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

The role of Hansenula polymorpha MIG1 homologues in catabolite repression and pexophagy

In the methanol-utilizing yeast Hansenula polymorpha, glucose and ethanol trigger the repression of peroxisomal enzymes at the transcriptional level, and rapid and selective degradation of methanol-induced peroxisomes by means of a process termed pexophagy. In this report we demonstrate that deficiency in the putative H. polymorpha homologues of transcriptional repressors Mig1 (HpMig1 and HpMig2), as well as HpTup1, partially and differentially affects the repression of peroxisomal alcohol oxidase by sugars and ethanol. As reported earlier, deficiency in HpTup1 leads to impairment of glucose- or ethanol-induced macropexophagy. In H. polymorpha mig1mig2 double-deletion cells, macropexophagy was also substantially impaired, whereas micropexophagy became a dominant mode of autophagic degradation. Our findings suggest that homologues of the elements of the Saccharomyces cerevisiae main repression pathway have pleiotropic functions in H. polymorpha.

Identification of Hexose Transporter-Like Sensor HXS1 and Functional Hexose Transporter HXT1 in the Methylotrophic Yeast Hansenula polymorpha▿

We identified in the methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) a novel hexose transporter homologue gene, HXS1 (hexose sensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene, HXT1 (hexose transporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the Saccharomyces cerevisiae transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an S. cerevisiae hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host, HXS1 is expressed at moderately low level and is required for glucose induction of the H. polymorpha functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However, HXS1 deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the hxs1Δ deletion strain. Our combined results suggest that in the Crabtree-negative yeast H. polymorpha, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression

A New Yeast Peroxin, Pex36, a Functional Homolog of Mammalian PEX16, Functions in the ER-to-Peroxisome Traffic of Peroxisomal Membrane Proteins.

Peroxisomal membrane proteins (PMPs) traffic to peroxisomes by two mechanisms: direct insertion from the cytosol into the peroxisomal membrane and indirect trafficking to peroxisomes via the endoplasmic reticulum (ER). In mammals and yeast, several PMPs traffic via the ER in a Pex3- and Pex19-dependent manner. In Komagataella phaffii (formerly called Pichia pastoris) specifically, the indirect traffic of Pex2, but not of Pex11 or Pex17, depends on Pex3, but all PMPs tested for indirect trafficking require Pex19. In mammals, the indirect traffic of PMPs also requires PEX16, a protein that is absent in most yeast species. In this study, we isolated PEX36, a new gene in K. phaffii, which encodes a PMP. Pex36 is required for cell growth in conditions that require peroxisomes for the metabolism of certain carbon sources. This growth defect in cells lacking Pex36 can be rescued by the expression of human PEX16, Saccharomyces cerevisiae Pex34, or by overexpression of the endogenous K. phaffii Pex25. Pex36 is not an essential protein for peroxisome proliferation, but in the absence of the functionally redundant protein, Pex25, it becomes essential and less than 20% of these cells show import-incompetent, peroxisome-like structures (peroxisome remnants). In the absence of both proteins, peroxisome biogenesis and the intra-ER sorting of Pex2 and Pex11C are seriously impaired, likely by affecting Pex3 and Pex19 function

Gene ontology categories of the genes regulated by HpHap4-B and ScYap1.

<p>Gene ontology categories of the genes regulated by HpHap4-B and ScYap1.</p

qRT-PCR of <i>HpHAP4-A</i> and <i>HpHAP4-B</i> regulation of gene expression in <i>H. polymorpha.</i>

<p>The P(H1) value indicates the probability that the difference between the sample and control groups is due only by chance and was analysed with the REST software (Qiagen, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112263#s2" target="_blank">Material and Methods</a> for more details). Statistically significant results are shown in bold. The case of AAD2* is not conclusive since the data were obtained in two different experiments (two qRT-PCRs, regulated and reproducible and a third one, performed later, which was not).</p><p>qRT-PCR of <i>HpHAP4-A</i> and <i>HpHAP4-B</i> regulation of gene expression in <i>H. polymorpha.</i></p

Analysis of transcriptomic data of the <i>Sc</i>Δ<i>hap4</i> strains.

<p>The analysed data are the normalized log2 ratios (intensity in WT strain versus intensity in the mutant strains) obtained from the microarray experiments (four biological replicates per condition) in the <i>ScΔhap4</i> genetic background. The WT strain is defined as <i>ScΔhap4</i> plus the empty plasmid pBFG1 and the three mutant strains as <i>ScΔhap4</i>plus pBFG1 containing the <i>ScHap4</i>, the <i>HpHAP4-A</i> or the <i>HpHAP4-B</i> genes. A: Heat map clustergram. This figure reveals the changes in gene expression between experiments. The experiments and the genes are clustered in a tree from their Pearson correlation coefficient values. Gene expression level is represented on a heat map by colour level (green when overexpressed and red when underexpressed compared to the WT strain). B: Presentation of the experiments in the 3D space of the principal components. The two axes of the figure are the three first principal components determined by Principal Component Analysis (PCA). They explain, respectively, 36%, 27% and 16% of the variance.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p