Figure 4.

Mutation T210A in Snf1 impairs interaction with Snf4 in the two-hybrid system. (A) Strains were GGY1∷171 or CTY10–5d. Fusion proteins were expressed from pRJ57 and pRJ58 (23), pSD4 and pSG1 (gifts of Z. Xue & T. Melese, Columbia University, New York; see ref. 23), and pGAD-SNF1T210A (19). Transformants were grown in SC + 2% galactose/2% glycerol/2% ethanol/0.05% glucose. Values are the average β-gal activity of three to four transformants. GBD, GAL4 DNA-binding domain; LexA₈₇, LexA DNA-binding domain. (B) Immunoblot of GAD-Snf1 and GAD-Snf1T210A proteins. Protein extracts were prepared from representative transformants assayed in (A) carrying (lanes 1 and 2) GBD-Snf4, (lanes 3 and 4) LexA₈₇-Snf4, and (lanes 5 and 6) LexA-Snf4. Proteins (50 μg) were separated by an SDS/7.5% polyacrylamide gel and detected by immunoblotting by using affinity-purified anti-Snf1 antibodies (1) and enhanced chemiluminescence with ECL reagents (Amersham).