FIG 2.

Distinct regulation and functions of two substrates of Ssp1. (A) Comparison of Cdr2-pT166 and Ssp2-pT189 at different glucose levels. Cells were shifted from 3% glucose to different glucose levels for 10 min or 3 h, and then whole-cell extracts were blotted. No tag, strain without integrated epitope tags. (B) Physical tethering of Ssp1 and Ssp2 induces Ssp2-pT189 in high glucose. GBP binds to GFP with nanomolar affinity. The indicated strains were grown in high (2%) or low (0.1%) glucose, and whole-cell extracts were probed with the indicated antibodies. (C) Comparison of Ssp2-pT189 levels in whole-cell extracts from wild-type and pom1Δ cells grown under 2% or 0.1% glucose levels. (D) Phosphorylation of Ssp2-T189 but not Cdr2-T166 is required for cell growth under low-glucose conditions. Tenfold serial dilutions of the indicated strains were spotted on EMM4S with 2% or 0.1% glucose. Amk2 and Cbs2 are β and γ subunits of the AMPK heterotrimer, respectively. (E) Detection of Ssp2-pT189 in whole-cell extracts from the indicated strains grown in 0.1% or 2% glucose. The asterisks indicate background bands, and the arrowhead marks a specific Ssp2-pT189 band. (F) Cell lengths of dividing, septated cells of the indicated strains (means ± standard deviations [SD]; n > 50 for each value). The P values from two-tailed Student's t tests are indicated with brackets for specific pairs.