Search

It was recently shown that the nematode-infecting fungus

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. This article is part of a Special Issue entitled: Peroxisomes.

Contrary to earlier findings, preperoxisomal membrane structures form in yeast cells lacking the peroxin Pex3 and are competent to mature into functional peroxisomes upon Pex3 reintroduction., We demonstrate that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells. Notably, pex3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex—Pex13 and Pex14—as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in pex3 cells. Instead, we find that reintroduced Pex3 sorts to the preperoxisomal structures in pex3 cells, after which these structures mature into normal peroxisomes.

We demonstrated that in the yeast Hansenula polymorpha peroxisome fission and degradation are coupled processes that are important to remove intra-organellar protein aggregates. Protein aggregates were formed in peroxisomes upon synthesis of a mutant catalase variant. We showed that the introduction of these aggregates in the peroxisomal lumen had physiological disadvantages as it affected growth and caused enhanced levels of reactive oxygen species. Formation of the protein aggregates was followed by asymmetric peroxisome fission to separate the aggregate from the mother organelle. Subsequently, these small, protein aggregate-containing organelles were degraded by autophagy. In line with this observation we showed that the degradation of the protein aggregates was strongly reduced in dnm1 and pex11 cells in which peroxisome fission is reduced. Moreover, this process was dependent on Atg1 and Atg11.

Genetic engineering of fungal cell factories mainly focuses on manipulating enzymes of the product pathway or primary metabolism. However, despite the use of strong promoters or strains containing the genes of interest in multiple copies, the desired strongly enhanced enzyme levels are often not obtained.Here we present a novel strategy to improve penicillin biosynthesis by Penicillium chrysogenum by reducing reactive and toxic metabolic by-products, 2-oxoaldehydes. This was achieved by overexpressing the genes encoding glyoxalase I and II, which resulted in a 10% increase in penicillin titers relative to the control strain.The protein levels of two key enzymes of penicillin biosynthesis, isopenicillin N synthase and isopenicillin N acyltransferase, were increased in the glyoxalase transformants, whereas their transcript levels remained unaltered. These results suggest that directed intracellular reduction of 2-oxoaldehydes prolongs the functional lifetime of these enzymes. (C) 2013 Elsevier Inc. All rights reserved.

Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.

The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture. (C) 2012 Elsevier Ltd. All rights reserved.

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.

Hansenula polymorpha is an important yeast in industrial biotechnology. In addition, it is extensively used in fundamental research devoted to unravel the principles of peroxisome biology and nitrate assimilation. Here we present an overview of key components of the genetic toolbox for H. polymorpha. In addition, we present new selection markers that we recently implemented in H. polymorpha. We describe novel strategies for the efficient creation of targeted gene deletions and integrations in H. polymorpha. For this, we generated a similar to 80 mutant, deficient in non-homologous end joining, resulting in strongly enhanced efficiency of gene targeting relative to the parental strain. Finally, we show the implementation of Gateway technology and a single-step PCR strategy to create deletions in H. polymorpha.

Evidence is accumulating that damaged components of eukaryotic cells are removed by autophagic degradation (e. g., mitophagy). Here we show that peroxisomes that are damaged by the abrupt removal of the membrane protein Pex3 are massively and rapidly degraded even when the cells are placed at peroxisome-inducing conditions and hence need the organelles for growth. Pex3 degradation was induced by a temperature shift using Hansenula polymorpha pex3 Delta cells producing a Pex3 fusion protein containing an N-terminal temperature sensitive degron sequence. The massive peroxisome degradation process, associated with Pex3 degradation, showed properties of both micro-and macropexophagy and was dependent on Atg1 and Ypt7. This mode of peroxisome degradation is of physiological significance as it was also observed at conditions that excessive ROS is formed from peroxisome metabolism, i.e., when methanol-grown wild-type cells are exposed to methanol excess conditions.

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.

Peroxisomes are ubiquitous organelles surrounded by a single membrane that display a variety of metabolic functions. These vary with the organism in which they occur and with environmental conditions. Peroxisomes multiply by division of existing organelles and can be formed from ER.The peroxisomal membrane, akin to the organelle itself, is a very dynamic structure that obtains building blocks from the ER. It can form diverse organized structures - lipid domains - that can be involved in regulation of various vesicle fusion processes. Additionally, this membrane may undergo extensive changes in lipid composition. We recently showed that upon proliferation the peroxisomal membrane changes its curvature in response to the activity of the peroxisomal membrane protein Pex11. Tubulation of the organelle may be important for efficient recruitment of GTPases from the dynamin protein family that is involved in organelle fission. (C) 2011 Elsevier Ltd. All rights reserved.

We have investigated the significance of autophagy in the production of the β-lactam antibiotic penicillin (PEN) by the filamentous fungus Penicillium chrysogenum . In this fungus PEN production is compartmentalized in the cytosol and in peroxisomes. We demonstrate that under PEN-producing conditions significant amounts of cytosolic and peroxisomal proteins are degraded via autophagy. Morphological analysis, based on electron and fluorescence microscopy, revealed that this phenomenon might contribute to progressive deterioration of late subapical cells. We show that deletion of the P. chrysogenum ortholog of Saccharomyces cerevisiae serine-threonine kinase atg1 results in impairment of autophagy. In P. chrysogenum atg1 cells, a distinct delay in cell degeneration is observed relative to wild-type cells. This phenomenon is associated with an increase in the enzyme levels of the PEN biosynthetic pathway and enhanced production levels of this antibacterial compound.

De novo peroxisome formation in peroxisome-deficient yeast cells requires Rho1 and the Pex11 family protein Pex25., We identified two proteins, Pex25 and Rho1, which are involved in reintroduction of peroxisomes in peroxisome-deficient yeast cells. These are, together with Pex3, the first proteins identified as essential for this process. Of the three members of the Hansenula polymorpha Pex11 protein family—Pex11, Pex25, and Pex11C—only Pex25 was required for reintroduction of peroxisomes into a peroxisome-deficient mutant strain. In peroxisome-deficient pex3 cells, Pex25 localized to structures adjacent to the ER, whereas in wild-type cells it localized to peroxisomes. Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes. Interestingly, pex11 pex25 double deletion cells, in which both peroxisome fission (due to the deletion of PEX11) and reintroduction (due to deletion of PEX25) was blocked, did display a peroxisome-deficient phenotype. Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11. Reintroduction in the presence of Pex25 required the function of the GTPase Rho1. These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.

We describe an alternative peroxisome formation pathway in yeast pex3 and pex19 cells, which relies on the existence of small peroxisomal remnants that are present in these cells. This groundbreaking result challenges current models prescribing that peroxisomes derive de novo from the ER. Our data also has major implications for the sorting pathway of specific peroxisomal membrane proteins (PMPs). We propose a novel sorting pathway for the PMPs Pex13 and Pex14 that is independent of the known Pex3/Pex19 machinery.

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.

d-Amino acid oxidase (DAO) is an important flavo-enzyme that catalyzes the oxidative deamination of d-amino acids into the corresponding alpha-keto acid, ammonia and H(2)O(2). We identified two amino acid oxidases in the methylotrophic yeast Pichia pastoris: Dao1p, which preferentially uses d-alanine as a substrate, and Dao2p, which uses d-aspartate as a preferred substrate. Dao1p has a molecular mass of 38.2 kDa and a pH optimum of 9.6. This enzyme was localized to peroxisomes, albeit a typical peroxisomal targeting signal is lacking. Interestingly, P. pastoris mutant strains, defective in the enzyme pyruvate carboxylase, showed a pronounced growth defect on d-alanine, concomitant with a significant reduction in Dao1p activity relative to the wild-type control. This indicates that pyruvate carboxylase functions in import and/or activation of P. pastoris Dao1p.

Peroxisomes are cell organelles that are present in almost all eukaryotic cells and involved in a large range of metabolic pathways. The organelles are highly dynamic in nature: their number and enzyme content is highly variable and continuously adapts to prevailing environmental conditions. This review summarizes recent relevant developments in research on processes that are involved in the regulation of peroxisome abundance and maintenance. These processes include fission of the organelles, formation of new peroxisomes from the endoplasmic reticulum, autophagic degradation and segregation/inheritance during cell division.

By genome analysis, we previously identified Pex14/17p as a putative novel peroxin of Penicillium chrysogenum. Here, we show that Pex14/17p is a component of the peroxisomal membrane that is essential for efficient peroxisomal targeting signal 1 and peroxisomal targeting signal 2 matrix protein import, implying that the protein is indeed a genuine peroxin. Additionally, a PEX14/17 deletion strain is affected in conidiospore formation. Pex14/17p has properties of both Pex14p and Pex17p, in that the N-terminus of this protein is similar to the highly conserved Pex5p-binding region present in the N-termini of Pex14p proteins, whereas its C-terminus shows weak similarity to yeast Pex17p proteins. We have identified a novel motif in both Pex17p and Pex14/17p that is absent in Pex14p. We show that an N-terminally truncated, but not a C-terminally truncated, Pex14/17p is able to complement both the matrix protein import and sporulation defects of a Delta pex14/17 strain, implying that it is the Pex17p-related portion of the protein that is crucial for its function as a peroxin. Possibly, this compensates for the fact that P. chrysogenum lacks an authenthic Pex17p. We also show that, in P. chrysogenum, Pex14/17p plays a role in making the penicillin biosynthesis process more efficient.

Peroxisomes are unique organelles which display properties of autonomous organelles, as they can multiply by fission of pre-existing ones. Peroxisomes, however, can also develop from the endoplasmic reticulum (ER). This process has convincingly been shown in peroxisome-deficient yeast cells, upon reintroduction of the corresponding gene. Whether peroxisomes also are formed from the ER in wild-type cells that contain peroxisomes is still under debate. Also, the existence of vesicular transport pathways between peroxisomes and the ER is still unresolved. Several new proteins and pathways that play a role in peroxisome proliferation have been identified in the last few years. A surprising finding was that proteins well known for their function in mitochondrial fission (Fis1, Dnm1) are responsible for peroxisome fission as well. In this contribution we discuss recent advancements in research on peroxisome proliferation.

We have cloned and characterized the Hansenula polymorpha PEX11 gene. Our morphological data are consistent with previous observations that peroxisome proliferation can be regulated by modulating Pex11p levels. Surprisingly, pex11 cells also showed a defect in peroxisome retention in mother cells during vegetative cell reproduction. Until now, Saccharomyces cerevisiae Inp1p has been the only peroxisomal protein that has been shown to play a role in the organelle retention process. H. polymorpha inp1 cells are also affected in peroxisome retention, like pex11 cells. We show by time-lapse imaging that Inp1-green fluorescent protein localization varies during the cell cycle and that the protein is normally recruited to peroxisomes in pex11 cells. Taken together, our data show that H. polymorpha Pex11p is not only important for peroxisome proliferation but is also required for proper peroxisome segregation during cell division.

Degradation processes are important for optimal functioning of eukaryotic cells. The two major protein degradation pathways in eukaryotes are the ubiquitin-proteasome pathway and autophagy. This contribution focuses on autophagy. This process is important for survival of cells during nitrogen starvation conditions but also has a house keeping function in removing exhausted, redundant or unwanted cellular components. We present an overview of the molecular mechanism involved in three major autophagy pathways: chaperone mediated autophagy, microautophagy and macroautophagy. Various recent reports indicate that autophagy plays a crucial role in human health and disease. Examples are presented of lysosomal storage diseases and the role of autophagy in cancer, neurodegenerative diseases, defense against pathogens and cell death. (C) 2008 Elsevier B.V. All rights reserved.

We analyzed the role of the peroxisomal peroxiredoxin Pmp20 of the yeast Hansenula polymorpha. Cells of a PMP20 disruption strain (pmp20) grew normally on substrates that are not metabolized by peroxisomal enzymes, but showed a severe growth defect on methanol, the metabolism of which involves a hydrogen peroxide producing peroxisomal oxidase. This growth defect was paralleled by leakage of peroxisomal matrix proteins into the cytosol. Methanol-induced pmp20 cells accumulated enhanced levels of reactive oxygen species and lipid peroxidation products. Moreover, the fatty acid composition of methanol-induced pmp20 cells differed relative to WT controls, suggesting an effect on fatty acid homeostasis. Plating assays and FACS-based analysis of cell death markers revealed that pmp20 cells show loss of clonogenic efficiency and membrane integrity, when cultured on methanol. We conclude that the absence of the peroxisomal peroxiredoxin leads to loss of peroxisome membrane integrity and necrotic cell death. (C) 2008 Elsevier Inc. All Fights reserved

We show that Mdv1 and Caf4, two components of the mitochondrial fission machinery in Saccharomyces cerevisiae, also function in peroxisome proliferation. Deletion of MDV1, CAF4 or both, however, had only a minor effect on peroxisome numbers at peroxisome-inducing growth conditions, most likely related to the fact that Vps1--and not Dnm1--is the key player in peroxisome fission in this organism. In contrast, in Hansenula polymorpha, which has only a Dnm1-dependent peroxisome fission machinery, deletion of MDV1 led to a drastic reduction of peroxisome numbers. This phenotype was accompanied by a strong defect in mitochondrial fission. The MDV1 paralog CAF4 is absent in H. polymorpha. In wild-type H. polymorpha, cells Dnm1-mCherry and green fluorescent protein (GFP)-Mdv1 colocalize in spots that associate with both peroxisomes and mitochondria. Furthermore, Fis1 is essential to recruit Mdv1 to the peroxisomal and mitochondrial membrane. However, formation of GFP-Mdv1 spots--and related to this normal organelle fission--is strictly dependent on the presence of Dnm1. In dnm1 cells, GFP-Mdv1 is dispersed over the surface of peroxisomes and mitochondria. Also, in H. polymorpha mdv1 or fis1 cells, the number of Dnm1-GFP spots is strongly reduced. These spots still associate to organelles but are functionally inactive.

Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT(C103S)) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein. Co-expression of the penDE(C103S) and wild-type penDE genes in P. chrysogenum (Wis54-DE(C103S) strain) led to a decrease in benzylpenicillin levels. Changes in the wild-type IAT processing profile (P subunit formation) were observed in the Wis54-DE(C103S) strain, suggesting a regulatory role of the unprocessed IAT(C103S) in the processing of the wild-type IAT. This was confirmed in Escherichia coli, where a delay in the processing of IAT in presence of the unprocessable IAT(C103S) was observed. Our results indicate that IAT is post-translationally regulated by its preprotein, which interferes with the self-processing. (c) 2008 Elsevier Inc. All rights reserved.

We demonstrated that formaldehyde can be efficiently coutilized by an engineered Saccharomyces cerevisiae strain that expresses Hansenula polymorpha genes encoding formaldehyde dehydrogenase ( FLD1 ) and formate dehydrogenase ( FMD ), in contrast to wild-type strains. Initial chemostat experiments showed that the engineered strain coutilized formaldehyde with glucose, but these mixed-substrate cultures failed to reach steady-state conditions and did not exhibit an increased biomass yield on glucose. Subsequent transcriptome analyses of chemostat cultures of the engineered strain, grown on glucose-formaldehyde mixtures, indicated that the presence of formaldehyde in the feed caused biotin limitations. Further transcriptome analysis demonstrated that this biotin inactivation was prevented by using separate formaldehyde and vitamin feeds. Using this approach, steady-state glucose-limited chemostat cultures were obtained that coutilized glucose and formaldehyde. Coutilization of formaldehyde under these conditions resulted in an enhanced biomass yield of the glucose-limited cultures. The biomass yield was quantitatively consistent with the use of formaldehyde as an auxiliary substrate that generates NADH and subsequently, via oxidative phosphorylation, ATP. On an electron pair basis, the biomass yield increase observed with formaldehyde was larger than that observed previously for formate, which is tentatively explained by different modes of formate and formaldehyde transport in S. cerevisiae .

We show that the dynamin-like proteins Dnm1p and Vps1p are not required for re-introduction of peroxisomes in Hansenula polymorpha pex3 cells upon complementation with PEX3-GFP. Instead, Dnm1p, but not Vps1p, plays a crucial role in organelle proliferation via fission. In H. polymorpha DNM1 deletion cells (dnm1) a single peroxisome is present that forms long extensions, which protrude into developing buds and divide during cytokinesis. Budding pex11.dnm1 double deletion cells lack these peroxisomal extensions, suggesting that the peroxisomal membrane protein Pex11p is required for their formation. Life cell imaging revealed that fluorescent Dnm1p-GFP spots fluctuate between peroxisomes and mitochondria. On the other hand Pex11p is present over the entire organelle surface, but concentrates during fission at the basis of the organelle extension in dnm1 cells. Our data indicate that peroxisome fission is the major pathway for peroxisome multiplication in H. polymorpha. (C) 2007 Elsevier B.V. All rights reserved.

Pex14 was initially identified as a peroxisomal membrane protein that is involved in docking of the soluble receptor proteins Pex5 and Pex7, which are required for import of PTS1- or PTS2-containing peroxisomal matrix proteins. However, Hansenula polymorpha Pex14 is also required for selective degradation of peroxisomes (pexophagy). Previously we showed that Pex1, Pex4, Pex6 and Pex8 are not required for this process. Here we show that also in the absence of various other peroxins, namely Pex2, Pex10, Pex12, Pex13 and Pex17, pexophagy can normally occur. These peroxins are, like Pex14, components of the peroxisomal translocon. Our data confirm that Pex14 is the sole peroxin that has a unique dual function in two apparent opposite processes, namely peroxisome formation and selective degradation.

We have analyzed the highly conserved N-terminus of Hansenula polymorpha Pex14p for its function in peroxisomal matrix protein import. The region comprising aa 10-54 of HpPex14p is predicted to contain three alpha-helices. Its alpha-helical structure was confirmed by CD analysis of a synthetic peptide, corresponding to residues 8-58. Deletion of aa 1-21 of HpPex14p, but not of aa 1-9, completely abolished PTS1 and PTS2 matrix protein import. An extensive mutational analysis of the first a-helix (aa 10-21) demonstrated that its secondary structure, as well as residues Phe20 and Leu21, are essential for PTS1 and PTS2 matrix protein import. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

We report on the rerouting of peroxisomal alcohol oxidase (AO) to the secretory pathway of Hansenula polymorpha. Using the leader sequence of the Saccharomyces cerevisiae mating factor alpha (MF alpha) as sorting signal, AO was correctly sorted to the endoplasmic reticulum (ER), which strongly proliferated in these cells. The MF alpha presequence, but not the prosequence, was cleaved from the protein. AO protein was present in the ER as monomers that lacked FAD, and hence was enzymatically inactive. Furthermore, the recombinant AO protein was subject to gradual degradation, possibly because the protein did not fold properly. However, when the S. cerevisiae invertase signal sequence (ISS) was used, secretion of AO protein was observed in conjunction with bulk of the protein being localized to the ER. The amount of secreted AO protein increased with increasing copy numbers of the AO expression cassette integrated into the genome. The secreted AO protein was correctly processed and displayed enzyme activity.

We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host-vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine beta-lyase. With this new host-vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein-PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL-SKL produced in H. polymorpha displays normal enzymatic activities.

ATG genes encode proteins that are required for macroautophagy, the Cvt pathway and/or pexophagy. Using the published Atg protein sequences, we have screened protein and DNA databases to identify putative functional homologs (orthologs) in 21 fungal species (yeast and filamentous fungi) of which the genome sequences were available. For comparison with Atg proteins in higher eukaryotes, also an analysis of Arabidopsis thaliana and Homo sapiens databases was included. This analysis demonstrated that Atg proteins required for non-selective macroautophagy are conserved from yeast to man, stressing the importance of this process in cell survival and viability. The A. thaliana and human genomes encode multiple proteins highly similar to specific fungal Atg proteins (paralogs), possibly representing cell type-specific isoforms. The Atg proteins specifically involved in the Cvt pathway and/or pexophagy showed poor conservation, and were generally not present in A. thaliana and man. Furthermore, Atg19, the receptor of Cvt cargo, was only detected in Saccharomyces cerevisiae. Nevertheless, Atg11, a protein that links receptor-bound cargo (peroxisomes, the Cvt complex) to the autophagic machinery was identified in all yeast species and filamentous fungi under study. This suggests that in fungi an organism-specific form of selective autophagy may occur, for which specialized Atg proteins have evolved.

SummaryThe mitochondrial contact site and cristae organizing system (MICOS) is a conserved multi-subunit complex crucial for maintaining the characteristic architecture of mitochondria. Studies with deletion mutants identified Mic10 and Mic60 as core subunits of MICOS. Mic60 has been studied in detail; however, topogenesis and function of Mic10 are unknown. We report that targeting of Mic10 to the mitochondrial inner membrane requires a positively charged internal loop, but no cleavable presequence. Both transmembrane segments of Mic10 carry a characteristic four-glycine motif, which has been found in the ring-forming rotor subunit of F1Fo-ATP synthases. Overexpression of Mic10 profoundly alters the architecture of the inner membrane independently of other MICOS components. The four-glycine motifs are dispensable for interaction of Mic10 with other MICOS subunits but are crucial for the formation of large Mic10 oligomers. Our studies identify a unique role of Mic10 oligomers in promoting the formation of inner membrane crista junctions.

The capacity to use methanol as sole source of carbon and energy is restricted to relatively few yeast species. This may be related to the low efficiency of methanol metabolism in yeast, relative to that of prokaryotes. This contribution describes the details of methanol metabolism in yeast and focuses on the significance of compartmentalization of this metabolic pathway in peroxisomes.

Yeast and filamentous fungi are important model organisms in microbody research. The value of these organisms as models for higher eukaryotes is underscored by the observation that the principles of various aspects of microbody biology are strongly conserved from lower to higher eukaryotes. This has allowed to resolve various peroxisome-related functions, including peroxisome biogenesis disorders in man. This paper summarizes the major advances in microbody research using fungal systems and specifies specific properties and advantages/disadvantages of the major model organisms currently in use.

We showed before that the two oppositely directed processes of peroxisome biogenesis and selective peroxisome degradation (macropexophagy) converge at the peroxisomal membrane protein Pex14p. Here we show that this protein is not required for peroxisomal degradation during nitrogen starvation-induced general autophagy, thereby limiting its function to the selective degradation process. Pex14p is present in two forms, namely an unmodified (Pex14p) and a phosphorylated form (Pex14pPi) that are differently induced during peroxisome proliferation. The data suggest that Pex14p is required for peroxisome biogenesis during organelle proliferation and Pex14pPi in macropexophagy. Finally, we show that macropexophagy is not coupled to normal peroxisome assembly and is required in only catalytic amounts to allow initiation of the selective peroxisome degradation process.

The reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p was examined. Using a Pex3-green fluorescent protein (Pex3-GFP) fusion protein, expressed under the control of an inducible promoter, it was observed that, initially on induction of Pex3-GFP synthesis, GFP fluorescence was localized to the endoplasmic reticulum and the nuclear envelope. Subsequently, a single organelle developed per cell that increased in size and multiplied by division. At these stages, GFP fluorescence was confined to peroxisomes. Fractionation experiments on homogenates of pex3 cells, in which the endoplasmic reticulum and nuclear envelope were marked with GFP, identified a small amount of GFP in peroxisomes present in the initial stage of peroxisome reassembly. Our data suggest a crucial role for the endoplasmic reticulum/nuclear envelope in peroxisome reintroduction on complementation of pex3 cells by the PEX3 gene.

The peroxin Pex19p is important for the formation of functional peroxisomal membranes. Here we show that Hansenula polymorpha Pex19p is also required for peroxisome inheritance. Peroxisome inheritance is partly defective when Pex19p farnesylation is blocked, whereas deletion of PEX19 resulted in a severe defect in partitioning of peroxisomal structures. Time lapse imaging revealed that in newly formed buds, which had not inherited a peroxisome from the mother cell, new peroxisomes are formed that derive from the nuclear envelope/endoplasmic reticulum. This process was impaired upon deletion of EMP24 and ERP3, genes that encode p24 proteins. p24 Proteins are components of coated vesicles that mediate trafficking between the endoplasmic reticulum and Golgi apparatus. In an H. polymorpha wild-type background, deletion of EMP24 and ERP3 resulted in a strong reduction of organelle number in conjunction with an increase in the size of individual peroxisomes. This observation suggests that p24 proteins also play a role in peroxisome development in wild-type H. polymorpha cells.

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G0 state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.

Yeasts combine the ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. Yeasts thus provide attractive platforms for the production of recombinant proteins. Here, four important species are presented and compared: the methylotrophic Hansenula polymorpha and Pichia pastoris, distinguished by an increasingly large track record as industrial platforms, and the dimorphic species Arxula adeninivorans and Yarrrowia lipolytica, not yet established as industrial platforms, but demonstrating promising technological potential, as discussed in this article.

The enzyme acetyl-CoA:isopenicillin N acyltransferase (IAT) is a peroxisomal enzyme that mediates the final step of penicillin biosynthesis in the filamentous fungi Penicillium chrysogenum and Aspergillus nidulans. However, the precise role of peroxisomes in penicillin biosynthesis is still not clear. To be able to use the power of yeast genetics to solve the function of peroxisomes in penicillin biosynthesis, we introduced IAT in the yeast Hansenula polymorpha. To this purpose, the P. chrysogenum penDE gene, encoding IAT, was amplified from a cDNA library to eliminate the three introns and introduced in H. polymorpha. In this organism IAT protein was produced as a 40 kDa pre-protein and, as in P. chrysogenum, processed into an 11 and 29 kDa subunit, although the efficiency of processing seemed to be slightly reduced relative to P. chrysogenum. The P. chrysogenum IAT, produced in H. polymorpha, is normally localized in peroxisomes and in cell-free extracts IAT activity could be detected. This is a first step towards the introduction of the penicillin biosynthesis pathway in H. polymorpha.

We have analyzed the functions of two vacuolar t-SNAREs, Vam3p and Vam7p, in peroxisome degradation in the methylotrophic yeast Hansenula polymorpha. A Hp-vam7 mutant was strongly affected in peroxisome degradation by selective macropexophagy as well as non-selective microautophagy. Deletion of Hp-Vam3p function had only a minor effect on peroxisome degradation processes. Both proteins were located at the vacuolar membrane, with Hp-Vam7p also having a partially cytosolic location. Previously, in baker's yeast Vam3p and Vam7p have been demonstrated to be components of a t-SNARE complex essential for vacuole biogenesis. We speculate that the function of this complex in macropexophagy includes a role in membrane fusion processes between the outer membrane layer of sequestered peroxisomes and the vacuolar membrane. Our data suggest that Hp-Vam3p may be functionally redundant in peroxisome degradation. Remarkably, deletion of Hp-VAM7 also significantly affected peroxisome biogenesis and resulted in organelles with multiple, membrane-enclosed compartments. These morphological defects became first visible in cells that were in the mid-exponential growth phase of cultivation on methanol, and were correlated with accumulation of electron-dense extensions that were connected to mitochondria.

Current industrial production of beta-lactam antibiotics, using the filamentous fungus Penicillium chrysogenum, is the result of many years of strain improvement by classical mutagenesis. More efficient production strains showed significant increases in the number and volume fraction of microbodies in their cells, organelles that harbor key enzymes involved in the biosynthesis of beta-lactam antibiotics. We have isolated the P. chrysogenum cDNA encoding Pc-Pex11p, a peroxin that is involved in microbody abundance. We demonstrate that overproduction of Pc-Pex11p in P. chrysogenum results in massive proliferation of tubular-shaped microbodies and a 2- to 2.5-fold increase in the level of penicillin in the culture medium. Notably, Pc-Pex11p-overproduction did not affect the levels of the enzymes of the penicillin biosynthetic pathway. Our results suggest that the stimulating effect of enhanced organelle numbers may reflect an increase in the fluxes of penicillin and/or its precursors across the now much enlarged microbody membrane.

Pichia pastoris and Hansenula polymorpha are methylotrophic yeasts capable of utilizing methanol, as a sole source of carbon and energy. Growth of these yeast species on methanol requires the synthesis of cytosolic and peroxisomal enzymes combined with the proliferation of peroxisomes. Peroxisomes are also abundantly present in the alkane-utilizing yeast Yarrowia lipolytica upon growth of cells on oleic acid. This feature has made these yeast species attractive model systems to dissect the molecular mechanisms controlling peroxisome biogenesis. We have found that upon glucose- or ethanol-induced catabolite inactivation, metabolically superfluous peroxisomes are rapidly and selectively degraded within the vacuole by a process called pexophagy, the selective removal of peroxisomes by autophagy-like processes. Utilizing several genetic screens, we have identified a number of genes that are essential for pexophagy. In this review, we will summarize our current knowledge of the molecular events of pexophagy.

Exposure of Hansenula polymorpha cells, grown in batch cultures on methanol at 37 °C, to a cold treatment (18 °C) is paralleled by a rapid degradation of peroxisomes present in these cells. Remarkably, the events accompanying organelle degradation at 18 °C are similar to those of selective glucose-induced peroxisome degradation in wild-type cells, described before. This observation was strengthened by the finding that cold-induced peroxisome degradation was not observed in mutants impaired in selective peroxisome degradation (Atg− mutants). Biochemical data indicated that the onset of peroxisome degradation was not triggered by the inactivation of peroxisome function due to the fall in temperature. We show that our findings have implications in case of fluorescence microscopy studies that are generally not conducted at physiological temperatures and thus may lead to strong morphological alterations unless proper precautions are taken.

Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.