I.J. van der Klei

Autophagy: Principles and significance in health and disease

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.

Hansenula polymorpha Pex1p and Pex6p are peroxisome-associated AAA proteins that functionally and physically interact

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

The methylotrophic yeast Hansenula polymorpha: a versatile cell factory.

The development of heterologous overexpression systems for soluble proteins has greatly advanced the study of the structure/function relationships of these proteins and their biotechnological and pharmaceutical applications. In this paper we present an overview on several aspects of the use of the methylotrophic yeast Hansenula polymorpha as a host for heterologous gene expression. H. polymorpha has been successfully exploited as a cell factory for the large-scale production of such components. Stable, engineered strains can be obtained by site-directed integration of expression cassettes into the genome, for which various constitutive and inducible promoters are available to control the expression of the foreign genes. New developments have now opened the way to additional applications of H. polymorpha, which are unprecedented for other organisms. Most importantly, it may be the organism of choice for reliable, large-scale production of heterologous membrane proteins, using inducible intracellular membranes and targeting sequences to specifically insert these proteins stably into these membranes. Furthermore, the use of H. polymorpha offers the possibility to accumulate the produced components into specific compartments, namely peroxisomes. These organelles are massively induced during growth of the organism on methanol and may occupy up to 80% of the cell volume. Accumulation inside peroxisomes prevents undesired modifications (e.g. proteolytic processing or glycosylation) and is also in particular advantageous when proteins are produced which are toxic or harmful for the host.

The Hansenula polymorpha PDD1 gene product, essential for the selective degradation of peroxisomes, is a homologue of Saccharomyces cerevisiae Vps34p

Via functional complementation we have isolated the Hansenula polymorpha PDD1 gene essential for selective, macroautophagic peroxisome degradation. HpPDD1 encodes a 116 kDa protein with high similarity (42% identity) to Saccharomyces cerevisiae Vps34p, which has been implicated in vacuolar protein sorting and endocytosis. Western blotting experiments revealed that HpPDD1 is expressed constitutively. In a H. polymorpha pdd1 disruption strain peroxisome degradation is fully impaired. Sequestered peroxisomes, typical for the first stage of peroxisome degradation in H. polymorpha, were never observed, suggesting that HpPdd1p plays a role in the tagging of redundant peroxisomes and/or sequestration of these organelles from the cytosol. Possibly, HpPdd1p is the functional homologue of ScVps34p, because—like S. cerevisiae vps34 mutants—H. polymorpha pdd1 mutants are temperature‐sensitive for growth and are impaired in the sorting of vacuolar carboxypeptidase Y. Moreover, HpPdd1p is associated to membranes, as was also observed for ScVps34p. Copyright

A Pichia pastoris VPS15 homologue is required in selective peroxisome autophagy

Abstract Methylotrophic yeasts contain large peroxisomes during growth on methanol. Upon exposure to excess glucose or ethanol these organelles are selectively degraded by autophagy. Here we describe the cloning of a Pichia pastoris gene (PpVPS15) involved in peroxisome degradation, which is homologous to Saccharomyces cerevisiae VPS15. In methanol-grown cells of a P. pastoris VPS15 deletion strain, the levels of peroxisomal marker enzymes remained high after addition of excess glucose or ethanol. Electron microscopic studies revealed that the organelles were not taken up by vacuoles, suggesting that PpVPS15 is required at an early stage in peroxisome degradation.

Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha

Abstract. We have cloned the Hansenula polymorpha BIP gene from genomic DNA using a PCR-based strategy. H. polymorpha BIP encodes a protein of 665 amino acids, which shows very high homology to Saccharomyces cerevisiae KAR2p. KAR2p belongs to the Hsp70 family of molecular chaperones and resides in the endoplasmic reticulum (ER)-lumen. H. polymorpha BiP contains a putative N-terminal signal sequence of 30 amino acids together with the conserved –HDEL sequence, the typical ER retention signal, at the extreme C-terminus. We have analysed the effect of BIP overexpression, placing the gene under control of the strong alcohol oxidase promoter (PMOX), on the secretion of artificially produced Aspergillus niger glucose oxidase (GOX) by H. polymorpha. BiP overproduction did not lead to any growth defects of the cells; at the subcellular level, proliferation of ER-like vesicles was observed. However, artificially enhanced BiP levels strongly affected GOX secretion and led to accumulation of this protein in the ER-like vesicles. This was not simply due to the high BiP overproduction, because it was also observed under conditions of low PMOX induction during growth of cells on glycerol. Vacuolar carboxypeptidase Y was properly sorted to its target organelle in the BiP overproducing strains.

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.

Physiological role of microbodies in the yeast Trichosporon cutaneum during growth on ethylamine as the source of energy, carbon and nitrogen

Compartmentation of the metabolism of ethylamine in Trichosporon cutaneum X4 was studied in cells, grown on this compound as the sole source of energy, carbon, and nitrogen. Transfer experiments indicated that an amine oxidase is involved in the early metabolism of ethylamine. The synthesis of this enzyme was induced by primary amines and was subject to partial carbon catabolite repression. Repression by ammonium ions was not observed. Adaptation of glucose-grown cells to growth on ethylamine was associated with the development of many microbodies, which developed from already existing organelles present in the inoculum cells and multiplied by division. Cytochemical experiments indicated that the organelles contained amine oxidase and catalase. Therefore, they were considered to play a key role in the metabolism of ethylamine. The physiological significance of the microbodies was investigated by fractionation studies of homogenized protoplasts from ethylamine-grown cells by differential- and sucrose-gradient centrifugation of subcellular organelles. Intact microbodies were only obtained when the isolation procedure was performed at pH 5.8 in the absence of Mg2+-ions. Analysis of the different fractions indicated that the key enzymes of the glyoxylate cycle, namely isocitrate lyase and malate synthase, cosedimented together with catalase and amine oxidase. In addition, activities of malate dehydrogenase, glutamate:oxaloacetate aminotransferase (GOT) and (NAD-dependent) glutamate dehydrogenase were detected in these fractions. Electron microscopy revealed that they mainly contained microbodies. Cytochemical experiments indicated that the above enzymes were all present in the same organelle. These findings suggest that microbodies of ethylamine-grown T. cutaneum X4 produce aspartate, so allowing NADH generated in the oxidation of malate by malate dehydrogenase to be quantitatively reoxidized inside the organelles in a series of reactions involving GOT and glutamate dehydrogenase. Aspartase and fumarase were not detected in the microbodies; activities of these two enzymes were present in the cytoplasm.

The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pak1p, involved in peroxisome integrity

Abstract 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.

Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha

We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre‐gMDH) was cloned in an H. polymorpha expression vector downstream of the inducible H. polymorpha alcohol oxidase promoter. During methylotrophic growth, pre‐gMDH was synthesized and imported into peroxisomes, where it was enzymatically active. The apparent molecular mass of the protein located in H. polymorpha peroxisomes was equal to that of pre‐gMDH (41 kDa), indicating that N‐terminal processing of the transit peptide had not occurred in the yeast.