ATP-Dependent Steps in Peroxisomal Protein Import.

Peroxisomes are organelles that play a central role in lipid metabolism and cellular redox homeostasis. The import of peroxisomal matrix proteins by peroxisomal targeting signal (PTS) receptors is an ATP-dependent mechanism. However, the energy-dependent steps do not occur early during the binding of the receptor-cargo complex to the membrane but late, because they are linked to the peroxisomal export complex for the release of the unloaded receptor. The first ATP-demanding step is the cysteine-dependent monoubiquitination of the PTS receptors, which is required for recognition by the AAA+ peroxins. They execute the second ATP-dependent step by extracting the ubiqitinated PTS receptors from the membrane for release back to the cytosol. After deubiquitination, the PTS receptors regain import competence and can facilitate further rounds of cargo import. Here, we give a general overview and discuss recent data regarding the ATP-dependent steps in peroxisome protein import.

Diverse Cellular Roles of Autophagy.

Macroautophagy is an intracellular degradation system that delivers diverse cytoplasmic materials to lysosomes via autophagosomes. Recent advances have enabled identification of several selective autophagy substrates and receptors, greatly expanding our understanding of the cellular functions of autophagy. In this review, we describe the diverse cellular functions of macroautophagy, including its essential contribution to metabolic adaptation and cellular homeostasis. We also discuss emerging findings on the mechanisms and functions of various types of selective autophagy.

Abiotic Stress Signaling and Responses in Plants.

As sessile organisms, plants must cope with abiotic stress such as soil salinity, drought, and extreme temperatures. Core stress-signaling pathways involve protein kinases related to the yeast SNF1 and mammalian AMPK, suggesting that stress signaling in plants evolved from energy sensing. Stress signaling regulates proteins critical for ion and water transport and for metabolic and gene-expression reprogramming to bring about ionic and water homeostasis and cellular stability under stress conditions. Understanding stress signaling and responses will increase our ability to improve stress resistance in crops to achieve agricultural sustainability and food security for a growing world population.

The physiological functions of human peroxisomes.

Peroxisomes are subcellular organelles that play a central role in human physiology by catalyzing a range of unique metabolic functions. The importance of peroxisomes for human health is exemplified by the existence of a group of usually severe diseases caused by an impairment in one or more peroxisomal functions. Among others these include the Zellweger spectrum disorders, X-linked adrenoleukodystrophy, and Refsum disease. To fulfill their role in metabolism, peroxisomes require continued interaction with other subcellular organelles including lipid droplets, lysosomes, the endoplasmic reticulum, and mitochondria. In recent years it has become clear that the metabolic alliance between peroxisomes and other organelles requires the active participation of tethering proteins to bring the organelles physically closer together, thereby achieving efficient transfer of metabolites. This review intends to describe the current state of knowledge about the metabolic role of peroxisomes in humans, with particular emphasis on the metabolic partnership between peroxisomes and other organelles and the consequences of genetic defects in these processes. We also describe the biogenesis of peroxisomes and the consequences of the multiple genetic defects therein. In addition, we discuss the functional role of peroxisomes in different organs and tissues and include relevant information derived from model systems, notably peroxisomal mouse models. Finally, we pay particular attention to a hitherto underrated role of peroxisomes in viral infections.

Peroxisomes Get Loud: A Redox Antidote to Hearing Loss.

Pejvakin (PJVK), a protein originally identified in Persian families with sensorineural hearing loss, regulates peroxisomal dynamics and the antioxidant defense triggered by noise exposure in hair cells and auditory neurons of the inner ear. These findings bring peroxisomes to the forefront of noise-induced hearing loss research.

Close encounters of the lysosome-peroxisome kind.

Lysosomes provide a major source for cellular cholesterol; however, most of this cholesterol is trafficked to the plasma membrane via unknown mechanisms. Chu et al. identify an unexpected role for peroxisomes in the transport of cholesterol from the lysosome to the plasma membrane via a lysosome-peroxisome membrane contact site.

Hypervulnerability to Sound Exposure through Impaired Adaptive Proliferation of Peroxisomes.

A deficiency in pejvakin, a protein of unknown function, causes a strikingly heterogeneous form of human deafness. Pejvakin-deficient (Pjvk(-/-)) mice also exhibit variable auditory phenotypes. Correlation between their hearing thresholds and the number of pups per cage suggest a possible harmful effect of pup vocalizations. Direct sound or electrical stimulation show that the cochlear sensory hair cells and auditory pathway neurons of Pjvk(-/-) mice and patients are exceptionally vulnerable to sound. Subcellular analysis revealed that pejvakin is associated with peroxisomes and required for their oxidative-stress-induced proliferation. Pjvk(-/-) cochleas display features of marked oxidative stress and impaired antioxidant defenses, and peroxisomes in Pjvk(-/-) hair cells show structural abnormalities after the onset of hearing. Noise exposure rapidly upregulates Pjvk cochlear transcription in wild-type mice and triggers peroxisome proliferation in hair cells and primary auditory neurons. Our results reveal that the antioxidant activity of peroxisomes protects the auditory system against noise-induced damage.

Cholesterol transport through lysosome-peroxisome membrane contacts.

Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases.

Beyond rare disorders: A new era for peroxisomal pathophysiology.

Metabolism is emerging as a central influencer of multiple disease states in humans. Peroxisomes are central metabolic organelles whose decreased function gives rise to severe peroxisomal diseases. Recently, it is becoming clear that, beyond such rare inborn errors, the deterioration of peroxisomal functions contributes to multiple and prevalent diseases such as cancer, viral infection, diabetes, and neurodegeneration. Despite the clear importance of peroxisomes in common pathophysiological processes, research on the mechanisms underlying their contributions is still sparse. Here, we highlight the timeliness of focusing on peroxisomes in current research on central, abundant, and society-impacting human pathologies. As peroxisomes are now coming into the spotlight, it is clear that intensive research into these important organelles will enable a better understanding of their contribution to human health, serving as the basis to develop new diagnostic and therapeutic approaches to prevent and treat human diseases.

PEX5 translocation into and out of peroxisomes drives matrix protein import.

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal enzymes are imported from the cytosol by the receptor PEX5, which interacts with a docking complex in the peroxisomal membrane and then returns to the cytosol after monoubiquitination by a membrane-embedded ubiquitin ligase. The mechanism by which PEX5 shuttles between cytosol and peroxisomes and releases cargo inside the lumen is unclear. Here, we use Xenopus egg extract to demonstrate that PEX5 accompanies cargo completely into the lumen, utilizing WxxxF/Y motifs near its N terminus that bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix that binds to the lumenal side of the ubiquitin ligase. The N terminus then emerges in the cytosol for monoubiquitination. Finally, PEX5 is extracted from the lumen, resulting in the unfolding of the receptor and cargo release. Our results reveal the unique mechanism by which PEX5 ferries proteins into peroxisomes.

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.

Motors, anchors, and connectors: orchestrators of organelle inheritance.

Organelle inheritance is a process whereby organelles are actively distributed between dividing cells at cytokinesis. Much valuable insight into the molecular mechanisms of organelle inheritance has come from the analysis of asymmetrically dividing cells, which transport a portion of their organelles to the bud while retaining another portion in the mother cell. Common principles apply to the inheritance of all organelles, although individual organelles use specific factors for their partitioning. Inheritance factors can be classified as motors, which are required for organelle transport; anchors, which immobilize organelles at distinct cell structures; or connectors, which mediate the attachment of organelles to motors and anchors. Here, we provide an overview of recent advances in the field of organelle inheritance and highlight how motor, anchor, and connector molecules choreograph the segregation of a multicopy organelle, the peroxisome. We also discuss the role of organelle population control in the generation of cellular diversity.

Peroxisome formation and maintenance are dependent on the endoplasmic reticulum.

Looks can be deceiving. Although peroxisomes appear to be simple organelles, their formation and maintenance pose unique challenges for the cell. The birth of new peroxisomes starts at the endoplasmic reticulum (ER), which delivers lipids and membrane proteins. To form a new peroxisomal compartment, ER-derived preperoxisomal vesicles carrying different membrane proteins fuse, allowing the assembly of the peroxisomal translocon. To complete formation, peroxisomes import their soluble proteins directly from the cytosol using the newly assembled translocon. Together with the ER-derived biogenic route, peroxisomal fission and segregation subsequently maintain the cellular peroxisome population. In this review we highlight the latest insights on the life cycle of peroxisomes and show how the new cell biology concept of peroxisome formation affects our thinking about peroxisome-related diseases and their evolutionary past. The future challenge lies in the identification of all the proteins involved in this elaborate biogenic process and the dissection of their mechanism of action.

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.

Acetyl-CoA Derived from Hepatic Peroxisomal ß-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 Activation.

Autophagy is activated by prolonged fasting but cannot overcome the ensuing hepatic lipid overload, resulting in fatty liver. Here, we describe a peroxisome-lysosome metabolic link that restricts autophagic degradation of lipids. Acyl-CoA oxidase 1 (Acox1), the enzyme that catalyzes the first step in peroxisomal ß-oxidation, is enriched in liver and further increases with fasting or high-fat diet (HFD). Liver-specific Acox1 knockout (Acox1-LKO) protected mice against hepatic steatosis caused by starvation or HFD due to induction of autophagic degradation of lipid droplets. Hepatic Acox1 deficiency markedly lowered total cytosolic acetyl-CoA levels, which led to decreased Raptor acetylation and reduced lysosomal localization of mTOR, resulting in impaired activation of mTORC1, a central regulator of autophagy. Dichloroacetic acid treatment elevated acetyl-CoA levels, restored mTORC1 activation, inhibited autophagy, and increased hepatic triglycerides in Acox1-LKO mice. These results identify peroxisome-derived acetyl-CoA as a key metabolic regulator of autophagy that controls hepatic lipid homeostasis.

Vacuolar degradation of plant organelles.

Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of the pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.

Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

Mitofusin-mediated contacts between mitochondria and peroxisomes regulate mitochondrial fusion.

Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.

Biochemically distinct vesicles from the endoplasmic reticulum fuse to form peroxisomes.

As a rule, organelles in eukaryotic cells can derive only from pre-existing organelles. Peroxisomes are unique because they acquire their lipids and membrane proteins from the endoplasmic reticulum (ER), whereas they import their matrix proteins directly from the cytosol. We have discovered that peroxisomes are formed via heterotypic fusion of at least two biochemically distinct preperoxisomal vesicle pools that arise from the ER. These vesicles each carry half a peroxisomal translocon complex. Their fusion initiates assembly of the full peroxisomal translocon and subsequent uptake of enzymes from the cytosol. Our findings demonstrate a remarkable mechanism to maintain biochemical identity of organelles by transporting crucial components via different routes to their final destination.

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.