly, the fifth candidate, OCIA domain-containing protein 1, was previously described as decreasing mitochondrial network formation. In this work, we confirmed its peroxisomal localization and further observed a reduction in peroxisome numbers in response to OCIAD1 overexpression. Hence, OCIAD1 appears to be a novel protein, which has an impact on both mitochondrial and peroxisomal maintenance.

ProSE-Pero: Peroxisomal Protein Localization Identification Model Based on Self-Supervised Multi-Task Language Pre-Training Model.

Peroxisomes are membrane-bound organelles that contain one or more types of oxidative enzymes. Aberrant localization of peroxisomal proteins can contribute to the development of various diseases. To more accurately identify and locate peroxisomal proteins, we developed the ProSE-Pero model. We employed three methods based on deep representation learning models to extract the characteristics of peroxisomal proteins and compared their performance. Furthermore, we used the SVMSMOTE balanced dataset, SHAP interpretation model, variance analysis (ANOVA), and light gradient boosting machine (LightGBM) to select and compare the extracted features. We also constructed several traditional machine learning methods and four deep learning models to train and test our model on a dataset of 160 peroxisomal proteins using tenfold cross-validation. Our proposed ProSE-Pero model achieves high performance with a specificity (Sp) of 93.37%, a sensitivity (Sn) of 82.41%, an accuracy (Acc) of 95.77%, a Matthews correlation coefficient (MCC) of 0.8241, an F1 score of 0.8996, and an area under the curve (AUC) of 0.9818. Additionally, we extended our method to identify plant vacuole proteins and achieved an accuracy of 91.90% on the independent test set, which is approximately 5% higher than the latest iPVP-DRLF model. Our model surpasses the existing In-Pero model in terms of peroxisomal protein localization and identification. Additionally, our study showcases the proficient performance of the pre-trained multitasking language model ProSE in extracting features from protein sequences. With its established validity and broad generalization, our model holds considerable potential for expanding its application to the localization and identification of proteins in other organelles, such as mitochondria and Golgi proteins, in future investigations.

Bi-allelic ACBD6 variants lead to a neurodevelopmental syndrome with progressive and complex movement disorders.

The acyl-CoA-binding domain-containing protein 6 (ACBD6) is ubiquitously expressed, plays a role in the acylation of lipids and proteins and regulates the N-myristoylation of proteins via N-myristoyltransferase enzymes (NMTs). However, its precise function in cells is still unclear, as is the consequence of ACBD6 defects on human pathophysiology. Using exome sequencing and extensive international data sharing efforts, we identified 45 affected individuals from 28 unrelated families (consanguinity 93%) with bi-allelic pathogenic, predominantly loss-of-function (18/20) variants in ACBD6. We generated zebrafish and Xenopus tropicalis acbd6 knockouts by CRISPR/Cas9 and characterized the role of ACBD6 on protein N-myristoylation with myristic acid alkyne (YnMyr) chemical proteomics in the model organisms and human cells, with the latter also being subjected further to ACBD6 peroxisomal localization studies. The affected individuals (23 males and 22 females), aged 1-50 years, typically present with a complex and progressive disease involving moderate-to-severe global developmental delay/intellectual disability (100%) with significant expressive language impairment (98%), movement disorders (97%), facial dysmorphism (95%) and mild cerebellar ataxia (85%) associated with gait impairment (94%), limb spasticity/hypertonia (76%), oculomotor (71%) and behavioural abnormalities (65%), overweight (59%), microcephaly (39%) and epilepsy (33%). The most conspicuous and common movement disorder was dystonia (94%), frequently leading to early-onset progressive postural deformities (97%), limb dystonia (55%) and cervical dystonia (31%). A jerky tremor in the upper limbs (63%), a mild head tremor (59%), parkinsonism/hypokinesia developing with advancing age (32%) and simple motor and vocal tics were among other frequent movement disorders. Midline brain malformations including corpus callosum abnormalities (70%), hypoplasia/agenesis of the anterior commissure (66%), short midbrain and small inferior cerebellar vermis (38% each) as well as hypertrophy of the clava (24%) were common neuroimaging findings. Acbd6-deficient zebrafish and Xenopus models effectively recapitulated many clinical phenotypes reported in patients including movement disorders, progressive neuromotor impairment, seizures, microcephaly, craniofacial dysmorphism and midbrain defects accompanied by developmental delay with increased mortality over time. Unlike ACBD5, ACBD6 did not show a peroxisomal localization and ACBD6-deficiency was not associated with altered peroxisomal parameters in patient fibroblasts. Significant differences in YnMyr-labelling were observed for 68 co- and 18 post-translationally N-myristoylated proteins in patient-derived fibroblasts. N-myristoylation was similarly affected in acbd6-deficient zebrafish and X. tropicalis models, including Fus, Marcks and Chchd-related proteins implicated in neurological diseases. The present study provides evidence that bi-allelic pathogenic variants in ACBD6 lead to a distinct neurodevelopmental syndrome accompanied by complex and progressive cognitive and movement disorders.

ACOX-driven peroxisomal heterogeneity and functional compartmentalization in brown adipocytes of hypothyroid rats.

We previously demonstrated that hypothyroidism increases peroxisomal biogenesis in rat brown adipose tissue (BAT). We also showed heterogeneity in peroxisomal origin and their unique structural association with mitochondria and/or lipid bodies to carry out β-oxidation, contributing thus to BAT thermogenesis. Distinctive heterogeneity creates structural compartmentalization within peroxisomal population, raising the question of whether it is followed by their functional compartmentalization regarding localization/colocalization of two main acyl-CoA oxidase (ACOX) isoforms, ACOX1 and ACOX3. ACOX is the first and rate-limiting enzyme of peroxisomal β-oxidation, and, to date, their protein expression patterns in BAT have not been fully defined. Therefore, we used methimazole-induced hypothyroidism to study ACOX1 and ACOX3 protein expression and their tissue immunolocalization. Additionally, we analysed their specific peroxisomal localization and colocalization in parallel with peroxisomal structural compartmentalization in brown adipocytes. Hypothyroidism caused a linear increase in ACOX1 expression, while a temporary decrease in ACOX3 levels is only recovered to the control level at day 21. Peroxisomal ACOX1 and ACOX3 localization and colocalization patterns entirely mirrored heterogeneous peroxisomal biogenesis pathways and structural compartmentalization, e.g. associations with mitochondria and/or lipid bodies. Hence, different ACOX isoforms localization/colocalization creates distinct functional heterogeneity of peroxisomes and drives their functional compartmentalization in rat brown adipocytes.

Molecular Cloning, Subcellular Localization, and Abiotic Stress Induction Analysis of a Polyamine Oxidase Gene from Oryza sativa

Abiotic stress is a major bottleneck for crop productivity. To counter abiotic stresses, plants have developed several strategies, and the accumulation of polyamine (PA) serving as an osmolyte is one of them. The cellular pool of PAs is primarily regulated by polyamine oxidases (PAOs) either by terminal catabolism or by back conversion (BC) of polyamines. The role of PAO in abiotic stress tolerance has also been reported. Polyamine oxidases are primarily localized in the cytosol, cell wall, and apoplastic regions; however, lately, their peroxisomal localization has also been demonstrated. In this study, we reported the detection of polyamine oxidase isoform 3 from Oryza sativa (OsPAO3) in peroxisome as early as 12 h post-transformation under in vitro conditions using fluorescent microscopy. The gene was also found to be significantly upregulated by salinity, dehydration, cold, and heat stress. We have also demonstrated that the expression of OsPAO3 gene was mediated by a second messenger, calcium. The upregulation of OsPAO3 by salinity, dehydration, cold, and heat stresses suggests that it could be a suitable candidate for providing tolerance to plants against abiotic stress combination or stress matrix, which is a common feature in agricultural field conditions. Furthermore, the data provided here would be valuable in understanding the abiotic stress-mediated signal transduction network of PAOs.

Molecular cloning of glutathione reductase from Oryza sativa, demonstrating its peroxisomal localization and upregulation by abiotic stresses.

Abiotic stress is a major constraint on crop productivity and in the agricultural field, multiple abiotic stresses act synchronously leading to substantial damage to plants. A common after-effect of abiotic stress-induced damage in plants is an increased concentration of reactive oxygen species (ROS) leading to oxidative damage. Glutathione reductase (GR) plays a significant role in curtailing ROS. Apart from the GR enzyme, the peroxisome as an organelle also plays a significant role in ROS homeostasis. Here, we report a peroxisome localized GR, whose expression was found to be upregulated by various abiotic stresses. The in silico analysis also revealed that the peroxisomal localization of GR could be a common phenomenon in angiosperms, suggesting that it could be a suitable candidate against abiotic stress combinations.

Identification and Expression Profiling of Two Saudi Arabia Catalase Genes from Wheat and Barley in Response to Abiotic and Hormonal Stresses.

Catalase is a crucial enzyme in antioxidant defense systems protecting eukaryotes from oxidative stress. These proteins are present in almost all living organisms and play important roles in controlling plant responses to biotic and abiotic stresses by catalyzing the decomposition of H2O2. Despite their importance, little is known about their expression in the majority of monocotyledonous species. Here, we isolated and characterized two novel catalase genes from Triticum turgidum and Hordeum vulgare, designated as TtCAT1 and HvCAT1, respectively. Phylogenetic analysis revealed that TtCAT1 and HvCAT1 presented 492 aa and shared an important identity with other catalase proteins belonging to subfamily 1. Using bioinformatic analysis, we predicted the 3D structure models of TtCAT1 and HvCAT1. Interestingly, analysis showed that the novel catalases harbor a peroxisomal targeting signal (PTS1) located at their C-terminus portion, as shown for other catalase proteins. In addition, this motif is responsible for the in silico peroxisomal localization of both proteins. Finally, RT-qPCR analysis showed that TtCAT1 and HvCAT1 are highly expressed in leaves in normal conditions but faintly in roots. Moreover, both genes are upregulated after the application of different stresses such as salt, osmotic, cold, heavy metal, and hormonal stresses. The positive responses of TtCAT1 and HvCAT1 to the various stimuli suggested that these proteins can help to protect both species against environmental stresses.

Fusarium verticillioides Pex7/20 mediates peroxisomal PTS2 pathway import, pathogenicity, and fumonisin B1 biosynthesis.

Fusarium verticillioides, a well-known fungal pathogen that causes severe disease in maize and contaminates the grains with fumonisin B1 (FB1) mycotoxin, affects the yield and quality of maize worldwide. The intrinsic roles of peroxisome targeting signal (PTS)-containing proteins in phytopathogens remain elusive. We therefore explored the regulatory role and other biological functions of the components of PTS2 receptor complex, FvPex7 and FvPex20, in F. verticillioides. We found that FvPex7 directly interacts with the carboxyl terminus of FvPex20 in F. verticillioides. PTS2-containing proteins are recognized and bound by the FvPex7 receptor or the FvPex7-Pex20 receptor complex in the cytoplasm, but the peroxisome localization of the PTS2-Pex7-Pex20 complex is only determined by Pex20 in F. verticillioides. However, we observed that some putative PTS2 proteins that interact with Pex7 are not transported into the peroxisomes, but a PTS1 protein that interacts with Pex5 was detected in the peroxisomes. Furthermore, ΔFvpex7pex20 as well as ΔFvpex7pex5 double mutants exhibited reduced pathogenicity and FB1 biosynthesis, along with defects in conidiation. The PTS2 receptor complex mutants (ΔFvpex7pex20) grew slowly on minimal media and showed reduced sensitivity to cell wall and cell membrane stress-inducing agents compared to the wild type. Taken together, we conclude that the PTS2 receptor complex mediates peroxisome matrix proteins import and contributes to pathogenicity and FB1 biosynthesis in F. verticillioides. KEY POINTS: • FvPex7 directly interacts with FvPex20 in F. verticillioides. • vThe PTS2 receptor complex is essential for the importation of PTS2-containing matrix protein into peroxisomes in F. verticillioides. • Fvpex7/pex20 is involved in pathogenicity and FB1 biosynthesis in F. verticillioides.

Image-Based Analysis Revealing the Molecular Mechanism of Peroxisome Dynamics in Plants.

Peroxisomes are present in eukaryotic cells and have essential roles in various biological processes. Plant peroxisomes proliferate by de novo biosynthesis or division of pre-existing peroxisomes, degrade, or replace metabolic enzymes, in response to developmental stages, environmental changes, or external stimuli. Defects of peroxisome functions and biogenesis alter a variety of biological processes and cause aberrant plant growth. Traditionally, peroxisomal function-based screening has been employed to isolate Arabidopsis thaliana mutants that are defective in peroxisomal metabolism, such as lipid degradation and photorespiration. These analyses have revealed that the number, subcellular localization, and activity of peroxisomes are closely related to their efficient function, and the molecular mechanisms underlying peroxisome dynamics including organelle biogenesis, protein transport, and organelle interactions must be understood. Various approaches have been adopted to identify factors involved in peroxisome dynamics. With the development of imaging techniques and fluorescent proteins, peroxisome research has been accelerated. Image-based analyses provide intriguing results concerning the movement, morphology, and number of peroxisomes that were hard to obtain by other approaches. This review addresses image-based analysis of peroxisome dynamics in plants, especially A. thaliana and Marchantia polymorpha.

Elucidating the role of NUDT19 in renal lipid metabolism

Coenzyme A (CoA) is an essential cofactor that plays a key role in fuel metabolism, as it is required for glucose oxidation and the synthesis and oxidation of fatty acids, amino acids, and ketone bodies. Three Nudix hydrolases, NUDT7, NUDT8, and NUDT19, hydrolyze CoA species at the phosphodiester bond, potentially contributing to the regulation of specific metabolic processes in the subcellular compartments where they reside. NUDT19 is found in the peroxisomes and is highly expressed in kidney proximal tubule cells (PTC). In vitro, NUDT19 readily hydrolyzes short and medium chain acyl‐CoAs, malonyl‐ and succinyl‐CoA, as well as free CoA. This substrate preference, combined with the peroxisomal localization of NUDT19, suggests a potential role in the regulation of peroxisomal lipid metabolism. To investigate the physiological function of NUDT19, we obtained global Nudt19‐/‐ mice and fed them a high fat diet (HFD) or a low fat control diet (CD) for 15 weeks. On either diet, Nudt19‐/‐ mice exhibit higher total CoA levels compared to wild type mice, indicating that NUDT19 contributes to the regulation of this cofactor. HFD feeding led to a higher albumin‐to‐creatinine ratio in the Nudt19‐/‐ mice compared to wild type mice, indicating a decrease in kidney function. Nudt19‐/‐ mice also exhibited elevated serum triglycerides and a higher body weight, not associated with changes in bone density or adiposity. In mice fed the HFD, deletion of Nudt19 caused only a few changes to the kidney cortex proteome, with only 8 proteins significantly altered, including 3 involved in peroxisomal and mitochondria fatty acid oxidation, which were increased in the kidneys of the Nudt19‐/‐ mice. Measurement of mitochondrial and peroxisomal fatty acid oxidation using 14C‐palmitate in kidney cortex slides did not reveal any differences between genotypes. Conversely, untargeted metabolomics analysis on whole kidneys revealed a general decrease in multiple lipid classes including lysophospholipids, monoacylglycerols, and long chain free fatty acids, without changes in triglycerides levels. Combined, the data support the conclusion that deletion of Nudt19 alters lipid metabolism.

Pls1 Is a Peroxisomal Matrix Protein with a Role in Regulating Lysine Biosynthesis.

Peroxisomes host essential metabolic enzymes and are crucial for human health and survival. Although peroxisomes were first described over 60 years ago, their entire proteome has not yet been identified. As a basis for understanding the variety of peroxisomal functions, we used a high-throughput screen to discover peroxisomal proteins in yeast. To visualize low abundance proteins, we utilized a collection of strains containing a peroxisomal marker in which each protein is expressed from the constitutive and strong TEF2 promoter. Using this approach, we uncovered 18 proteins that were not observed in peroxisomes before and could show their metabolic and targeting factor dependence for peroxisomal localization. We focus on one newly identified and uncharacterized matrix protein, Ynl097c-b, and show that it localizes to peroxisomes upon lysine deprivation and that its localization to peroxisomes depends on the lysine biosynthesis enzyme, Lys1. We demonstrate that Ynl097c-b affects the abundance of Lys1 and the lysine biosynthesis pathway. We have therefore renamed this protein Pls1 for Peroxisomal Lys1 Stabilizing 1. Our work uncovers an additional layer of regulation on the central lysine biosynthesis pathway. More generally it highlights how the discovery of peroxisomal proteins can expand our understanding of cellular metabolism.

Peroxisomal ATP Uptake Is Provided by Two Adenine Nucleotide Transporters and the ABCD Transporters.

Peroxisomes are essential organelles involved in various metabolic processes, including fatty acid β-oxidation. Their metabolic functions require a controlled exchange of metabolites and co-factors, including ATP, across the peroxisomal membrane. We investigated which proteins are involved in the peroxisomal uptake of ATP in the yeast Saccharomyces cerevisiae. Using wild-type and targeted deletion strains, we measured ATP-dependent peroxisomal octanoate β-oxidation, intra-peroxisomal ATP levels employing peroxisome-targeted ATP-sensing reporter proteins, and ATP uptake in proteoliposomes prepared from purified peroxisomes. We show that intra-peroxisomal ATP levels are maintained by different peroxisomal membrane proteins each with different modes of action: 1) the previously reported Ant1p protein, which catalyzes the exchange of ATP for AMP or ADP, 2) the ABC transporter protein complex Pxa1p/Pxa2p, which mediates both uni-directional acyl-CoA and ATP uptake, and 3) the mitochondrial Aac2p protein, which catalyzes ATP/ADP exchange and has a dual localization in both mitochondria and peroxisomes. Our results provide compelling evidence for a complementary system for the uptake of ATP in peroxisomes.

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol.

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90–100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.

Pex7 selectively imports PTS2 target proteins to peroxisomes and is required for anthracnose disease development in Colletotrichum scovillei

Pex7 is a shuttling receptor that imports matrix proteins with a type 2 peroxisomal targeting signal (PTS2) to peroxisomes. The Pex7-mediated PTS2 protein import contributes to crucial metabolic processes such as the fatty acid β-oxidation and glucose metabolism in a number of fungi, but cellular roles of Pex7 between the import of PTS2 target proteins and metabolic processes have not been fully understood. In this study, we investigated the functional roles of CsPex7, a homolog of the yeast Pex7, by targeted gene deletion in the pepper anthracnose fungus Colletotrichum scovillei. CsPex7 was required for carbon source utilization, scavenging of reactive oxygen species, conidial production, and disease development in C. scovillei. The expression of fluorescently tagged PTS2 signal of hexokinases and 3-ketoacyl-CoA thiolases showed that peroxisomal localization of the hexokinase CsGlk1 PTS2 is dependent on CsPex7, but those of the 3-ketoacyl-CoA thiolases are independent on CsPex7. In addition, GFP-tagged CsPex7 proteins were intensely localized to the peroxisomes on glucose-containing media, indicating a role of CsPex7 in glucose utilization. Collectively, these findings indicate that CsPex7 selectively recognizes specific PTS2 signal for import of PTS2-containing proteins to peroxisomes, thereby mediating peroxisomal targeting efficiency of PTS2-containing proteins in C. scovillei. On pepper fruits, the ΔCspex7 mutant exhibited significantly reduced virulence, in which excessive accumulation of hydrogen peroxide was observed in the pepper cells. We think the reduced virulence results from the abnormality in hydrogen peroxide metabolism of the ΔCspex7 mutant. Our findings provide insight into the cellular roles of CsPex7 in PTS2 protein import system.

Phylogeny and subcellular localization analyses reveal distinctions in monocot and eudicot class IV acyl-CoA-binding proteins.

Plant class IV ACBPs diverged with the split of monocots and eudicots. Difference in the subcellular localization supported the functional variation of plant class IV ACBP. Acyl-CoA-binding proteins (ACBPs) are divided into class I-IV in plants. Class IV ACBPs are kelch motif containing proteins that are specific to plants. The currently known subcellular localizations of plant class IV ACBPs are either in the cytosol (Arabidopsis) or in the peroxisomes (rice). However, it is not clear whether peroxisomal localization of class IV ACBP is a shared character that distinguishes eudicots and monocots. Here, the phylogeny of class IV ACBPs from 73 plant species and subcellular localization of class IV ACBPs from six monocots and eudicots were conducted. Phylogenetic analysis of 112 orthologues revealed that monocot class IV ACBPs were basal to the monophyletic clade formed by eudicots and basal angiosperm. Transient expression of GFP fusions in onion epidermal cells demonstrated that monocot maize (Zea mays), wheat (Triticum aestivum), and sorghum (Sorghum bicolor) and eudicot poplar (Populus trichocarpa) all contained at least one peroxisomal localized class IV ACBP, while orthologues from cucumber (Cucumis sativus L.) and soybean (Glycine max) were all cytosolic. Combining the location of Arabidopsis and rice class IV ACBPs, it indicates that maintaining at least one peroxisomal class IV ACBP could be a shared feature within the tested monocots, while cytosolic class IV ACBPs would be preferred in the tested eudicots. Furthermore, the interaction between OsACBP6 and peroxisomal ATP-binding cassette (ABC) transporter provided clues for the functional mechanism of OsACBP6.