Identification of Sortin2 hypersensitive mutants. (A) Primary screen: 4827 haploid deletion strains were challenged with 1% DMSO (DMSO) or Sortin2 at 2 μg/ml (4.7 μM) (Sortin2). Culture media was analyzed for the presence of CPY by dot blot assay. Clones secreting significant amounts of CPY compared to the negative control were selected as putative mutants. The arrowheads show the primary hits. (B) Secondary screen: 243 putative mutants were tested on DMSO and varying concentrations of Sortin2. Threshold was defined as the concentration of Sortin2 that had no residual CPY secretion on a wild type parental line (wt, 2 μg/ml). For CPY secretor strains, the strain was considered as hypersensitive to Sortin2 if the CPY secretion in the presence of Sortin2 was in excess of that displayed in the presence of DMSO. Different scenarios are shown as examples of the secondary screen results: a false positive strain (mam1), a Sortin2 hypersensitive strain (ubp3), and two Sortin2 hypersensitive strains secreting CPY on control conditions (hur1 and vps55).
Norambuena et al. BMC Chemical Biology 2008 8:1 doi:10.1186/1472-6769-8-1