FIG. 2.
Afr1 is a Glc7-targeting subunit. (A) Two-hybrid analysis of Afr1 and Glc7. Three representative transformants are shown for each interaction tested. Dark color reflects activation of the lacZ reporter gene. The plasmids used are as follows: for AFR1, pBTM116-AFR1; for afr1-Δ474, pBTM116-afr1-Δ474; for afr1VA/FA, pJB20; for GLC7, pHH148 or pCDV471 (used for BNI4-GLC7 two-hybrid analysis); vector, pACTII; for BNI4, pAR16. Units of β-galactosidase activity are reported on the right. (B) FRET analysis of Afr1 and Glc7. MATa strains JB241-5B (Afr1-mCitrine, Glc7-mCFP), JB241-19A (Afr1-mCitrine), and JB229-2B (Glc7-mCFP) were treated with 10−7 M α-factor for 2 h and imaged with CFP, YFP, and FRET filter sets. Bar, 5 μm. Arrows mark regions where FRET signal was observed.