Biomedical subjects

Jan A K W Kiel

Publications and source records attributed to Jan A K W Kiel.

At least 19 recordsLinked to original sources

PubMed2007

ATG genes involved in non-selective autophagy are conserved from yeast to man, but the selective Cvt and pexophagy pathways also require organism-specific genes.

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.

Amino Acid Sequence↗

PubMed2007

A peroxisomal lon protease and peroxisome degradation by autophagy play key roles in vitality of Hansenula polymorpha cells.

In eukaryote cells various mechanisms exist that are responsible for the removal of non-functional proteins. Here we show that in the yeast Hansenula polymorpha (H. polymorpha) a peroxisomal Lon protease, Pln, plays a role in degradation of unfolded and non-assembled peroxisomal matrix proteins. In addition, we demonstrate that whole peroxisomes are constitutively degraded by autophagy during normal vegetative growth of WT cells. Deletion of both H. polymorpha PLN and ATG1, required for autophagy, resulted in a significant increase in peroxisome numbers, paralleled by a decrease in cell viability relative to WT cells. Also, in these cells and in cells of PLN and ATG1 single deletion strains, the intracellular levels of reactive oxygen species had increased relative to WT controls. The enhanced generation of reactive oxygen species may be related to an uneven distribution of peroxisomal catalase activities in the mutant cells, as demonstrated by cytochemistry. We speculate that in the absence of HpPln or autophagy unfolded and non-assembled peroxisomal matrix proteins accumulate, which can form aggregates and lead to an imbalance in hydrogen peroxide production and degradation in some of the organelles.

Autophagy↗

PubMed2006

Pexophagy: autophagic degradation of peroxisomes.

The abundance of peroxisomes within a cell can rapidly decrease by selective autophagic degradation (also designated pexophagy). Studies in yeast species have shown that at least two modes of peroxisome degradation are employed, namely macropexophagy and micropexophagy. During macropexophagy, peroxisomes are individually sequestered by membranes, thus forming a pexophagosome. This structure fuses with the vacuolar membrane, resulting in exposure of the incorporated peroxisome to vacuolar hydrolases. During micropexophagy, a cluster of peroxisomes is enclosed by vacuolar membrane protrusions and/or segmented vacuoles as well as a newly formed membrane structure, the micropexophagy-specific membrane apparatus (MIPA), which mediates the enclosement of the vacuolar membrane. Subsequently, the engulfed peroxisome cluster is degraded. This review discusses the current state of knowledge of pexophagy with emphasis on studies on methylotrophic yeast species.

Animals↗

PubMed2006

PEX genes in fungal genomes: common, rare or redundant.

PEX genes encode proteins, termed peroxins, that are required for the biogenesis and proliferation of microbodies (peroxisomes). We have screened the available protein and DNA databases to identify putative peroxin orthologs in 17 fungal species (yeast and filamentous fungi) and in humans. This analysis demonstrated that most peroxins are present in all fungi under study. Only Pex16p is absent in most yeast species, with the exception of Yarrowia lipolytica, but this peroxin is present in all filamentous fungi. Furthermore, we found that the Y. lipolytica PEX9 gene, a putative orphan gene, might encode a Pex26p ortholog. In addition, in the genomes of Saccharomyces cerevisiae and Candida glabrata, several PEX genes appear to have been duplicated, exemplified by the presence of paralogs of the peroxins Pex5p and Pex21p, which were absent in other organisms. In all organisms, we observed multiple paralogs of the peroxins involved in organelle proliferation. These proteins belong to two groups of peroxins that we propose to designate the Pex11p and Pex23p families. This redundancy may complicate future studies on peroxisome biogenesis and proliferation in fungal species.

Amino Acid Sequence↗

PubMed2005

Pexophagy: the selective autophagy of peroxisomes.

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.

Autophagy↗

PubMed2005

The Hansenula polymorpha ATG25 gene encodes a novel coiled-coil protein that is required for macropexophagy.

We have isolated the Hansenula polymorpha ATG25 gene, which is required for glucose-induced selective peroxisome degradation by macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein colocalizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). In cells of a constructed ATG25 deletion strain (atg25) peroxisomes are constitutively degraded by nonselective microautophagy, a process that in WT H. polymorpha is only observed at nitrogen limitation conditions. This suggests that nonselective microautophagy is deregulated in H. polymorpha atg25 cells.

Autophagy↗

PubMed2005

Hansenula polymorpha Vam7p is required for macropexophagy.

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.

Molecular Sequence Data↗

PubMed2005

Obstruction of polyubiquitination affects PTS1 peroxisomal matrix protein import.

Pex4p is an ubiquitin-conjugating enzyme that functions at a late stage of peroxisomal matrix protein import. Here we show that in the methylotrophic yeast Hansenula polymorpha production of a mutant form of ubiquitin (Ub(K48R)) has a dramatic effect on PTS1 matrix protein import. This effect was not observed in cells lacking Pex4p, in which the peroxisome biogenesis defect was largely suppressed. These findings provide the first indication that the function of Pex4p in matrix protein import involves polyubiquitination. We also demonstrate that the production of Ub(K48R) in H. polymorpha results in enhanced Pex5p degradation. A similar observation was made in cells in which the PEX4 gene was deleted. We demonstrate that in both strains Pex5p degradation was due to ubiquitination and subsequent degradation by the proteasome. This process appeared to be dependent on a conserved lysine residue in the N-terminus of Pex5p (Lys21) and was prevented in a Pex5p(K21R) mutant. We speculate that the degradation of Pex5p by the proteasome is important to remove receptor molecules that are stuck at a late stage of the Pex5p-mediated protein import pathway.

Amino Acid Sequence↗

PubMed2005

Atg8 is essential for macropexophagy in Hansenula polymorpha.

We have isolated a peroxisome-degradation-deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis. Sequencing revealed that the mutant was affected in the HpATG8 gene. HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogen-limitation induced microautophagy. Fluorescence microscopy revealed that a GFP.Atg8 fusion protein was located close to the vacuole. After induction of macropexophagy, the GFP.Atg8 containing spot extended to engulf an individual peroxisome. In cells of a constructed deletion strain, sequestration of individual organelles was never completed; analysis of series of serial sections revealed that invariably a minor diaphragm-like opening remained. We hypothesize that H. polymorpha Atg8 facilitates sealing of the sequestering membranes during selective peroxisome degradation.

Amino Acid Sequence↗

PubMed2004

Overproduction of a single protein, Pc-Pex11p, results in 2-fold enhanced penicillin production by Penicillium chrysogenum.

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.

Amino Acid Sequence↗

PubMed2004

Atg21p is essential for macropexophagy and microautophagy in the yeast Hansenula polymorpha.

ATG genes are required for autophagy-related processes that transport proteins/organelles destined for proteolytic degradation to the vacuole. Here, we describe the identification and characterisation of the Hansenula polymorpha ATG21 gene. Its gene product Hp-Atg21p, fused to eGFP, had a dual location in the cytosol and in peri-vacuolar dots. We demonstrate that Hp-Atg21p is essential for two separate modes of peroxisome degradation, namely glucose-induced macropexophagy and nitrogen limitation-induced microautophagy. In atg21 cells subjected to macropexophagy conditions, sequestration of peroxisomes tagged for degradation is initiated but fails to complete.

Autophagy↗

PubMed2004

Ubiquitination of the peroxisomal targeting signal type 1 receptor, Pex5p, suggests the presence of a quality control mechanism during peroxisomal matrix protein import.

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.

Fungal Proteins↗

PubMed2004

Microautophagy and macropexophagy may occur simultaneously in Hansenula polymorpha.

We subjected methanol-grown cells of wild type Hansenula polymorpha simultaneously to nitrogen depletion and excess glucose conditions. Both treatments induce the degradation of peroxisomes, either selective (via excess glucose) or non-selective (via nitrogen limitation). Our combined data strongly suggest that both processes occur simultaneously under these conditions. The implications of these findings on studies of autophagy and related transport pathways to the vacuole in yeast are discussed.

Autophagy↗

PubMed2004

Hansenula polymorpha Swi1p and Snf2p are essential for methanol utilisation.

We have cloned the Hansenula polymorpha SWI1 and SNF2 genes by functional complementation of mutants that are defective in methanol utilisation. These genes encode proteins similar to Saccharomyces cerevisiae Swi1p and Snf2p, which are subunits of the SWI/SNF complex. This complex belongs to the family of nucleosome-remodeling complexes that play a role in transcriptional control of gene expression. Analysis of the phenotypes of constructed H. polymorpha SWI1 and SNF2 disruption strains indicated that these genes are not necessary for growth of cells on glucose, sucrose, or various organic nitrogen sources which involve the activity of peroxisomal oxidases. Both disruption strains showed a moderate growth defect on glycerol and ethanol, but were fully blocked in methanol utilisation. In methanol-induced cells of both disruption strains, two peroxisomal enzymes involved in methanol metabolism, alcohol oxidase and dihydroxyacetone synthase, were hardly detectable, whereas in wild-type cells these proteins were present at very high levels. We show that the reduction in alcohol oxidase protein levels in H. polymorpha SWI1 and SNF2 disruption strains is due to strongly reduced expression of the alcohol oxidase gene. The level of Pex5p, the receptor involved in import of alcohol oxidase and dihydroxyacetone synthase into peroxisomes, was also reduced in both disruption strains compared to that in wild-type cells.

Alcohol Oxidoreductases↗

PubMed2004

Hansenula polymorpha Tup1p is important for peroxisome degradation.

In the yeast Hansenula polymorpha peroxisomes are selectively degraded upon a shift of cells from methanol to glucose-containing media. We identified the H. polymorpha TUP1 gene by functional complementation of the peroxisome degradation deficient mutant pdd2-4. Tup1 proteins function in transcriptional repression of specific sets of genes involved in various cellular processes. Our combined data indicate that H. polymorpha TUP1 is involved in regulation of the switch between peroxisome biogenesis and selective degradation. The initial DNA fragment that complemented H. polymorpha pdd2-4 contained a second gene, encoding H. polymorpha Vps4p. Deletion of the VPS4 gene did not affect selective peroxisome degradation.

Amino Acid Sequence↗

PubMed2004

Penicillium chrysogenum Pex5p mediates differential sorting of PTS1 proteins to microbodies of the methylotrophic yeast Hansenula polymorpha.

We have isolated the Penicillium chrysogenum pex5 gene encoding the receptor for microbody matrix proteins containing a type 1 peroxisomal targeting signal (PTS1). Pc-pex5 contains 2 introns and encodes a protein of approximately 75 kDa. P. chrysogenum pex5 disruptants appear to be highly unstable, show poor growth, and are unable to sporulate asexually. Furthermore, pex5 cells mislocalize a fluorescent PTS1 reporter protein to the cytosol. Pc-pex5 was expressed in a PEX5 null mutant of the yeast Hansenula polymorpha. Detailed analysis demonstrated that the PTS1 proteins dihydroxyacetone synthase and catalase were almost fully imported into microbodies. Surprisingly, alcohol oxidase, which also depends on Pex5p for import into microbodies, remained mainly in the cytosol. Thus, P. chrysogenum Pex5p has a different specificity of cargo recognition than its H. polymorpha counterpart. This was also suggested by the observation that Pc-Pex5p sorted a reporter protein fused to various functional PTS1 signals with different efficiencies.

Alcohol Oxidoreductases↗

PubMed2004

Uptake of the beta-lactam precursor alpha-aminoadipic acid in Penicillium chrysogenum is mediated by the acidic and the general amino acid permease.

External addition of the beta-lactam precursor alpha-aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular alpha-aminoadipic acid concentration and an increase in penicillin production. The exact route for alpha-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes, PcGAP1 and PcDIP5, of P. chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of alpha-aminoadipic acid, although PcGap1 showed a higher affinity for alpha-aminoadipic acid than PcDip5 (K(m) values, 230 and 800 microM, respectively). Leucine strongly inhibits alpha-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the alpha-aminoadipic acid flux in beta-lactam-producing P. chrysogenum. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the alpha-aminoadipic acid flux.

2-Aminoadipic Acid↗

PubMed2003

Macropexophagy in Hansenula polymorpha: facts and views.

The hallmark of eukaryotic cells is compartmentalization of distinct cellular functions into specific organelles. This necessitates the cells to run energetically costly mechanisms to precisely control maintenance and function of these compartments. One of these continuously controls organelle activity and abundance, a process termed homeostasis. Yeast peroxisomes are favorable model systems for studies of organelle homeostasis because both the proliferation and degradation of these organelles can be readily manipulated. Here, we highlight recent achievements in regulation of peroxisome turnover in yeast, in particular Hansenula polymorpha, with a focus on directions of future research.

Autophagy↗