Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2.

The wine and beer yeast Dekkera bruxellensis thrives in environments that are harsh and limiting, especially in concentrations with low oxygen and high ethanol. Its different strains' chromosomes greatly vary in number (karyotype). This study isolates two novel centromeric loci (CEN1 and CEN2), which support both the yeast's autonomous replication and the stable maintenance of plasmids. In the sequenced genome of the D. bruxellensis strain CBS 2499, CEN1 and CEN2 are each present in one copy. They differ from the known "point" CEN elements, and their biological activity is retained within ~900-1300 bp DNA segments. CEN1 and CEN2 have features of both "point" and "regional" centromeres: They contain conserved DNA elements, ARSs, short repeats, one tRNA gene, and transposon-like elements within less than 1 kb. Our discovery of a miniature inverted-repeat transposable element (MITE) next to CEN2 is the first report of such transposons in yeast. The transformants carrying circular plasmids with cloned CEN1 and CEN2 undergo a phenotypic switch: They form fluffy colonies and produce three times more biofilm. The introduction of extra copies of CEN1 and CEN2 promotes both genome rearrangements and ploidy shifts, with these effects mediated by homologous recombination (between circular plasmid and genome centromere copy) or by chromosome breakage when integrated. Also, the proximity of the MITE-like transposon to CEN2 could translocate CEN2 within the genome or cause chromosomal breaks, so promoting genome dynamics. With extra copies of CEN1 and CEN2, the yeast's enhanced capacities to rearrange its genome and to change its gene expression could increase its abilities for exploiting new and demanding niches.

Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases.

In the present study, we modified xylose uptake properties of a recombinant xylose-utilizing yeast Saccharomyces cerevisiae by expression of heterologous and homologous permease-encoding genes. In a mutant yeast strain with the main seven hexose transporter genes deleted, and engineered for xylose utilization, we screened an expression cDNA library of the filamentous fungus Trichoderma reesei (Hypocrea jecorina) for enhanced growth on xylose plates. One cDNA clone with significant homology to fungal sugar transporters was obtained, but when the clone was retransformed into the host, it did not support significant growth on xylose. However, during a long liquid culture of the strain carrying the cDNA clone, adaptive mutations apparently occurred in the host, which led to growth on xylose but not on glucose. The new transporter homologue, Trxlt1 thus appears to code for a protein specific for xylose uptake. In addition, xylose-transporting properties of some homologous hexose transporters were studied. All of them, i.e., Hxt1, Hxt2, Hxt4, and Hxt7 were capable of xylose uptake. Their affinities for xylose varied, K (m) values between 130 and 900 mM were observed. The single-Hxt strains showed a biphasic growth mode on xylose, alike the Trxlt1 harboring strain. The initial, slow growth was followed by a long lag and finally by exponential growth.

Observation of the Yarrowia lipolytica peroxisome-vacuole dynamics by fluorescence microscopy with a single filter set.

We constructed the fusion of peroxisomal acyl-CoA oxidase 3 and the enhanced yellow fluorescent protein (EYFP) for fluorescent labeling of Yarrowia lipolytica peroxisomes. Using the spectral overlap between EYFP and FM4-64, we developed a procedure for simultaneous observation of Y. lipolytica peroxisomes and vacuoles with the single fluorescein isothiocyanate filter set. Using this procedure we were able to follow the Y. lipolytica peroxisome-vacuole dynamics under pexophagy conditions and show that Y. lipolytica peroxisomes are degraded in the vacuoles by a macropexophagic mechanism.

Pexophagy: the selective autophagy of peroxisomes.

Pichia pastoris and Hansenula polymorpha are methylotrophic yeasts capable of utilizing methanol, as a sole source of carbon and energy. Growth of these yeast species on methanol requires the synthesis of cytosolic and peroxisomal enzymes combined with the proliferation of peroxisomes. Peroxisomes are also abundantly present in the alkane-utilizing yeast Yarrowia lipolytica upon growth of cells on oleic acid. This feature has made these yeast species attractive model systems to dissect the molecular mechanisms controlling peroxisome biogenesis. We have found that upon glucose- or ethanol-induced catabolite inactivation, metabolically superfluous peroxisomes are rapidly and selectively degraded within the vacuole by a process called pexophagy, the selective removal of peroxisomes by autophagy-like processes. Utilizing several genetic screens, we have identified a number of genes that are essential for pexophagy. In this review, we will summarize our current knowledge of the molecular events of pexophagy.

Trs85 is required for macroautophagy, pexophagy and cytoplasm to vacuole targeting in Yarrowia lipolytica and Saccharomyces cerevisiae.

Yarrowia lipolytica was recently introduced as a new model organism to study peroxisome degradation in yeasts. Transfer of Y. lipolytica cells from oleate/ethylamine to glucose/ammonium chloride medium leads to selective macroautophagy of peroxisomes. To decipher the molecular mechanisms of macropexophagy we isolated mutants of Y. lipolytica defective in the inactivation of peroxisomal enzymes under pexophagy conditions. Through this analysis we identified the gene YlTRS85, the ortholog of Saccharomyces cerevisiae TRS85 that encodes the 85 kDa subunit of transport protein particle (TRAPP). A parallel genetic screen in S. cerevisiae also identified the trs85 mutant. Here, we report that Trs85 is required for nonspecific autophagy, pexophagy and the cytoplasm to vacuole targeting pathway in both yeasts.

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 (Ser(85)) 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.

Identification of intragenic mutations in the Hansenula polymorpha PEX6 gene that affect peroxisome biogenesis and methylotrophic growth.

Two interacting AAA ATPases, Pex1p and Pex6p, are indispensable for peroxisome biogenesis in different organisms. Mutations affecting corresponding genes are the most common cause of the peroxisome biogenesis disorders in humans. By UV mutagenesis of the Hansenula polymorpha pex6 mutant, deficient in peroxisome biogenesis, we isolated a conditional cold-sensitive strain with restored ability to grow in methanol medium at 37 degrees C but not at 28 degrees C. Sequencing of the pex6 allele revealed a point mutation in the first AAA module of the PEX6 gene that leads to substitution of a conserved amino acid residue (G737E). An additional intragenic mutation identified in the cold-sensitive pex6 allele leads to a conserved amino acid substitution in the second AAA domain (R1000G). Electron microscopic analysis revealed restored peroxisomes in methanol-induced cold-sensitive pex6 cells at both permissive and restrictive temperatures. If separated, the secondary mutation did not affect methylotrophic growth. Our data suggest that H. polymorpha Pex6p may have a complex function in peroxisome biogenesis in which identified amino acid residues are involved.

Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica.

Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.

Vacuolar accumulation and extracellular extrusion of electrophilic compounds by wild-type and glutathione-deficient mutants of the methylotrophic yeast Hansenula polymorpha.

The methylotrophic yeast Hansenula polymorpha CBS4732 leu2 detoxifies electrophilic xenobiotics by glutathione (GSH)-dependent accumulation in vacuoles, as shown by fluorescence microscopy. GSH-dependent and GSH-independent export of xenobiotic derivatives were also demonstrated by high-performance liquid chromatography (HPLC). Conjugates of GSH and N-acetylcysteine with monobromobimane and N-[1-pyrene]maleimide were observed among the HPLC fractions, along with unidentified derivatives.

GSH2, a gene encoding gamma-glutamylcysteine synthetase in the methylotrophic yeast Hansenula polymorpha.

The GSH2 gene, encoding Hansenula polymorpha gamma-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121.