Search

Organelle division and segregation are important in cellular homeostasis. Peroxisomes (POs) and mitochondria share a core division machinery and mechanism of membrane scission. The division of each organelle is interdependent not only on the other but also on other organelles, reflecting the dynamic communication between subcellular compartments, even as they coordinate the exchange of metabolites and signals. We highlight common and unique mechanisms involved in the fission of these organelles under the premise that much can be gleaned regarding the division of one organelle based on information available for the other.

How environmental cues influence peroxisome proliferation, particularly through organelles, remains largely unknown. Yeast peroxisomes metabolize fatty acids (FA), and methylotrophic yeasts also metabolize methanol. NADH and acetyl-CoA, produced by these pathways enter mitochondria for ATP production and for anabolic reactions. During the metabolism of FA and/or methanol, the mitochondrial oxidative phosphorylation (OXPHOS) pathway accepts NADH for ATP production and maintains cellular redox balance. Remarkably, peroxisome proliferation in Pichia pastoris was abolished in NADH-shuttling- and OXPHOS mutants affecting complex I or III, or by the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), indicating ATP depletion causes the phenotype. We show that mitochondrial OXPHOS deficiency inhibits expression of several peroxisomal proteins implicated in FA and methanol metabolism, as well as in peroxisome division and proliferation. These genes are regulated by the Snf1 complex (SNF1), a pathway generally activated by a high AMP/ATP ratio. In OXPHOS mutants, Snf1 is activated by phosphorylation, but Gal83, its interacting subunit, fails to translocate to the nucleus. Phenotypic defects in peroxisome proliferation observed in the OXPHOS mutants, and phenocopied by the Δgal83 mutant, were rescued by deletion of three transcriptional repressor genes (MIG1, MIG2, and NRG1) controlled by SNF1 signaling. Our results are interpreted in terms of a mechanism by which peroxisomal and mitochondrial proteins and/or metabolites influence redox and energy metabolism, while also influencing peroxisome biogenesis and proliferation, thereby exemplifying interorganellar communication and interplay involving peroxisomes, mitochondria, cytosol, and the nucleus. We discuss the physiological relevance of this work in the context of human OXPHOS deficiencies.

Despite major advances in our understanding of players and mechanisms involved in peroxisome biogenesis and peroxisome degradation, very few studies have focused on unraveling the multi-layered connections between, and the coordination of, these two opposing processes that regulate peroxisome homeostasis. The intersection between these processes also provides exciting avenues for future research. This review highlights the links between peroxisome biogenesis and degradation, incorporating an integrative approach that is critical not only for a mechanistic understanding, but also for manipulating the balance between these processes in relevant disease models.

The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.

Damaged or redundant peroxisomes and their luminal cargoes are removed by pexophagy, a selective autophagy pathway. In yeasts, pexophagy depends mostly on the pexophagy receptors, such as Atg30 for Pichia pastoris and Atg36 for Saccharomyces cerevisiae, the autophagy scaffold proteins, Atg11 and Atg17, and the core autophagy machinery. In P. pastoris, the receptors for peroxisomal matrix proteins containing peroxisomal targeting signals (PTSs) include the PTS1 receptor, Pex5, and the PTS2 receptor and co-receptor, Pex7 and Pex20, respectively. These shuttling receptors are predominantly cytosolic and only partially peroxisomal. It remains unresolved as to whether, when and how the cytosolic pools of peroxisomal receptors, as well as the peroxisomal matrix proteins, are degraded under pexophagy conditions. These cytosolic pools exist both in normal and mutant cells impaired in peroxisome biogenesis. We report here that Pex5 and Pex7, but not Pex20, are degraded by an Atg30-independent, selective autophagy pathway. To enter this selective autophagy pathway, Pex7 required its major PTS2 cargo, Pot1. Similarly, the degradation of Pex5 was inhibited in cells missing abundant PTS1 cargoes, such as alcohol oxidases and Fox2 (hydratase-dehydrogenase-epimerase). Furthermore, in cells deficient in PTS receptors, the cytosolic pools of peroxisomal matrix proteins, such as Pot1 and Fox2, were also removed by Atg30-independent, selective autophagy, under pexophagy conditions. In summary, the cytosolic pools of PTS receptors and their cargoes are degraded via a pexophagy-independent, selective autophagy pathway under pexophagy conditions. These autophagy pathways likely protect cells from futile enzymatic reactions that could potentially cause the accumulation of toxic cytosolic products.Abbreviations: ATG: autophagy related; Cvt: cytoplasm to vacuole targeting; Fox2: hydratase-dehydrogenase-epimerase; PAGE: polyacrylamide gel electrophoresis; Pot1: thiolase; PMP: peroxisomal membrane protein; Pgk1: 3-phosphoglycerate kinase; PTS: peroxisomal targeting signal; RADAR: receptor accumulation and degradation in the absence of recycling; RING: really interesting new gene; SDS: sodium dodecyl sulphate; TCA, trichloroacetic acid; Ub: ubiquitin; UPS: ubiquitin-proteasome system Vid: vacuole import and degradation.

Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.

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

Peroxisomal membrane proteins (PMPs) traffic to peroxisomes by two mechanisms: direct insertion from the cytosol into the peroxisomal membrane and indirect trafficking to peroxisomes via the endoplasmic reticulum (ER). In mammals and yeast, several PMPs traffic via the ER in a Pex3- and Pex19-dependent manner. In Komagataella phaffii (formerly called Pichia pastoris) specifically, the indirect traffic of Pex2, but not of Pex11 or Pex17, depends on Pex3, but all PMPs tested for indirect trafficking require Pex19. In mammals, the indirect traffic of PMPs also requires PEX16, a protein that is absent in most yeast species. In this study, we isolated PEX36, a new gene in K. phaffii, which encodes a PMP. Pex36 is required for cell growth in conditions that require peroxisomes for the metabolism of certain carbon sources. This growth defect in cells lacking Pex36 can be rescued by the expression of human PEX16, Saccharomyces cerevisiae Pex34, or by overexpression of the endogenous K. phaffii Pex25. Pex36 is not an essential protein for peroxisome proliferation, but in the absence of the functionally redundant protein, Pex25, it becomes essential and less than 20% of these cells show import-incompetent, peroxisome-like structures (peroxisome remnants). In the absence of both proteins, peroxisome biogenesis and the intra-ER sorting of Pex2 and Pex11C are seriously impaired, likely by affecting Pex3 and Pex19 function.

Most proteins destined for the peroxisomal matrix depend on the peroxisomal targeting signals (PTSs), which require the PTS receptor PEX5, whose deficiency causes fatal human peroxisomal biogenesis disorders (PBDs). TRIM37 gene mutations cause muscle-liver-brain-eye (mulibrey) nanism. We found that TRIM37 localizes in peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with its C-terminal 51 amino acids (CT51), which is required for PTS protein import. PEX5 mutations (K464A or ΔCT51), or TRIM37 depletion or mutation, reduce PEX5 abundance by promoting its proteasomal degradation, thereby impairing its functions in cargo binding and PTS protein import in human cells. TRIM37 or PEX5 depletion induces apoptosis and enhances sensitivity to oxidative stress, underscoring the cellular requirement for functional peroxisomes. Therefore, TRIM37-mediated ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import, suggesting that mulibrey nanism is a new PBD.

The peroxins Pex19 and Pex3 play an indispensable role in peroxisomal membrane protein (PMP) biogenesis, peroxisome division, and inheritance. Pex19 plays multiple roles in these processes, but how these functions relate to the structural organization of the Pex19 domains is unresolved. To this end, using deletion mutants, we mapped the Pex19 regions required for peroxisome biogenesis in the yeast Pichia pastoris Surprisingly, import-competent peroxisomes still formed when Pex19 domains previously believed to be required for biogenesis were deleted, although the peroxisome size was larger than that in wild-type cells. Moreover, these mutants exhibited a delay of 14-24 h in peroxisome biogenesis. The shortest functional N-terminal (NTCs) and C-terminal constructs (CTCs) were Pex19 (aa 1-150) and Pex19 (aa 89-300), respectively. Deletions of the N-terminal Pex3-binding site disrupted the direct interactions of Pex19 with Pex3, but preserved interactions with a membrane peroxisomal targeting signal (mPTS)-containing PMP, Pex10. In contrast, deletion of the C-terminal mPTS-binding domain of Pex19 disrupted its interaction with Pex10 while leaving the Pex19-Pex3 interactions intact. However, Pex11 and Pex25 retained their interactions with both N- and C-terminal deletion mutants. NTC-CTC co-expression improved growth and reversed the larger-than-normal peroxisome size observed with the single deletions. Pex25 was critical for peroxisome formation with the CTC variants, and its overexpression enhanced their interactions with Pex3 and aided the growth of both NTC and CTC Pex19 variants. In conclusion, physical segregation of the Pex3- and PMP-binding domains of Pex19 has provided novel insights into the modular architecture of Pex19. We define the minimum region of Pex19 required for peroxisome biogenesis and a unique role for Pex25 in this process.

Pexophagy is a selective autophagy process that degrades damaged and/or superfluous peroxisomes in the yeast vacuole or in mammalian lysosomes. The molecular mechanisms of pexophagy are well studied in yeast. Peroxisomes can be rapidly induced by oleate in the budding yeast, Saccharomyces cerevisiae, and by oleate or methanol in the methylotrophic yeast, Pichia pastoris. A number of peroxisomal matrix enzymes, such as 3-ketoacyl CoA thiolase (thiolase) and alcohol oxidase (AOX), are upregulated correspondingly to meet metabolic demands of the cells. Removal of these peroxisome-inducing carbon sources creates conditions wherein peroxisomes are superfluous and results in pexophagy and the degradation of these peroxisomal matrix enzymes. In this chapter, we discuss different assays to monitor pexophagy in yeast. These assays rely on tracking the localization of the BFP-SKL protein (a peroxisomally targeted version of the blue fluorescent protein) by microscopy, biochemical analysis of the degradation of peroxisomal matrix proteins, thiolase and AOX, and/or measuring the reduction of AOX activity during pexophagy.

Pexophagy is a selective autophagy process that degrades damaged and/or superfluous peroxisomes in the yeast vacuole or in mammalian lysosomes. The molecular mechanisms of pexophagy are well studied in yeast. Peroxisomes can be rapidly induced by oleate in the budding yeast, Saccharomyces cerevisiae, and by oleate or methanol in the methylotrophic yeast, Pichia pastoris. A number of peroxisomal matrix enzymes, such as 3-ketoacyl CoA thiolase (thiolase) and alcohol oxidase (AOX), are upregulated correspondingly to meet metabolic demands of the cells. Removal of these peroxisome-inducing carbon sources creates conditions wherein peroxisomes are superfluous and results in pexophagy and the degradation of these peroxisomal matrix enzymes. In this chapter, we discuss different assays to monitor pexophagy in yeast. These assays rely on tracking the localization of the BFP-SKL protein (a peroxisomally targeted version of the blue fluorescent protein) by microscopy, biochemical analysis of the degradation of peroxisomal matrix proteins, thiolase and AOX, and/or measuring the reduction of AOX activity during pexophagy.

Peroxisomes proliferate by growth and division of pre-existing peroxisomes or could arise de novo. Though the de novo pathway of peroxisome biogenesis is a more recent discovery, several studies have highlighted key mechanistic details of the pathway. The endoplasmic reticulum (ER) is the primary source of lipids and proteins for the newly-formed peroxisomes. More recently, an intricate sorting process functioning at the ER has been proposed, that segregates specific PMPs first to peroxisome-specific ER domains (pER) and then assembles PMPs selectively into distinct pre-peroxisomal vesicles (ppVs) that later fuse to form import-competent peroxisomes. In addition, plausible roles of the three key peroxins Pex3, Pex16 and Pex19, which are also central to the growth and division pathway, have been suggested in the de novo process. In this review, we discuss key developments and highlight the unexplored avenues in de novo peroxisome biogenesis.

Peroxisomes are essential organelles required for proper cell function in all eukaryotic organisms. They participate in a wide range of cellular processes including the metabolism of lipids and generation, as well as detoxification, of hydrogen peroxide (H2O2). Therefore, peroxisome homoeostasis, manifested by the precise and efficient control of peroxisome number and functionality, must be tightly regulated in response to environmental changes. Due to the existence of many physiological disorders and diseases associated with peroxisome homoeostasis imbalance, the dynamics of peroxisomes have been widely examined. The increasing volume of reports demonstrating significant involvement of the autophagy machinery in peroxisome removal leads us to summarize current knowledge of peroxisome degradation in mammalian cells. In this review we present current models of peroxisome degradation. We particularly focus on pexophagy-the selective clearance of peroxisomes through autophagy. We also critically discuss concepts of peroxisome recognition for pexophagy, including signalling and selectivity factors. Finally, we present examples of the pathological effects of pexophagy dysfunction and suggest promising future directions.

During de novo peroxisome biogenesis, importomer complex proteins sort via two preperoxisomal vesicles (ppVs). However, the sorting mechanisms segregating peroxisomal membrane proteins to the preperoxisomal endoplasmic reticulum (pER) and into ppVs are unknown. We report novel roles for Pex3 and Pex19 in intra-endoplasmic reticulum (ER) sorting and budding of the RING-domain peroxins (Pex2, Pex10, and Pex12). Pex19 bridged the interaction at the ER between Pex3 and RING-domain proteins, resulting in a ternary complex that was critical for the intra-ER sorting and subsequent budding of the RING-domain peroxins. Although the docking subcomplex proteins (Pex13, Pex14, and Pex17) also required Pex19 for budding from the ER, they sorted to the pER independently of Pex3 and Pex19 and were spatially segregated from the RING-domain proteins. We also discovered a unique role for Pex3 in sorting Pex10 and Pex12, but with the docking subcomplex. Our study describes an intra-ER sorting process that regulates segregation, packaging, and budding of peroxisomal importomer subcomplexes, thereby preventing their premature assembly at the ER.

Protein ubiquitylation in mammals is known to trigger selective autophagy of peroxisomes through a process termed pexophagy. The physiological peroxisomal target for pexophagy-related ubiquitylation has been controversial, but two studies have now identified the protein PEX5 as the real candidate.

Pexophagy is a process that selectively degrades peroxisomes by autophagy. The Pichia pastoris pexophagy receptor Atg30 is recruited to peroxisomes under peroxisome proliferation conditions. During pexophagy, Atg30 undergoes phosphorylation, a prerequisite for its interactions with the autophagy scaffold protein Atg11 and the ubiquitin-like protein Atg8. Atg30 is subsequently shuttled to the vacuole along with the targeted peroxisome for degradation. Here, we defined the binding site for Atg30 on the peroxisomal membrane protein Pex3 and uncovered a role for Pex3 in the activation of Atg30 via phosphorylation and in the recruitment of Atg11 to the receptor protein complex. Pex3 is classically a docking protein for other proteins that affect peroxisome biogenesis, division, and segregation. We conclude that Pex3 has a role beyond simple docking of Atg30 and that its interaction with Atg30 regulates pexophagy in the yeast P. pastoris.

Peroxisomal matrix protein import uses two peroxisomal targeting signals (PTSs). Most matrix proteins use the PTS1 pathway and its cargo receptor, Pex5. The PTS2 pathway is dependent on another receptor, Pex7, and its coreceptor, Pex20. We found that during the matrix protein import cycle, the stability and dynamics of Pex7 differ from those of Pex5 and Pex20. In Pichia pastoris, unlike Pex5 and Pex20, Pex7 is constitutively degraded in wild-type cells but is stabilized in pex mutants affecting matrix protein import. Degradation of Pex7 is more prevalent in cells grown in methanol, in which the PTS2 pathway is nonessential, in comparison with oleate, suggesting regulation of Pex7 turnover. Pex7 must shuttle into and out of peroxisomes before it is polyubiquitinated and degraded by the proteasome. The shuttling of Pex7, and consequently its degradation, is dependent on the receptor recycling pathways of Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. We also found that blocking the export of Pex20 from peroxisomes inhibits PTS1-mediated import, suggesting sharing of limited components in the export of PTS receptors/coreceptors. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20, exemplifying a novel interdependence of the PTS1 and PTS2 pathways.

Autophagy is a membrane trafficking pathway that sequesters proteins and organelles into autophagosomes. The selectivity of this pathway is determined by autophagy receptors, such as the Pichia pastoris autophagy-related protein 30 (Atg30), which controls the selective autophagy of peroxisomes (pexophagy) through the assembly of a receptor protein complex (RPC). However, how the pexophagic RPC is regulated for efficient formation of the phagophore, an isolation membrane that sequesters the peroxisome from the cytosol, is unknown. Here we describe a new, conserved acyl-CoA-binding protein, Atg37, that is an integral peroxisomal membrane protein required specifically for pexophagy at the stage of phagophore formation. Atg30 recruits Atg37 to the pexophagic RPC, where Atg37 regulates the recruitment of the scaffold protein, Atg11. Palmitoyl-CoA competes with Atg30 for Atg37 binding. The human orthologue of Atg37, acyl-CoA-binding domain containing protein 5 (ACBD5), is also peroxisomal and is required specifically for pexophagy. We suggest that Atg37/ACBD5 is a new component and positive regulator of the pexophagic RPC.

In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1-110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.