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. 2021 Nov 24;41(1):e108813. doi: 10.15252/embj.2021108813

Figure 4. The T557I point mutation in Sir3 abolishes Sae2‐Sir3 interaction.

Figure 4

  1. Schematic representation of the assay used to screen for SIR3 mutants deficient for Sae2 interaction while maintaining interaction with Sir4. The SIR3SaID fragment (464–728) was mutagenized by PCR, cloned in the pACT2 two‐hybrid plasmid and transformed into the reporter strain along with plasmids expressing LexA‐BD‐SAE2C and GAL4‐BD‐SIR4C fusion proteins. The reporter strain (yKD1991) bears a Gal4 binding sequence (Gal4BD) upstream of a HIS3 reporter gene, and a LexA binding sequence (LexABD) precedes a LacZ reporter gene. Transformants in which the gal4‐BD‐Sir4C and Sir3SaID‐gal4‐AD fragments interact were selected for HIS3 expression on ‐HIS + 3AT medium and subsequently screened for LacZ expression upon X‐gal coloration. Cells showing no LacZ expression were collected and the mutated sir3SaID‐GAL4‐AD was retrieved and sequenced.
  2. Representative images of two‐hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C.
  3. Representative images of two‐hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C.