BackgroundMethylotrophic yeast species (e.g. Hansenula polymorpha, Pichia pastoris) can grow on methanol as sole source of carbon and energy. These organisms are important cell factories for the production of recombinant proteins, but are also used in fundamental research as model organisms to study peroxisome biology. During exponential growth on glucose, cells of H. polymorpha typically contain a single, small peroxisome that is redundant for growth while on methanol multiple, enlarged peroxisomes are present. These organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism.In this study, changes in the transcriptional profiles during adaptation of H. polymorpha cells from glucose- to methanol-containing media were investigated using DNA-microarray analyses.ResultsTwo hours after the shift of cells from glucose to methanol nearly 20% (1184 genes) of the approximately 6000 annotated H. polymorpha genes were significantly upregulated with at least a two-fold differential expression. Highest upregulation (> 300-fold) was observed for the genes encoding the transcription factor Mpp1 and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. Upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal β-oxidation.A moderate increase in transcriptional levels (up to 4-fold) was observed for several PEX genes, which are involved in peroxisome biogenesis. Only PEX11 and PEX32 were higher upregulated. In addition, an increase was observed in expression of the several ATG genes, which encode proteins involved in autophagy and autophagy processes. The strongest upregulation was observed for ATG8 and ATG11.Approximately 20% (1246 genes) of the genes were downregulated. These included glycolytic genes as well as genes involved in transcription and translation.ConclusionTranscriptional profiling of H. polymorpha cells shifted from glucose to methanol showed the expected downregulation of glycolytic genes together with upregulation of the methanol utilisation pathway. This serves as a confirmation and validation of the array data obtained. Consistent with this, also various PEX genes were upregulated. The strong upregulation of ATG genes is possibly due to induction of autophagy processes related to remodeling of the cell architecture required to support growth on methanol. These processes may also be responsible for the enhanced peroxisomal β-oxidation, as autophagy leads to recycling of membrane lipids. The prominent downregulation of transcription and translation may be explained by the reduced growth rate on methanol (td glucose 1 h vs td methanol 4.5 h).

We have cloned and characterized the Hansenula polymorpha PER9 gene by functional complementation of the per9-1 mutant of H. polymorpha, which is defective in peroxisome biogenesis. The predicted product, Per9p, is a polypeptide of 52 kDa with sequence similarity to Pas3p, a protein involved in peroxisome biogenesis in Saccharomyces cerevisiae. In a per9 disruption strain (⌬per9), peroxisomal matrix and membrane proteins are present at wild-type levels. The matrix proteins accumulated in the cytoplasm. However, the location of the membrane proteins remained obscure; fully induced ⌬per9 cells lacked residual peroxisomal vesicles ("ghosts"). Analysis of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced during growth of cells on peroxisome-inducing substrates. The highest expression levels were observed in cells grown on methanol. Localization studies revealed that Per9p is an integral membrane protein of the peroxisome. Targeting studies suggested that Per9p may be sorted to the peroxisome via the endoplasmic reticulum. Overexpression of PER9 induced a significant increase in the number of peroxisomes per cell, a result that suggests that Per9p may be involved in peroxisome proliferation and/or membrane biosynthesis. When PER9 expression was placed under the control of a strongly regulatable promoter and switched off, peroxisomes were observed to disintegrate over time in a manner that suggested that Per9p may be required for maintenance of the peroxisomal membrane.Peroxisomes are cell organelles that are present in virtually all eukaryotic cells. They perform specific metabolic functions that are often related to the developmental stage and/or the organism in which they occur (1). The metabolic importance of peroxisomes in humans is demonstrated by the fact that the absence of the organelles leads to severe abnormalities, followed by an early death (e.g. Zellweger syndrome (2)). Consequently, many studies are now devoted to unravel the molecular mechanisms of peroxisome biogenesis and function. Yeasts are excellent model systems for such studies having the advantages that (i) the induction and protein composition of peroxisomes can readily be manipulated by varying growth conditions and (ii) in the absence of peroxisomes, yeasts are viable (3, 4). Hence, peroxisome-deficient mutants have been isolated from different yeast species (4), and the corresponding genes are being cloned and characterized.In yeast, peroxisomes normally develop by growth and fission from pre-existing ones. Peroxisomal matrix proteins are nuclear-encoded, synthesized in the cytoplasm, and directed to the organelle by topogenic signals (PTSs).1 Two PTSs have been identified and are located either at the extreme C terminus (PTS1) or the N terminus of the protein (PTS2) (4). Our knowledge on the sorting of peroxisomal membrane proteins is still limited, and consensus topogenic sequences have yet to be identified (5).In our laboratory, we use the methylotrophic yeast Hansenula po...

Pex3p is a peroxisomal membrane protein that is essential for peroxisome biogenesis. Here, we show that a conserved stretch of positively charged amino acids (Arg 11 -X-Lys-Lys-Lys 15 ) in the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into its target membrane. Despite the strong conservation, this sequence shows a high degree of redundancy. Substitution of either Arg 11 , Lys 13 , Lys 14 , or Lys 15 with uncharged or negatively charged amino acids did not interfere with Pex3p location and function. However, a mutant Pex3p, carrying negatively charged amino acids at position 13 and 15 (K13E/K15E), caused moderate but significant defects in peroxisome assembly and matrix protein import. Additional changes in the N terminus of Pex3p, e.g. replacing three or four of the positively charged amino acids with negatively charged ones, led to a typical pex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cytosol and absence of peroxisomal remnants. Also, in these cases, the mutant Pex3p levels were reduced. Remarkably, mutant Pex3p proteins were mislocalized to mitochondria or the cytosol, depending on the nature of the mutation. Furthermore, in case of reduced amounts of Pex3p, the levels of other peroxisomal membrane proteins, e.g. Pex10p and Pex14p, were also diminished, suggesting that Pex3p maybe involved in the recruitment or stabilization of these proteins (in the membrane).

We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14‐1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome‐inducing growth conditions. In cells of both pex14‐1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild‐type H.polymorpha cells resulted in a peroxisome‐deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import.

BackgroundIn research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed.ResultsA number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance.ConclusionEven in cases where 90 % of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25 %. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality.

Genome2D is a software tool that enables bacterial transcriptome data to be visualized on chromosome maps.

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.

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 yku80 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.