Figure 5.

Hrr25p is not the only Crz1p kinase. (A) Crz1p phosphorylation state differs in vivo and in vitro. For the in vitro assay, 0.1 µg recombinant GST-Crz1p was incubated for 30 min ± 0.03 µg GST-Hrr25 and 200 µM ATP. For the in vivo experiment, wild-type yeast (YPH499) expressing GST-Crz1p (pLMB119) were treated for 30 min with 2 µg/mL FK506 or solvent, and extracts were prepared. Proteins were analyzed by SDS-PAGE followed by Western blotting with an α-GST antibody. (B) GST-Crz1p is phosphorylated to a lesser extent by Hrr25p-depleted extracts. Five micrograms recombinant GST-Crz1p immobilized on glutathione sepharose was incubated with 250 µg extract prepared from wild-type (YPH499) or KKY387 cells grown in galactose (expressing Hrr25p) or in glucose (Hrr25p-depleted), γ³²P-ATP, and an ATP regenerating system, and incubated for 40 min. Samples were analyzed by SDS-PAGE and autoradiography. The lower panel shows the depletion of Hrr25p from the extracts by Western blot using an α-HA antibody.