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. 2019 Mar 4;218(3):961–976. doi: 10.1083/jcb.201807154

Figure 3.

Figure 3.

Rab5 GTPases and their GTP loading are necessary for efficient phosphorylation and activation of Ypk1 by TORC2. (A) WT (BY4741) or otherwise isogenic yMLT9 (muk1Δ vps9Δ), yMLT3 (vps9Δ), or yMLT5 (muk1Δ) cells, all expressing Ypk17A-myc from its native promoter on a CEN plasmid (pFR252), were grown to mid-exponential phase and treated with ethanol or an equivalent volume of the same solvent containing AbA (1.8 µM final concentration). After incubation for 2 h, the cells were harvested, and extracts were prepared, resolved by Phos-tag SDS-PAGE, transferred to a nitrocellulose filter, stained with Ponceau S, and then analyzed by immunoblotting (IB), as in Materials and methods. (B) As in A, except WT (BY4741) and yMLT42 (vps21Δ ypt52Δ ypt53Δ) cells were compared. (C) Cultures of WT (BY4741) and JTY6142 (ypk1Δ) cells were plated in fivefold serial dilutions on growth medium in the absence (–) or presence (+) of AbA (40 nM final concentration). (D) As in C, for WT (BY4741), yMLT9 (muk1Δ vps9Δ), yMLT42 (vps21Δ ypt52Δ ypt53Δ), and yMLT42 (ypt7Δ) cells. (E) As in C, for WT (BY4741) cells transformed with either empty multicopy vector (EV; pRS426) or the same vector expressing from its native promoter Msb3-FLAG (pMLT111).