Figure 1.

Schematic representation of the strategy used to study the FKBP–rapamycin–FRB complex. (Left) FKBP and FRB are fused to one of the two complementary fragments of murine DHFR (F[1,2] and F[3]) to generate FRB–F[1,2] and FKBP–F[3]. The addition of rapamycin induces the association of FKBP with FRB, which in turn drives the reconstitution of DHFR (F-[1,2] + F-[3]), allowing DHFR-negative cells expressing these constructs to grow in medium lacking nucleotides. (Right) The fluorescence assay is based on high-affinity binding of the specific DHFR inhibitor fMTX to reconstituted DHFR. fMTX passively crosses the cell membrane, binds to reconstituted DHFR, and is thus retained in the cell. Unbound fMTX is released rapidly from the cells by active transport. Detection of bound and retained fMTX can then be detected by fluorescence microscopy, FACS, or fluorescence spectroscopy.