FIG. 2.

Tpk1p phosphorylates residues within the Crz1p NLS. (A) Phosphorylation of Crz1p fragments by Tpk1p. Recombinant GST-Tpk1p was combined with 2 μg each of full-length GST-Crz1p (FL), GST-Crz1p^1-180 (I), GST-Crz1p^181-340 (II), GST-Crz1p^341-440 (III), or GST-Crz1p^441-678 (IV) and then incubated with ATP and [γ-³²P]ATP for 40 min at 30°C. Samples were analyzed by SDS-PAGE and autoradiography. Fragment III (residues 341 to 440) contains the NLS. (B) Sequence of the Crz1p NLS, extended from residues 422 to 429 (28). Putative PKA phosphorylation sites are underlined (19). Residues mutated to alanine are marked with an asterisk. (C) Mutation of residues 409, 410, 423, 427, and 429 disrupts Tpk1p phosphorylation of Crz1p in vitro. Wild-type and mutant versions of recombinant GST-Crz1p^341-440 were incubated with GST-Tpk1p, ATP, and [γ-³²P]ATP. Samples were analyzed by SDS-PAGE and imaged on a Typhoon scanner for quantitation.