IDENTIFICATION AND ANALYSIS OF A SACCHAROMYCES-CEREVISIAE COPPER HOMEOSTASIS GENE ENCODING A HOMEODOMAIN PROTEIN

Yeast metallothionein, encoded by the CUP1 gene, and its copper-depend ent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfona te mutant of a yeast strain in which CUP1 and ACE1 were deleted, we is olated a gene, designated CUP9, which permits yeast cells to grow at h igh concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chr omosome XVI, caused a loss of copper resistance in strains which posse ssed CUP1 and ACE1, as well as in the cup1 ace1 deletion strain. Measu rement of intracellular copper levels of the wild-type and cup9-1 muta nt demonstrated that total intracellular copper concentrations were un affected by CUP9. CUP9 mRNA levels were, however, down regulated by co pper when yeast cells were grown with glucose but not with lactate or glycerol-ethanol as the sole carbon source. This down regulation was i ndependent of the copper metalloregulatory transcription factor ACE1. The DNA sequence of CUP9 predicts an open reading frame of 306 amino a cids in which a 55-amino-acid sequence showed 47% identity with the ho meobox domain of the human proto-oncogene PBX1, suggesting that CUP9 i s a DNA-binding protein which regulates the expression of important co pper homeostatic genes.