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...

We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome‐deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Δpex1 or Δpex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co‐sedimented with the peroxisomal membrane protein HpPex3p in both wild‐type cells and in Δpex4, Δpex8 or Δpex14 cells. Both proteins are loosely membrane‐bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co‐overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane‐bound, but only partially co‐localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity. Copyright © 1999 John Wiley & Sons, Ltd.

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The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pak1p, involved in peroxisome integrity Klei, Ida J. van der; Heide, Meis van der; Baerends, Richard J.S.; Rechinger, Karl-Björn; Nicolay, Klaas; Kiel, Jan A.K.W.; Veenhuis, Marten Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 04-04-2019Abstract The Hansenula polymorpha per6-210 mutant is impaired in respect of growth on methanol (Mut -) and is characterized by aberrant peroxisome formation. The functionally complementing DNA fragment contains two open reading frames. The first encodes dihydroxyacetone kinase (DAK), a cytosolic enzyme essential for formaldehyde assimilation; the second ORF codes for a novel protein (Pak1p). We have demonstrated that per6-210 cells lack DAK activity, causing the Mut -phenotype, and have strongly reduced levels of Pak1p, resulting in peroxisomal defects. Sequence analysis revealed that per6-210 contains a mutation in the 3′ end of the DAK coding region, which overlaps with the promoter region of PAK1. Possibly this mutation also negatively affects PAK1 expression.

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 nitrogenlimitation 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.Key words: autophagy, methylotrophic yeast, peroxisome degradation, sequestration Received 28 September 2004, revised and accepted for publication 21 October 2004Proper cellular homeostasis involves the efficient re-use of cell components that have been hydrolyzed by the protein degradation machinery. In yeast, two major protein degradation machineries are known -the proteasome and vacuolar hydrolysis. The vacuole contains various hydrolytic enzymes involved in the degradation of cellular constituents.Degradation processes via the vacuole are termed autophagic processes. In yeast, several autophagic processes have been described. Non-selective autophagy is a process characterized by the bulk turnover of portions of the cytoplasm (cytosol plus organelles, and including peroxisomes) (1). However, degradation of yeast peroxisomes may also occur via a strictly selective autophagic process, termed macropexophagy (2,3). The methylotrophic yeast Hansenula polymorpha is an attractive model organism to study peroxisome degradation because the proliferation and degradation of these organelles can be readily prescribed by manipulation of the growth conditions. In H. polymorpha, macropexophagy is induced by exposing methanol-grown cells to new growth environments in which the organelles are redundant for growth (3). However, when the cells are transferred to nitrogen-starvation conditions, microautophagy is induced in this yeast (4). Molecular studies to unravel the principles of pexophagy in H. polymorpha revealed that both macropexophagy and autophagy share many components with other protein/ organelle sorting pathways to the vacuole, e.g. endocytosis, and the Vps and Cvt pathways (3). To circumvent confusion in protein nomenclature, a unified nomenclature has recently been introduced for genes involved in AuTophaGy-related processes (ATG genes) (5).In this study, we describe the isolation and characterization of the H. polymorpha ATG8 gene (previously designated AUT7). We show that a GFP.Atg8 fusion protein is observed as a peri-vacuolar fluoresc...

Organisation de Coopération et de Développement Économiques Organisation for Economic Co-operation and Development 20-Jul-2015 ___________________________________________________________________________________________ _____________ English -Or. English ENVIRONMENT DIRECTORATE BIODIVERSITY POLICY RESPONSE INDICATORS -ENVIRONMENT WORKING PAPER No. 90 by Christina Van Winkle and Katia Karousakis (OECD), Rosalind Bark (CSIRO) and Martijn van der Heide (LEI Wageningen UR) OECD Working Papers should not be reported as representing the official views of the OECD or of its member countries. The opinions expressed and arguments employed are those of the author(s).

Planning and conserving nature areas are challenging tasks in urbanized and intensively used countries like the Netherlands. This paper supports decision making and public policy debate about these tasks in both an empirical and a methodological way. Empirically, we explore policy alternatives by determining the potential consequences of different nature policy scenarios in the Netherlands. Methodologically, we employ a mixed monetary and non-monetary evaluation method known as multi-criteria cost-benefit analysis (MCCBA). We evaluate four new future directions of Dutch nature policy that address four dominant stakeholder demands: biodiversity conservation, the provision of ecosystem services, recreational potential as well as economic gains. To balance compact presentation of evaluation outcomes on the one hand and information richness of results on the other, we distinguish between two impact indicator sets: three “headline” and ten “elaborate” indicators. Using these indicators we discuss the quantitative assessment of the four nature policy scenarios by comparing them to two other scenarios, reflecting the 2010 stand-still baseline situation (2010) as well as a reference policy (Trend). In total, we evaluate six scenarios; four present new directions and two reflect existing or recently (2010) halted practices. Our findings first of all show that even in an urbanized country like the Netherlands, with its intensive competition among land use functions, serious gains in national and international biodiversity are possible. Second, we find that it is doubtful whether stimulating the provision of regulating ecosystem services in a country which applies intensive and profitable agricultural techniques is beneficial. Other countries or areas that are less suitable for intensive agricultural practices may be more logical for this. Finally we demonstrate that increasing urban recreational green space − a common challenge for many urban areas − can only be achieved at relatively high costs, while it does not seem to lead to relatively high scores on nature appreciation. Nature appreciation seems to be served better by wilder nature than by park-like nature.

Forests play a key role in a bio-based economy by providing renewable materials, mitigating climate change, and accommodating biodiversity. However, forests experience massive increases in stresses in their ecological and socioeconomic environments, threatening forest ecosystem services supply. Alleviating those stresses is hampered by conflicting and disconnected governance arrangements, competing interests and claims, and rapid changes in technology and social demands. Identifying which stresses threaten forest ecosystem services supply and which factors hamper their alleviation requires stakeholders' perceptions. Stakeholder-oriented stress tests for the supply of forest ecosystem services are therefore necessary but are not yet available. This perspective presents a roadmap to develop a stress test tailored to multiple stakeholders' needs and demands across spatial scales. We provide the Cascade and Resilience Rosetta, with accompanying performance-and resilience indicators, as tools to facilitate development of the stress test. The application of the stress test will facilitate the transition toward a bio-based economy in which healthy and diverse forests provide sustainable and resilient ecosystem services. ll