Copper sensitive mutants of <i>Hansenula polymorpha</i>

Abstract

Copper is an essential trace element as it is an important cofactor in electron transport and for many redox-actjve metalloenzymes. However, due to its redox activity, copper can be highly toxic when present in excess intracellularly, because of its ability to generate reactive oxygen species and damage proteins, membranes and nucleic acids. The thermotolerant yeast Hansenula polymorpha represents an interesting model for investigating the mechanisms evolved by cells in response to potentially cytotoxic metal ions. H. polymorpha can easily adapt to high copper concentrations by repeated culturing of wild type cells in the presence of successively increasing concentrations of copper sulphate. Moreover, this yeast is able to grow on vanadate concentrations in the millimolar range, while most cells are inhibited by micromolar concentrations of this metal ion. As part of an investigation programme concerning the understanding of the mechanisms responsible for heavy metal resistance in the yeast H. polymorpha, we have isolated 75 cos (copper sensitive) recessive mutants, 29 of which harbour pleiotropic mutations. Twenty-six of these that present vas (vanadate sensitive) cos or vas cos vpa (vacuolar proteolitic activity defective) phenotypes are part of the vas collection of mutants described in the accompanying abstract (see Tagliapietra Orciani et al.). The remaining 3 mutants present a cos vpa phenotype. All these mutants are currently being subjected to complementation analysis. Moreover, in two of them, one cos and one cos vpa, the wild type phenotype has been restored by means of transformation with a H. polymorpha genomic library. The complementing plasmids are under study