Histoplasma capsulatum requires peroxisomes for multiple virulence functions including siderophore biosynthesis.

Peroxisomes are versatile eukaryotic organelles essential for many functions in fungi, including fatty acid metabolism, reactive oxygen species detoxification, and secondary metabolite biosynthesis. A suite of Pex proteins (peroxins) maintains peroxisomes, while peroxisomal matrix enzymes execute peroxisome functions. Insertional mutagenesis identified peroxin genes as essential components supporting the intraphagosomal growth of the fungal pathogen Histoplasma capsulatum. Disruption of the peroxins Pex5, Pex10, or Pex33 in H. capsulatum prevented peroxisome import of proteins targeted to the organelle via the PTS1 pathway. This loss of peroxisome protein import limited H. capsulatum intracellular growth in macrophages and attenuated virulence in an acute histoplasmosis infection model. Interruption of the alternate PTS2 import pathway also attenuated H. capsulatum virulence, although only at later time points of infection. The Sid1 and Sid3 siderophore biosynthesis proteins contain a PTS1 peroxisome import signal and localize to the H. capsulatum peroxisome. Loss of either the PTS1 or PTS2 peroxisome import pathway impaired siderophore production and iron acquisition in H. capsulatum, demonstrating compartmentalization of at least some biosynthetic steps for hydroxamate siderophore biosynthesis. However, the loss of PTS1-based peroxisome import caused earlier virulence attenuation than either the loss of PTS2-based protein import or the loss of siderophore biosynthesis, indicating additional PTS1-dependent peroxisomal functions are important for H. capsulatum virulence. Furthermore, disruption of the Pex11 peroxin also attenuated H. capsulatum virulence independently of peroxisomal protein import and siderophore biosynthesis. These findings demonstrate peroxisomes contribute to H. capsulatum pathogenesis by facilitating siderophore biosynthesis and another unidentified role(s) for the organelle during fungal virulence. IMPORTANCE The fungal pathogen Histoplasma capsulatum infects host phagocytes and establishes a replication-permissive niche within the cells. To do so, H. capsulatum overcomes and subverts antifungal defense mechanisms which include the limitation of essential micronutrients. H. capsulatum replication within host cells requires multiple distinct functions of the fungal peroxisome organelle. These peroxisomal functions contribute to H. capsulatum pathogenesis at different times during infection and include peroxisome-dependent biosynthesis of iron-scavenging siderophores to enable fungal proliferation, particularly after activation of cell-mediated immunity. The multiple essential roles of fungal peroxisomes reveal this organelle as a potential but untapped target for the development of therapeutics.

Bioactive dipeptides enhance the tolerance of lager yeast to ethanol-oxidation cross-stress by regulating the multilevel defense system.

Although high gravity brewing technology has been widely used for beer industries due to its economic benefits, yeast cells are subjected to multiple environmental stresses throughout the fermentation process. Eleven bioactive dipeptides (LH, HH, AY, LY, IY, AH, PW, TY, HL, VY, FC) were selected to evaluate their effects on cell proliferation, cell membrane defense system, antioxidant defense system and intracellular protective agents of lager yeast against ethanol-oxidation cross-stress. Results showed that the multiple stresses tolerance and fermentation performance of lager yeast were enhanced by bioactive dipeptides. Cell membrane integrity was improved by bioactive dipeptides through altering the structure of macromolecular compounds of the cell membrane. Intracellular reactive oxygen species (ROS) accumulation was significantly decreased by bioactive dipeptides, especially for FC, decreasing by 33.1%, compared with the control. The decrease of ROS was closely related to the increase of mitochondrial membrane potential, intracellular antioxidant enzyme activities including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), and glycerol level. In addition, bioactive dipeptides could regulate the expression of key genes (GPD1, OLE1, SOD2, PEX11, CTT1, HSP12) to enhance the multilevel defense systems under ethanol-oxidation cross-stress. Therefore, bioactive dipeptides should be potentially efficient and feasible bioactive ingredients to improve the multiple stresses tolerance of lager yeast during high gravity fermentation.

Peroxisome biogenesis initiated by protein phase separation.

Peroxisomes are organelles that carry out ß-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction1. Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts2, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes3. Current models postulate a large pore formed by transmembrane proteins4; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as 'stickers' in associative polymer models of LLPS5,6. Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP-Pex13 and GFP-Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5-cargo. Our findings lead us to suggest a model in which LLPS of Pex5-cargo with Pex13 and Pex14 results in transient protein transport channels7.

Novel targeting assay uncovers targeting information within peroxisomal ABC transporter Pxa1.

The mechanism behind peroxisomal membrane protein targeting is still poorly understood, with only two yeast proteins believed to be involved and no consensus targeting sequence. Pex19 is thought to bind peroxisomal membrane proteins in the cytosol, and is subsequently recruited by Pex3 at the peroxisomal surface, followed by protein insertion via a mechanism that is as-yet-unknown. However, some peroxisomal membrane proteins still correctly sort in the absence of Pex3 or Pex19, suggesting that multiple sorting pathways exist. Here, we studied sorting of yeast peroxisomal ABC transporter Pxa1. Co-localisation analysis of Pxa1-GFP in a collection of 86 peroxisome-related deletion strains revealed that Pxa1 sorting requires Pex3 and Pex19, while none of the other 84 proteins tested were essential. To identify regions with peroxisomal targeting information in Pxa1, we developed a novel in vivo re-targeting assay, using a reporter consisting of the mitochondrial ABC transporter Mdl1 lacking its N-terminal mitochondrial targeting signal. Using this assay, we showed that the N-terminal 95 residues of Pxa1 are sufficient for retargeting this reporter to peroxisomes. Interestingly, truncated Pxa1 lacking residues 1-95 still localised to peroxisomes. This was confirmed via localisation of various Pxa1 truncation and deletion constructs. However, localisation of Pxa1 lacking residues 1-95 depended on the presence of its interaction partner Pxa2, indicating that this truncated protein does not contain a true targeting signal.

Comparison of human PEX knockout cell lines suggests a dual role of PEX1 in peroxisome biogenesis.

For the biogenesis and maintenance of peroxisomes several proteins, called peroxins, are essential. Malfunctions of these proteins lead to severe diseases summarized as peroxisome biogenesis disorders. The different genetic background of patient-derived cell lines and the residual expression of mutated PEX genes impede analysis of the whole spectrum of cellular functions of affected peroxins. To overcome these difficulties, we have generated a selected PEX knockout resource of HEK T-REx293 cells using the CRISPR/Cas9 technique. Comparative analyses of whole cell lysates revealed PEX-KO specific alterations in the steady-state level of peroxins and variations in the import efficacy of matrix proteins with a Type 2 peroxisomal targeting signal. One of the observed differences concerned PEX1 as in the complete absence of the protein, the number of peroxisomal ghosts is significantly increased. Upon expression of PEX1, import competence and abundance of peroxisomes was adjusted to the level of normal HEK cells. In contrast, expression of an alternatively spliced PEX1 isoform lacking 321 amino acids of the N-terminal region failed to rescue the peroxisomal import defects but reduced the number of peroxisomal vesicles. All in all, the data suggest a novel 'moonlighting' function of human PEX1 in the regulation of pre-peroxisomal vesicles.

The nexus between peroxisome abundance and chronological ageing in Saccharomyces cerevisiae.

Ageing is characterized by changes in several cellular processes, with dysregulation of peroxisome function being one of them. Interestingly, the most conserved function of peroxisomes, ROS homeostasis, is strongly associated with ageing and age-associated pathologies. Previous studies have identified a role for peroxisomes in the regulation of chronological lifespan in yeast. In this study, we report the effect of altered peroxisome number on the chronological lifespan of yeast in two different growth media conditions. Three mutants, pex11, pex25 and pex27, defective in peroxisome fission, have been thoroughly investigated for the chronological lifespan. Reduced chronological lifespan of all the mutants was observed in peroxisome-inducing growth conditions. Furthermore, the combined deletion pex11pex25 exhibited the most prominent reduction in lifespan. Interestingly altered peroxisomal phenotype upon ageing was observed in all the cells. Increased ROS accumulation and reduced catalase activity was exhibited by chronologically aged mutant cells. Interestingly, mutants with reduced number of peroxisomes concomitantly also exhibited an accumulation of free fatty acids and increased number of lipid droplets. Taken together, our results reveal a previously unrealized effect of fission proteins in the chronological lifespan of yeast.

Structure and function of the peroxisomal ubiquitin ligase complex.

Peroxisomes are membrane-bounded organelles that exist in most eukaryotic cells and are involved in the oxidation of fatty acids and the destruction of reactive oxygen species. Depending on the organism, they house additional metabolic reactions that range from glycolysis in parasitic protozoa to the production of ether lipids in animals and antibiotics in fungi. The importance of peroxisomes for human health is revealed by various disorders - notably the Zellweger spectrum - that are caused by defects in peroxisome biogenesis and are often fatal. Most peroxisomal metabolic enzymes reside in the lumen, but are synthesized in the cytosol and imported into the organelle by mobile receptors. The receptors accompany cargo all the way into the lumen and must return to the cytosol to start a new import cycle. Recycling requires receptor monoubiquitination by a membrane-embedded ubiquitin ligase complex composed of three RING finger (RF) domain-containing proteins: PEX2, PEX10, and PEX12. A recent cryo-electron microscopy (cryo-EM) structure of the complex reveals its function as a retro-translocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that assemble into an open channel. The N terminus of a receptor likely inserts into the pore from the lumenal side, and is then monoubiquitinated by one of the RFs to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitinated by the concerted action of the other two RFs and ultimately degraded. The new data provide mechanistic insight into a crucial step of peroxisomal protein import.

Enhanced production of acetyl-CoA-based products via peroxisomal surface display in Saccharomyces cerevisiae.

Colocalization of enzymes is a proven approach to increase pathway flux and the synthesis of nonnative products. Here, we develop a method for enzyme colocalization using the yeast peroxisomal membrane as an anchor point. Pathway enzymes were fused to the native Pex15 anchoring motif to enable display on the surface of the peroxisome facing the cytosol. The peroxisome is the sole location of ß-oxidation in Saccharomyces cerevisiae, and acetyl-CoA is a by-product that is exported in the form of acetyl-carnitine. To access this untapped acetyl-CoA pool, we surface-anchored the native peroxisomal/mitochondrial enzyme Cat2 to convert acetyl-carnitine to acetyl-CoA directly upon export across the peroxisomal membrane; this increased acetyl-CoA levels 3.7-fold. Subsequent surface attachment of three pathway enzymes - Cat2, a high stability Acc1 (for conversion of acetyl-CoA to malonyl-CoA), and the type III PKS 2-pyrone synthase - demonstrated the success of peroxisomal surface display for both enzyme colocalization and access to acetyl-CoA from exported acetyl-carnitine. Synthesis of the polyketide triacetic acid lactone increased by 21% over cytosolic expression at low gene copy number, and an additional 11-fold (to 766 mg/L) after further optimization. Finally, we explored increasing peroxisomal membrane area through overexpression of the peroxisomal biogenesis protein Pex11. Our findings establish peroxisomal surface display as an efficient strategy for enzyme colocalization and for accessing the peroxisomal acetyl-CoA pool to increase synthesis of acetyl-CoA-based products.

Peroxisome biogenesis and inter-organelle communication: an indispensable role for Pex11 and Pex30 family proteins in yeast.

Peroxisomes are highly dynamic organelles present in most eukaryotic cells. They also play an important role in human health and the optimum functioning of cells. An extensive repertoire of proteins is associated with the biogenesis and function of these organelles. Two protein families that are involved in regulating peroxisome number in a cell directly or indirectly are Pex11 and Pex30. Interestingly, these proteins are also reported to regulate the contact sites between peroxisomes and other cell organelles such as mitochondria, endoplasmic reticulum and lipid droplets. In this manuscript, we review our current knowledge of the role of these proteins in peroxisome biogenesis in various yeast species. Further, we also discuss in detail the role of these protein families in the regulation of inter-organelle contacts in yeast.

A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.

Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1-4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1-4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.

Ubiquitin-conjugating activity by PEX4 is required for efficient protein transport to peroxisomes in Arabidopsis thaliana.

Protein transport to peroxisomes requires various proteins, such as receptors in the cytosol and components of the transport machinery on peroxisomal membranes. The Arabidopsis apem (aberrant peroxisome morphology) mutant apem7 shows decreased efficiency of peroxisome targeting signal 1-dependent protein transport to peroxisomes. In apem7 mutants, peroxisome targeting signal 2-dependent protein transport is also disturbed, and plant growth is repressed. The APEM7 gene encodes a protein homologous to peroxin 4 (PEX4), which belongs to the ubiquitin-conjugating (UBC) protein family; however, the UBC activity of Arabidopsis PEX4 remains to be investigated. Here, we show using electron microscopy and immunoblot analysis using specific PEX4 antibodies and in vitro transcription/translation assay that PEX4 localizes to peroxisomal membranes and possesses UBC activity. We found that the substitution of proline with leucine by apem7 mutation alters ubiquitination of PEX4. Furthermore, substitution of the active-site cysteine residue at position 90 in PEX4, which was predicted to be a ubiquitin-conjugation site, with alanine did not restore the apem7 phenotype. Taken together, these findings indicate that abnormal ubiquitination in the apem7 mutant alters ubiquitin signaling during the process of protein transport, suggesting that the UBC activity of PEX4 is indispensable for efficient protein transport to peroxisomes.

The peroxisomal exportomer directly inhibits phosphoactivation of the pexophagy receptor Atg36 to suppress pexophagy in yeast.

Autophagy receptor (or adaptor) proteins facilitate lysosomal destruction of various organelles in response to cellular stress, including nutrient deprivation. To what extent membrane-resident autophagy receptors also respond to organelle-restricted cues to induce selective autophagy remains poorly understood. We find that latent activation of the yeast pexophagy receptor Atg36 by the casein kinase Hrr25 in rich media is repressed by the ATPase activity of Pex1/6, the catalytic subunits of the exportomer AAA+ transmembrane complex enabling protein import into peroxisomes. Quantitative proteomics of purified Pex3, an obligate Atg36 coreceptor, support a model in which the exportomer tail anchored to the peroxisome membrane represses Atg36 phosphorylation on Pex3 without assistance from additional membrane factors. Indeed, we reconstitute inhibition of Atg36 phosphorylation in vitro using soluble Pex1/6 and define an N-terminal unstructured region of Atg36 that enables regulation by binding to Pex1. Our findings uncover a mechanism by which a compartment-specific AAA+ complex mediating organelle biogenesis and protein quality control staves off induction of selective autophagy.

Increased peroxisome proliferation is associated with early yeast replicative ageing.

Peroxisomes are single membrane-bound organelles ubiquitously present in several cell types and are associated with cell and tissue-specific functions. Their role in cellular ageing is under investigation in various model systems. Metabolism of cellular reactive oxygen species is a universal function performed by these organelles. In this study, we investigated alterations in peroxisome number upon early replicative ageing of yeast cells. Increase in the number of peroxisomes in replicatively aged mother cells of wild-type yeast was observed when cultured in both peroxisome-inducing and non-inducing medium. Further, we investigated if this increase in peroxisome number in replicatively aged cells is due to enhanced peroxisome proliferation. For this, the number of peroxisomes in replicatively aged mother cells of pex11, pex25 and pex11pex25 was analysed. Increased percentage of aged cells was observed in pex25 and pex11pex25 cells cultured in peroxisome-inducing oleic acid medium. Interestingly, when cultured in oleic acid, young mother cells devoid of Pex11 showed reduced peroxisome proliferation compared to old mother cells. Induced activity of the antioxidant enzyme catalase and reduced accumulation of reactive oxygen species were reported in all studied strains when cultured in oleic acid medium. Further, our data also suggest that replicatively aged cells with increased peroxisome number also display mitochondrial dysfunction and fragmentation in all the strains studied. In conclusion, our data suggests a correlation between increase in peroxisome number and replicative age of yeast cells and interestingly this increase seems to be partly dependent on the fission proteins.

Ubiquitin ligase MARCH5 localizes to peroxisomes to regulate pexophagy.

Mitochondria and peroxisomes are independent but functionally closely related organelles. A few proteins have been characterized as dual-organelle locating proteins with distinct or similar roles on mitochondria and peroxisomes. MARCH5 is a mitochondria-associated ubiquitin ligase best known for its regulatory role in mitochondria quality control, fission, and fusion. Here, we used a proximity tagging system, PUP-IT, and identified new interacting proteins of MARCH5. Our data uncover that MARCH5 is a dual-organelle locating protein that interacts with several peroxisomal proteins. PEX19 binds the transmembrane region on MARCH5 and targets it to peroxisomes. On peroxisomes, MARCH5 binds and mediates the ubiquitination of PMP70. Furthermore, we find PMP70 ubiquitination and pexophagy induced by mTOR inhibition are blocked in the absence of MARCH5. Our study suggests novel roles of MARCH5 on peroxisomes.

Peroxisomal ß-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis.

To liberate fatty acids (FAs) from intracellular stores, lipolysis is regulated by the activity of the lipases adipose triglyceride lipase (ATGL), hormone-sensitive lipase and monoacylglycerol lipase. Excessive FA release as a result of uncontrolled lipolysis results in lipotoxicity, which can in turn promote the progression of metabolic disorders. However, whether cells can directly sense FAs to maintain cellular lipid homeostasis is unknown. Here we report a sensing mechanism for cellular FAs based on peroxisomal degradation of FAs and coupled with reactive oxygen species (ROS) production, which in turn regulates FA release by modulating lipolysis. Changes in ROS levels are sensed by PEX2, which modulates ATGL levels through post-translational ubiquitination. We demonstrate the importance of this pathway for non-alcoholic fatty liver disease progression using genetic and pharmacological approaches to alter ROS levels in vivo, which can be utilized to increase hepatic ATGL levels and ameliorate hepatic steatosis. The discovery of this peroxisomal ß-oxidation-mediated feedback mechanism, which is conserved in multiple organs, couples the functions of peroxisomes and lipid droplets and might serve as a new way to manipulate lipolysis to treat metabolic disorders.

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER.

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.

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.

The biochemical basis of mitochondrial dysfunction in Zellweger Spectrum Disorder.

Peroxisomal biogenesis disorders (PBDs) are genetic disorders of peroxisome biogenesis and metabolism that are characterized by profound developmental and neurological phenotypes. The most severe class of PBDs-Zellweger spectrum disorder (ZSD)-is caused by mutations in peroxin genes that result in both non-functional peroxisomes and mitochondrial dysfunction. It is unclear, however, how defective peroxisomes contribute to mitochondrial impairment. In order to understand the molecular basis of this inter-organellar relationship, we investigated the fate of peroxisomal mRNAs and proteins in ZSD model systems. We found that peroxins were still expressed and a subset of them accumulated on the mitochondrial membrane, which resulted in gross mitochondrial abnormalities and impaired mitochondrial metabolic function. We showed that overexpression of ATAD1, a mitochondrial quality control factor, was sufficient to rescue several aspects of mitochondrial function in human ZSD fibroblasts. Together, these data suggest that aberrant peroxisomal protein localization is necessary and sufficient for the devastating mitochondrial morphological and metabolic phenotypes in ZSDs.

Mitochondria as emergency landing for abandoned peroxins.

Zellweger spectrum disorder (ZSD) is the most severe peroxisomal biogenesis disorder (PBD). Why ZSD patients not only loose functional peroxisomes but also present with severe mitochondrial dysfunction was a long-standing mystery. In this issue, Nuebel et al (2021) identified that loss of peroxisomes leads to re-routing of peroxisomal proteins to mitochondria, thereby impairing mitochondrial structure and function. The findings provide the first molecular understanding of the mitochondrial-peroxisomal link in ZSD.

Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice.

The tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b/PEX5R) is an interaction partner and auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are key for rhythm generation in the brain and in the heart. Since TRIP8b is expressed in central neurons but not in cardiomyocytes, the TRIP8b-HCN interaction has been studied intensely in the brain, but is deemed irrelevant in the cardiac conduction system. Still, to date, TRIP8b has not been studied in the intrinsic cardiac nervous system (ICNS), a neuronal network located within epicardial fat pads. In vitro electrophysiological studies revealed that TRIP8b-deficient mouse hearts exhibit increased atrial refractory and atrioventricular nodal refractory periods, compared to hearts of wild-type littermates. Meanwhile, heart rate, sino-nodal recovery time, and ventricular refractory period did not differ between genotypes. Trip8b mRNA was detected in the ICNS by quantitative polymerase chain reaction. RNAscope in situ hybridization confirmed Trip8b localization in neuronal somata and nerve fibers. Additionally, we found a very low amount of mRNAs in the sinus node and atrioventricular node, most likely attributable to the delicate fibers innervating the conduction system. In contrast, TRIP8b protein was not detectable. Our data suggest that TRIP8b in the ICNS may play a role in the modulation of atrial electrophysiology beyond HCN-mediated sino-nodal control of the heart.