Abstract
The dimorphic yeast Yarrowia lipolytica is more resistant to high copper concentrations than Saccharomyces cerevisiae. This differential tolerance to copper ions has been observed in several strains arising from non-related isolates. To investigate the molecular basis of this resistance, we obtained several copper-sensitive mutants. By complementation of one of them, we isolated the YlCRF1 gene encoding for a copper-binding transcription factor of 411 amino acids homologous to ScAce1p, CgAmt1p, and ScMac1p. Naturally occurring copper-sensitive strains lack the CRF1 allele. The YlCRF1 transcript is not induced by the addition of copper to the medium. Gene disruption demonstrated that YlCRF1 is responsible for a 4- to 5-fold increase in Y. lipolytica copper tolerance. We further show that strain Deltacrf1 is more sensitive to cadmium but not to other metals. The role of YlCrf1p as a copper-sensitive transcription factor is supported by the finding that the protein is immunolocalized in the nucleus during growth in copper-supplemented but not in copper-free medium. However, in contrast to the S. cerevisiae strain mutated in the metallothionein transcription activator ACE1, Y. lipolytica strain Deltacrf1 is still able to increase metallothionein (MTP) mRNA levels in response to copper addition. CRF1 deletion does not affect superoxide dismutase (SOD) activity either. Our data suggest the existence of one or more different target genes for Crf1p, other than MTP or SOD1, and support its role as a novel copper-responsive transcription factor involved in metal detoxification.
Highlights
Copper is an essential element for life due to its ability to catalyze redox reactions, and as such is the ideal cofactor of many enzymes such as cytochrome c oxidase, Cu,Zn-superoxide dismutase (SOD)1, and several ATPases [1, 2]
Characterization of Copper Resistance in Y. lipolytica— S. cerevisiae strains exhibit a differential resistance to copper: the most sensitive strains fail to grow in 0.1 mM CuSO4, whereas resistant strains are able to grow in up to 1.75 mM CuSO4 [36]
We demonstrate that the YlCRF1 gene plays an essential role in this resistant phenotype and that variations in the copper resistance of different Y. lipolytica strains are correlated with naturally occurring modifications of the CRF1 allele
Summary
Yeast Cell Mutagenesis—Copper-sensitive mutants were screened after exposing a suspension of cells (1.4 ϫ 107 cells/ml in 0.85% NaCl) in a Petri dish to 2.82 J/s/m2 of UV light for 70 s Different dilutions of this suspension were plated onto YPD medium. To obtain spore-rich samples, two scrapings of the sporulated cultures were resuspended in 4 ml of 50 mM sodium citrate buffer, sonicated, and incubated for 3– 4 h in 2 ml of buffer containing cytohelicase (180 mg), -glucuronidase (12 mg), and cysteine (12 mg) at 37 °C This suspension was washed three times in 0.85% NaCl, resuspended in 20% glycerol/0.85% NaCl, and sonicated 4ϫ 30 s to kill vegetative cells. PMP7 contains a fragment (including the CRF1 gene) that complements the MP73 strain mutation and was obtained by digesting pMP3 with ClaI-BamHI and by cloning the 4-kb fragment at the same sites of pINA62 The insertion of this ClaI-BamHI fragment into the corresponding sites of pBluescript-SK generated the plasmid pMP13. MatA MatB MatA leu lys MatA leu lys ura MatB leu lys MatB leu lys ura3–18XPR2 MatB leuB spo MatA met A spo MatB adeA ura hisA MatA ade 1 MatB ade 1 leu lys xpr MatB ura uvs pro MatA lys MatB leu lys ura xpr2::LYS5 MatB lys3.12 MatB trp MatA leu lys ura crf MatA ura MatB leu lys ura xpr2::LYS5 MatA leu ura XPR2 MatB crf leu ura MatB crf leu ura LEU2-CRF1-LACZ MatB leu lys ura XPR2 crf1::LEU2
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