FIGURE 1:

Optimization of fluorogen binding to FAP-Ste2. (A) Cells (yAEA152) expressing FAP-Ste2 from the endogenous STE2 locus were grown to mid–exponential phase in BSM, incubated with fluorogen (0.4 mM final concentration) either on ice without agitation or at 30°C with agitation (1200 rpm) for the time periods indicated, washed and collected by brief centrifugation, and viewed by fluorescence microscopy (top panels) and bright field microscopy (bottom panels), as described under Materials and Methods. Scale bar, 5 μm. (B) As in A, except the cells were propagated in BSM buffered at the indicated pH values (with either 100 mM phosphate or 50 mM succinate, as appropriate), incubated with fluorogen for 15 min at 30°C, and then imaged. (C) Portions of the same culture as in A were incubated for 15 min at 30°C in the absence (–) or presence (+) of fluorogen, and then samples of a set of fivefold serial dilutions were spotted using a multiprong inoculator on an agar plate containing BSM, and, after incubation for 48 h at 30°C, the resulting growth was recorded.