Evaluation of FKBP and DHFR based destabilizing domains in Saccharomyces cerevisiae

Abstract

Two orthogonal destabilizing domains have been developed based on mutants of human FKBP12 as well as bacterial DHFR and these engineered domains have been used to control protein concentration in a variety of contexts in vitro and in vivo. FKBP12 based destabilizing domains cannot be rescued in the yeast Saccharomyces cerevisiae; ecDHFR based destabilizing domains are not degraded as efficiently in S. cerevisiae as in mammalian cells or Plasmodium, but provide a starting point for the development of domains with increased signal-to-noise in S. cerevisiae.

Figure 1

a) Flow cytometry data from Y7092 expressing yeGFP-DD_FKBP; untransformed Y7092 (red), YRR030 (Y7092 ho::P_SOD1:yeGFP-DD_FKBP::KANMX4 (blue), YRR030 + 5μM Shield-1 (green), YRR030 +100nM rapamycin (orange). YRR030 +1μM FK506 (green) is shown in the right panel, for clarity. b) Flow cytometry data from Y7092 expressing yeGFP-DD_DHFR; untransformed Y7092 (red), YRR009 (Y7092 ho::P_SOD1:yeGFP-DD_DHFR::NATMX4)(blue), YRR009 + 100μM TMP (green). c) Flow cytometry data from NIH 3T3 cells; untransduced (red), transduced with YFP-DD_DHFR (blue), transduced with YFP-DD_DHFR +10μM TMP (green).

Figure 2

a) Flow cytometry data from Y7092 grown at 37C (red), YRR009 grown at 37C (blue) and YRR009 +100μM TMP at 37C (green). b) Flow cytometry data from yeast grown to near saturation, Y7092 (red), YRR009 (blue) YRR009 +100μM TMP (green). c) same as in b), but grown in YPEG.

Figure 3

Cell-cycle analysis by flow cytometry; a) W303a cells were grown to overnight in 2x YPD, diluted to a starting concentration of A₆₀₀=0.05–0.1 and grown for two generations prior to addition of various concentrations of alpha factor 2hrs prior to fixation and staining (as indicated). b) W303a derivatives treated as in a), as indicated.