Gluing yeast peroxisomes - composition and function of membrane contact sites.

Membrane contact sites are defined as regions of close proximity between two membranes; this association is mediated by protein-protein and/or protein-lipid interactions. Contact sites are often involved in lipid transport, but also can perform other functions. Peroxisomal membrane contact sites have obtained little attention compared to those of other cell organelles. However, recent studies resulted in a big leap in our knowledge of the occurrence, composition and function of peroxisomal contact sites. Studies in yeast strongly contributed to this progress. In this Review, we present an overview of our current knowledge on peroxisomal membrane contact sites in various yeast species, including Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica. Yeast peroxisomes form contacts with almost all other cellular organelles and with the plasma membrane. The absence of a component of a yeast peroxisomal contact site complex results in a range of peroxisomal phenotypes, including metabolic and biogenesis defects and alterations in organelle number, size or position.

Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation in yeast.

The yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome-ER contacts. Upon deletion of PEX24 or PEX32 - and to a much lesser extent, of PEX23 or PEX29 - peroxisome-ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome-ER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome-ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.

Yeast cells contain a heterogeneous population of peroxisomes that segregate asymmetrically during cell division.

Here, we used fluorescence microscopy and a peroxisome-targeted tandem fluorescent protein timer to determine the relative age of peroxisomes in yeast. Our data indicate that yeast cells contain a heterogeneous population of relatively old and young peroxisomes. During budding, the peroxisome retention factor inheritance of peroxisomes protein 1 (Inp1) selectively associates to the older organelles, which are retained in the mother cells. Inp2, a protein required for transport of peroxisomes to the bud, preferentially associates to younger organelles. Using a microfluidics device, we demonstrate that the selective segregation of younger peroxisomes to the buds is carefully maintained during multiple budding events. The replicative lifespan of mother cells increased upon deletion of INP2, which resulted in the retention of all organelles in mother cells. These data suggest that, in wild-type yeast, transport of aged and deteriorated peroxisomes to the bud is prevented, whereas the young and vital organelles are preferably transported to the newly forming buds.

Peroxisome Maintenance Depends on De Novo Peroxisome Formation in Yeast Mutants Defective in Peroxisome Fission and Inheritance.

There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast Hansenula polymorpha, lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance. Also, in cells of mutants without the peroxisome inheritance protein Inp2 peroxisome segregation can still occur. In contrast, peroxisome fission and inheritance were not observed in cells of a pex11 inp2 double deletion strain. In buds of cells of this double mutant, new organelles likely appear de novo. Growth of pex11 inp2 cells on methanol, a growth substrate that requires functional peroxisomes, is retarded relative to the wild type control. Based on these observations we conclude that in H. polymorpha de novo peroxisome formation is a rescue mechanism, which is less efficient than organelle fission and inheritance to maintain functional peroxisomes.

Saccharomyces cerevisiae cells lacking Pex3 contain membrane vesicles that harbor a subset of peroxisomal membrane proteins.

Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.

The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission.

The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrate that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Similar observations were made for mammalian Pex11ß and the corresponding DLP Drp1, indicating that DLP activation by Pex11p is conserved. Our work identifies a previously unknown requirement for a GAP in DLP function.

The birth of yeast peroxisomes.

This contribution describes the phenotypic differences of yeast peroxisome-deficient mutants (pex mutants). In some cases different phenotypes were reported for yeast mutants deleted in the same PEX gene. These differences are most likely related to the marker proteins and methods used to detect peroxisomal remnants. This is especially evident for pex3 and pex19 mutants, where the localization of receptor docking proteins (Pex13, Pex14) resulted in the identification of peroxisomal membrane remnants, which do not contain other peroxisomal membrane proteins, such as the ring proteins Pex2, Pex10 and Pex12. These structures in pex3 and pex19 cells are the template for peroxisome formation upon introduction of the missing gene. Taken together, these data suggest that in all yeast pex mutants analyzed so far peroxisomes are not formed de novo but use membrane remnant structures as a template for peroxisome formation upon reintroduction of the missing gene. The relevance of this model for peroxisomal membrane protein and lipid sorting to peroxisomes is discussed.

Stress exposure results in increased peroxisomal levels of yeast Pnc1 and Gpd1, which are imported via a piggy-backing mechanism.

Saccharomyces cerevisiae glycerol phosphate dehydrogenase 1 (Gpd1) and nicotinamidase (Pnc1) are two stress-induced enzymes. Both enzymes are predominantly localised to peroxisomes at normal growth conditions, but were reported to localise to the cytosol and nucleus upon exposure of cells to stress. Import of both proteins into peroxisomes depends on the peroxisomal targeting signal 2 (PTS2) receptor Pex7. Gpd1 contains a PTS2, however, Pnc1 lacks this sequence. Here we show that Pnc1 physically interacts with Gpd1, which is required for piggy-back import of Pnc1 into peroxisomes. Quantitative fluorescence microscopy analyses revealed that the levels of both proteins increased in peroxisomes and in the cytosol upon exposure of cells to stress. However, upon exposure of cells to stress we also observed enhanced cytosolic levels of the control PTS2 protein thiolase, when produced under control of the GPD1 promoter. This suggests that these conditions cause a partial defect in PTS2 protein import, probably because the PTS2 import pathway is easily saturated.

Impaired biosynthesis of the non-bilayer lipids phosphatidylethanolamine or cardiolipin does not affect peroxisome biogenesis and proliferation in Saccharomyces cerevisiae.

The non-bilayer forming lipids cardiolipin (CL) and phosphatidylethanolamine (PE) modulate membrane curvature, facilitate membrane fusion and affect the stability and function of membrane proteins. Yeast peroxisomal membranes contain significant amounts of CL and PE. We analysed the effect of CL deficiency and PE depletion on peroxisome biogenesis and proliferation in Saccharomyces cerevisiae. Our data indicate that deletion of CRD1, which encodes cardiolipin synthase, does not affect peroxisome biogenesis or abundance, both at peroxisome repressing (glucose) or inducing (oleate) growth conditions. Analysis of strains deficient in one of the three PE biosynthesis pathways (psd1, psd2 or the triple deletion strain eki1 cki1 dpl1) revealed that in all three strains peroxisome numbers were reduced upon growth of cells on oleic acid, whereas the psd1 strain also showed a reduction in peroxisome abundance upon growth on glucose. Because PE is an intermediate of the phosphatidylcholine (PC) biosynthesis pathway, PE depletion affects PC formation. PC however can be synthesized by an alternative pathway when choline is supplemented to the growth medium. Because the addition of choline resulted in suppression of the peroxisome phenotypes in phosphatidylserine decarboxylase mutant strains, we conclude that peroxisome biogenesis and proliferation are not crucially dependent on CL or PE.

Membrane curvature during peroxisome fission requires Pex11.

Pex11 is a key player in peroxisome proliferation, but the molecular mechanisms of its function are still unknown. Here, we show that Pex11 contains a conserved sequence at the N-terminus that can adopt the structure of an amphipathic helix. Using Penicillium chrysogenum Pex11, we show that this amphipathic helix, termed Pex11-Amph, associates with liposomes in vitro. This interaction is especially evident when negatively charged liposomes are used with a phospholipid content resembling that of peroxisomal membranes. Binding of Pex11-Amph to negatively charged membrane vesicles resulted in strong tubulation. This tubulation of vesicles was also observed when the entire soluble N-terminal domain of Pex11 was used. Using mutant peptides, we demonstrate that maintaining the amphipathic properties of Pex11-Amph in conjunction with retaining its α-helical structure are crucial for its function. We show that the membrane remodelling capacity of the amphipathic helix in Pex11 is conserved from yeast to man. Finally, we demonstrate that mutations abolishing the membrane remodelling activity of the Pex11-Amph domain also hamper the function of full-length Pex11 in peroxisome fission in vivo.

Pex19p contributes to peroxisome inheritance in the association of peroxisomes to Myo2p.

During budding of yeast cells peroxisomes are distributed over mother cell and bud, a process that involves the myosin motor protein Myo2p and the peroxisomal membrane protein Inp2p. Here, we show that Pex19p, a peroxin implicated in targeting and complex formation of peroxisomal membrane proteins, also plays a role in peroxisome partitioning. Binding studies revealed that Pex19p interacts with the cargo-binding domain of Myo2p. We identified mutations in Myo2p that specifically reduced binding to Pex19p, but not to Inp2p. The interaction between Myo2p and Pex19p was also reduced by a mutation that blocked Pex19p farnesylation. Microscopy revealed that the Pex19p-Myo2p interaction is important for peroxisome inheritance, because mutations that affect this interaction hamper peroxisome inheritance in vivo. Together these data suggest that both Inp2p and Pex19p are required for proper association of peroxisomes to Myo2p.

The versatility of peroxisome function in filamentous fungi.

Peroxisomes are ubiquitous and versatile cell organelles. They consist of a single membrane that encloses a proteinaceous matrix. Conserved functions are fatty acid ß-oxidation and hydrogen peroxide metabolism. In filamentous fungi, many other metabolic functions have been identified. Also, they contain highly specialized peroxisome-derived structures termed Woronin bodies, which have a structural function in plugging septal pores in order to prevent cytoplasmic bleeding of damaged hyphae.In filamentous fungi peroxisomes play key roles in the production of a range of secondary metabolites such as antibiotics. Most likely the atlas of fungal peroxisomal metabolic pathways is still far from complete. Relative recently discovered functions include their role in biotin biosynthesis as well as in the production of several toxins, among which polyketides. Finally, in filamentous fungi peroxisomes are important for development and pathogenesis.In this contribution we present an overview of our current knowledge on fungal peroxisome formation as well as on their functional diversity.

Hansenula polymorpha cells lacking the ER-localized peroxins Pex23 or Pex29 show defects in mitochondrial function and morphology.

Pex23 family proteins localize to the endoplasmic reticulum and play a role in peroxisome and lipid body formation. The yeast Hansenula polymorpha contains four members: Pex23, Pex24, Pex29 and Pex32. We previously showed that loss of Pex24 or Pex32 results in severe peroxisomal defects, caused by reduced peroxisome-endoplasmic reticulum contact sites. We now analyzed the effect of the absence of all four Pex23 family proteins on other cell organelles. Vacuoles were normal in all four deletion strains. The number of lipid droplets was reduced in pex23 and pex29, but not in pex24 and pex32 cells, indicating that peroxisome and lipid droplet formation require different Pex23 family proteins in H. polymorpha. In pex23 and pex29 cells mitochondria were fragmented and clustered accompanied by reduced levels of the fusion protein Fzo1. Deletion of DNM1 suppressed the morphological phenotype of pex23 and pex29 cells, suggesting that mitochondrial fusion is affected. pex23 and pex29 cells showed retarded growth and reduced mitochondrial activities. The growth defect was partially suppressed by DNM1 deletion as well as by an artificial mitochondrion-endoplasmic reticulum tether. Hence, the absence of Pex23 family proteins may influence mitochondrion-endoplasmic reticulum contact sites.

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.

Overexpression of PEX14 results in mistargeting to mitochondria, accompanied by organelle fragmentation and clustering in human embryonic kidney cells.

Peroxisome biogenesis disorders are caused by pathogenic variants in genes involved in biogenesis and maintenance of peroxisomes. However, mitochondria are also often affected in these diseases. Peroxisomal membrane proteins, including PEX14, have been found to mislocalise to mitochondria in cells lacking peroxisomes. Recent studies indicated that this mislocalisation contributes to mitochondrial abnormalities in PEX3-deficient patient fibroblasts cells. Here, we studied whether mitochondrial morphology is also affected in PEX3-deficient HEK293 cells and whether PEX14 mislocalises to mitochondria in these cells. Using high-resolution imaging techniques, we show that although endogenous PEX14 mislocalises to mitochondria, mitochondrial morphology was normal in PEX3-KO HEK293 cells. However, we discovered that overexpression of tagged PEX14 in wild-type HEK293 cells resulted in its mitochondrial localisation, accompanied by altered mitochondrial morphology. Our data indicate that overexpression of tagged PEX14 alone directly or indirectly cause mitochondrial abnormalities in cells containing peroxisomes.

Peroxisome fission is associated with reorganization of specific membrane proteins.

Membrane remodeling is an important aspect in organelle biogenesis. We show that different peroxisome membrane proteins that play a role in organelle biogenesis and proliferation (Pex8, Pex10, Pex14, Pex25 and Pex11) are subject to spatiotemporal behavior during organelle development. Using fluorescence microscopy analysis of Hansenula polymorpha dnm1 cells that are blocked in the normal fission process, we show that green fluorescent protein (GFP) fusions of Pex8, Pex10, Pex14 and Pex25 show enhanced fluorescence at the organelle extensions that are formed in budding cells. In contrast, Pex11 fluorescence is enriched at the base of this extension on the mother organelle. A fusion protein of GFP with the transporter Pmp47, used as a control, did not show enhanced fluorescence at any specific region of the organelle. The concentration of specific peroxins at the peroxisome surface was lost upon deletion of PEX11 or the N-terminal domain of Pex11 that is involved in membrane remodeling. Comparable distribution patterns as in dnm1 cells were observed in wild-type cells where Pex8, Pex10, Pex14 and Pex25, but not Pex11, were especially present at newly formed organelles that migrated to the bud. We speculate that peroxin reorganization events result in enhanced levels of peroxins involved in peroxisome biogenesis in nascent organelles.

Peroxisomal proteostasis involves a Lon family protein that functions as protease and chaperone.

Proteins are subject to continuous quality control for optimal proteostasis. The knowledge of peroxisome quality control systems is still in its infancy. Here we show that peroxisomes contain a member of the Lon family of proteases (Pln). We show that Pln is a heptameric protein and acts as an ATP-fueled protease and chaperone. Hence, Pln is the first chaperone identified in fungal peroxisomes. In cells of a PLN deletion strain peroxisomes contain protein aggregates, a major component of which is catalase-peroxidase. We show that this enzyme is sensitive to oxidative damage. The oxidatively damaged, but not the native protein, is a substrate of the Pln protease. Cells of the pln strain contain enhanced levels of catalase-peroxidase protein but reduced catalase-peroxidase enzyme activities. Together with the observation that Pln has chaperone activity in vitro, our data suggest that catalase-peroxidase aggregates accumulate in peroxisomes of pln cells due to the combined absence of Pln protease and chaperone activities.

The relevance of the non-canonical PTS1 of peroxisomal catalase.

Catalase is sorted to peroxisomes via a C-terminal peroxisomal targeting signal 1 (PTS1), which binds to the receptor protein Pex5. Analysis of the C-terminal sequences of peroxisomal catalases from various species indicated that catalase never contains the typical C-terminal PTS1 tripeptide-SKL, but invariably is sorted to peroxisomes via a non-canonical sorting sequence. We analyzed the relevance of the non-canonical PTS1 of catalase of the yeast Hansenula polymorpha (-SKI). Using isothermal titration microcalorimetry, we show that the affinity of H. polymorpha Pex5 for a peptide containing -SKI at the C-terminus is 8-fold lower relative to a peptide that has a C-terminal -SKL. Fluorescence microscopy indicated that green fluorescent protein containing the -SKI tripeptide (GFP-SKI) has a prolonged residence time in the cytosol compared to GFP containing -SKL. Replacing the -SKI sequence of catalase into -SKL resulted in reduced levels of enzymatically active catalase in whole cell lysates together with the occurrence of catalase protein aggregates in the peroxisomal matrix. Moreover, the cultures showed a reduced growth yield in methanol-limited chemostats. Finally, we show that a mutant catalase variant that is unable to properly fold mislocalizes in protein aggregates in the cytosol. However, by replacing the PTS1 into -SKL the mutant variant accumulates in protein aggregates inside peroxisomes. Based on our findings we propose that the relatively weak PTS1 of catalase is important to allow proper folding of the enzyme prior to import into peroxisomes, thereby preventing the accumulation of catalase protein aggregates in the organelle matrix.

Pexophagy-linked degradation of the peroxisomal membrane protein Pex3p involves the ubiquitin-proteasome system.

Peroxisome autophagy, also known as pexophagy, describes the wholesale degradation of peroxisomes via the vacuole, when organelles become damaged or redundant. In the methylotrophic yeast Hansenula polymorpha, pexophagy is stimulated when cells growing on methanol are exposed to excess glucose. Degradation of the peroxisomal membrane protein Pex3p, a process that does not involve the vacuole, was shown to trigger pexophagy. In this contribution, we have characterised pexophagy-associated Pex3p degradation further. We show that Pex3p breakdown depends on ubiquitin and confirm that Pex3p is a target for ubiquitination. Furthermore, we identify a role for the peroxisomal E3 ligases Pex2p and Pex10p in Pex3p degradation, suggesting the existence of a ubiquitin-dependent pathway involved in removing proteins from the peroxisomal membrane.