Characterisation of Schizosaccharomyces pombe wis2+, a cyclophilin-40 homologue

• Main Author: Gaskell, Terri Louise
• Published: University of Edinburgh 2000
• Subjects: 579
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.651350

The work described in this thesis is concerned with further genetic and molecular analysis of <i>wis2⁺</i> The aim of this work was to establish a cellular function for <i>wis2⁺</i> and to explain the effect of overexpression in the triple mutant strain described above. Using an <i>in vitro</i> binding assay, a number of proteins that show a physical interaction with Wis2 were isolated. These proteins were identified by peptide sequencing. Five were found to be ribosomal proteins. Another, ~85kD, was identified as <i>S. pombe </i>Hsp90; the intensity of the interacting band is increased, and the interaction is more consistent, if the extract is made from <i>S. pombe </i>cells which have previously been heat shocked. Genetic analysis revealed that overexpressing of <i>wis2⁺</i> lowers the restrictive temperature of a number of <i>cdc2</i> mutant alleles which arrest at G2-M. This effect of enhancing a cell cycle block is in contrast to the situation in the <i>cdc25-22 wee1-10 win1-1 </i>background where the G2-M block is relieved by <i>wis2⁺</i> overexpression. An extensive genetic screen was conducted to isolate mutations that are synthetically lethal with <i>wis2</i><i>D, </i>with the aim of identifying functionally redundant genes or downstream targets of <i>wis2</i>. No synthetic lethal interactions were identified. Indirect immunofluorescence studies established that Wis2 is localised exclusively in the cytoplasm. Consistent with this, a consensus nuclear export signal was identified in the C-terminus of Wis2. Structure-function analysis showed that overexpression of the C-terminal domain of Wis2 alone has the same activity as full length Wis2 <i>in vivo</i>. Overexpression of the N-terminus alone had no observable effect in the genetic backgrounds tested. The C-terminal domain of Wis2 was shown to be responsible for the interaction with Hsp90 in the <i>in vitro</i> binding experiment; this is consistent with results from other systems where the TPR domain was found to be responsible for Hsp90 binding.