Diazaborine in yeast

For several years we are investigating the mechanism of action of the antibacterial drug diazaborine. In order to study the effects on eukaryotic organisms we explored the resistance mechanisms in baker`s yeast to this substance. We found that four yeast genes may mediate resistance: allelic forms of genes DRG1(AFG2), PDR1 and PDR3 and the wild type gene YAP1 when overexpressed. Recently we have identified another complementation group among the resistant mutants. The gene which is responsible for resistance in the new complementation group has yet to be isolated which we plan to perforrn in the forthcoming funding period. We could already identify one efflux-pump protein, Flr1p, which promotes diazaborine resistance. However, our results demonstrate that there must be either one or several other detoxifying proteins present which contribute to overall resistance. We will identify the gene (or the genes) coding for this protein (or these proteins) in the coming project period. We found that, for resistance to occur, Pdr1p, Pdr3p and Yap1p, influence each other. In a dpdr1 dpdr3 background overexpression of Yap1p does not mediate resistance. We will study how these proteins interact and activate diazaborine resistance. This will involve assays for joint promoter binding of these activators to the FLR1-promoter. We will also test whether there is a repressor protein involved in diazaborine resistance which complexes Pdr3p or Pdr1p and whether this complex formation may be influenced by increasing the intracellular Yap1p concentration. We will also look for posttranslational modifications in Pdr1p and Pdr3p as result of YAPI overexpression leading to an enhanced activity of these transcription activators.

In the present research project we investigated the mechanisms underlying the resistance phenomenon in baker`s yeast to the drug diazaborine. We concentrated on those resistance phenomena that were mediated by cellular detoxification events. We were able to discover two genetic networks which are involved in this type of resistance. The first is based on the yeast equivalent of the transcription factor AP1, Yap1, which is known to be involved in various types of stress response. We discovered a "gain of function" allele of gene YAP1 which mediates diazaborine resistance by activating the genes for the cytoplasmatic pump protein Flr1 and the vacuolar membrane protein Ycf1. The second network that we have discovered and which is mediating diazaborine resistance is dependent on the transcription factors Pdr1 and Pdr3. We have discovered in a previous project "gain of function" alleles of PDR1 and PDR3 that cause high level diazaborine resistance. In the present work we have shown that the two alleles activate the efflux pumps Pdr5 and Snq2 which are thus part of this detoxification network. Recently we were able to show that the two networks are connected in that the transcription factor allele PDR1-12 activates YAP1. We believe that this observation will enhance our understanding of the complex series of reactions leading to stress response in yeast.