The Hansenula polymorpha PER8 Gene Encodes a Novel Peroxisomal Integral Membrane Protein Involved in Proliferation

We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organdies, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organdies. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.

Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase

Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the catabolism of methanol as a carbon source and certain primary amines. such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris homologue, the H. polymorpha gene does not contain an intron. The predicted FLD1 product (Fld1p) is a protein of 380 amino acids (ca. 41 kDa) with 82% identity to P. pastoris Fld1p, 76% identity to the FLD protein sequence from n-alkane-assimilating yeast Candida maltosa and 63-64% identity to dehydrogenase class III enzymes from humans and other higher eukaryotes. The expression of FLD1 is strictly regulated and can be controlled at two expression levels by manipulation of the growth conditions. The gene is strongly induced under methylotrophic growth conditions; moderate expression is obtained under conditions in which a primary amine, e.g. methylamine, is used as nitrogen source. These properties render the FLD1 promoter of high interest for heterologous gene expression. The availability of the H. polymorpha FLD1 promoter provides an attractive alternative for expression of foreign genes besides the commonly used alcohol oxidase promoter. The sequence has been deposited in the GenBank/NCBI data library under Accession No. AF364077. Copyright (C) 2002 John Wiley Sons, Ltd.</p

A Pichia pastoris VPS15 homologue is required in selective peroxisome autophagy

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 ill 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

Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation

We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes. PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p. Similarities between Pex5p and Pex20p were noted in their protein interactions and dynamics during import, and both contain a conserved NH2-terminal domain. In the absence of the E2-like Pex4p or the AAA proteins Pex1p and Pex6p, Pex20p is degraded via polyubiquitylation of residue K19, and the K19R mutation causes accumulation of Pex20p in peroxisome remnants. Finally, either interference with K48-branched polyubiquitylation or removal of the conserved NH2-terminal domain causes accumulation of Pex20p in peroxisomes, mimicking a defect in its recycling to the cytosol. Our data are consistent with a model in which Pex20p enters peroxisomes and recycles back to the cytosol in an ubiquitin-dependent manner

A Hexose Transporter Homologue Controls Glucose Repression in the Methylotrophic Yeast Hansenula polymorpha

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser85) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker’s yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport

Determining the relative contribution of retinal disparity and blur cues to ocular accommodation in Down syndrome

Individuals with Down syndrome (DS) often exhibit hypoaccommodation alongside accurate vergence. This study investigates the sensitivity of the two systems to retinal disparity and blur cues, establishing the relationship between the two in terms of accommodative-convergence to accommodation (AC/A) and convergence-accommodation to convergence (CA/C) ratios. An objective photorefraction system measured accommodation and vergence under binocular conditions and when retinal disparity and blur cues were removed. Participants were aged 6–16 years (DS n = 41, controls n = 76). Measures were obtained from 65.9% of participants with DS and 100% of controls. Accommodative and vergence responses were reduced with the removal of one or both cues in controls (p < 0.007). For participants with DS, removal of blur was less detrimental to accommodative responses than removal of disparity; accommodative responses being significantly better when all cues were available or when blur was removed in comparison to when proximity was the only available cue. AC/A ratios were larger and CA/C ratios smaller in participants with DS (p < 0.00001). This study demonstrates that retinal disparity is the main driver to both systems in DS and illustrates the diminished influence of retinal blur. High AC/A and low CA/C ratios in combination with disparity-driven responses suggest prioritisation of vergence over accurate accommodation