Richard J.S. Baerends

The Hansenula polymorpha PER9 Gene Encodes a Peroxisomal Membrane Protein Essential for Peroxisome Assembly and Integrity

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 (Δper9), 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 Δper9 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.

Adaptation of Hansenula polymorpha to methanol: a transcriptome analysis

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 ?-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 ?-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).

The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis

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.

A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data

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: a visualization tool for the rapid analysis of bacterial transcriptome data

Genome2D is a Windows-based software tool for visualization of bacterial transcriptome and customized datasets on linear chromosome maps constructed from annotated genome sequences. Genome2D facilitates the analysis of transcriptome data by using different color ranges to depict differences in gene-expression levels on a genome map. Such output format enables visual inspection of the transcriptome data, and will quickly reveal transcriptional units, without prior knowledge of expression level cutoff values. The compiled version of Genome2D is freely available for academic or non-profit use from http://molgen.biol.rug.nl/molgen/research/molgensoftware.php.

Transcriptome analysis and related databases of Lactococcus lactis

Several complete genome sequences of Lactococcus lactis and their annotations will become available in the near future, next to the already published genome sequence of L. lactis ssp. lactis IL1403. This will allow intra-species comparative genomics studies as well as functional genomics studies aimed at a better understanding of physiological processes and regulatory networks operating in lactococci. This paper decribes the initial set-up of a DNA-microarray facility in our group, to enable transcriptome analysis of various Gram-positive bacteria, including a ssp. lactis and a ssp. cremoris strain of Lactococcus lactis. Moreover a global description will be given of the hardware and software requirements for such a set-up, highlighting the crucial integration of relevant bioinformatics tools and methods. This includes the development of MolGenIS, an information system for transcriptome data storage and retrieval, and LactococCyc, a metabolic pathway/genome database of Lactococcus lactis.

Normal peroxisome development from vesicles induced by truncated Hansenula polymorpha Pex3p

We show that the synthesis of the N-terminal 50 amino acids of Pex3p (Pex3p1–50) in Hansenula polymorpha pex3 cells is associated with the formation of vesicular membrane structures. Biochemical and ultrastructural findings suggest that the nuclear membrane is the donor membrane compartment of these vesicles. These structures also contain Pex14p and can develop into functional peroxisomes after subsequent reintroduction of the full-length Pex3p protein. We discuss the significance of this finding in relation to peroxisome reintroduction, e.g. in case peroxisomes are lost due to failure in inheritance.

A novel scheme to assess factors involved in the reproducibility of DNA-microarray data

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.ConclusionsEven 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 (very) lowly expressed genes. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality.

PEROXISOMAL REMNANTS IN PEROXISOME-DEFICIENT MUTANTS OF THE YEAST HANSENULA POLYMORHA

We have analyzed the presence of peroxisomal remnants (‘ghosts’) in three peroxisome‐deficient (per) mutants of the yeast Hansenula polymorpha, namely Δper4, Δper5 and Δper10. Under peroxisome‐inducing growth conditions peroxisomal membrane proteins (PMPs) were normally synthesized in cells of these mutants. In addition, these cells contained clusters of small membranous vesicles, which were absent in cells grown under peroxisome‐repressing growth conditions. These structures displayed typical peroxisomal properties in that they proliferated upon overproduction of Per8p, the H. polymorpha peroxisome proliferation factor. Moreover, in Δper4 and Δper5 these vesicles were susceptible to glucose‐induced proteolytic degradation.

Deviant Pex3p levels affect normal peroxisome formation in Hansenula polymorpha: high steady-state levels of the protein fully abolish matrix protein import.

PEX3 encodes a 52 kDa peroxisomal membrane protein (PMP), essential for peroxisome biogenesis in the yeast Hansenula polymorpha. The relation between Pex3p levels and peroxisome formation was studied in wild type (WT) and Δpex3 strains expressing additional copies of PEX3 under control of a substrate‐inducible promoter, namely the strong alcohol oxidase (PAOX) or the weaker amine oxidase (PAMO) promoter. In glucose‐grown Δpex3 cells, containing PAOX. PEX3, Pex3p was undetectable and peroxisomes were absent. After induction of these cells on methanol, peroxisomes were rapidly formed. At Pex3p levels up to 7–10 times the values observed in WT controls normal peroxisomes were present. However, at further enhanced Pex3p levels a general matrix protein import defect was observed. This phenomenon was paralleled by aberrant peroxisome assembly and the formation of numerous small vesicles. These vesicles contained Pex3p, together with other H. polymorpha PMPs, but lacked the major matrix proteins which has accumulated in the cytosol. The implications of our results on PEX3 gene regulation and functioning of the peroxisomal matrix protein import machinery in H. polymorpha are discussed.