(A) Appearance of a slower migration Pop2p in post-log phase. Yeast strain D24 (wild-type diploid) was inoculated into YPD media and cultivated at 24°C. At each culture time indicated, protein sample was prepared from the cells and Pop2p was detected by Western blot analysis. (B) The 97th threonine is responsible for the mobility shift. Sixteen mutant Pop2p, harboring threonine to alanine substitutions (one had a triple replacement) at the indicated positions, were introduced into the pop2Δ-3 (A2280) cells. The cells were grown to post-log phase and Pop2p was detected by Western blotting. (C) Schematic illustration of Pop2p including the amino acid sequence around the 97th threonine residue.

(A) Phosphorylation of Pop2p. Exponentially growing D24 cells in YPD medium were collected by centrifugation, washed once with YP medium, and resuspended in YP medium. At the times indicated, Pop2p from the cells was detected by Western blot analysis. (B) Dephosphorylation of Pop2p. Exponentially growing D24 cells in YPD medium were transferred to YP medium, and glucose was added to the culture to a final concentration of 20 mM. Pop2p from the cells was detected by Western blotting. Effects of various carbon sources (C), glucose analogs (D), and mutations of hexokinase genes (E) on Pop2p phosphorylation. Exponentially growing D24 cells (in A and B) or A1643 (hxk1Δ hxk2Δ mutant, in C) in YPD medium (YPD) were transferred to YP medium for 60 min (YP), and indicated carbon sources were added to the culture to a final concentration of 20 mM, unless otherwise stated. After 10 min, protein samples were prepared from the cells and Pop2p was detected by Western blotting. (Glc) Glucose; (Frc) fructose; (Gal) galactose; (Gly) glycerol; (EtOH) ethanol; (L-Glc) L-glucose; (6-D-Glc) 6-deoxyglucose; (2-D-Glc) 2-deoxyglucose.

Detection, purification, and identification of the Pop2p peptide kinase. (A) Detection of Pop2 peptide-specific phosphorylation activity in phosphocellulose column fraction. Lysate of stationary-phase yeast cells was loaded on a phosphocellulose (P11) column. Protein was eluted with the NaCl concentrations indicated. The Pop2 peptide and T97A peptide kinase activities of each fraction were determined. (B) Elution profile of the final step of the Pop2 peptide kinase purification (ceramic hydroxyapatite chromatography). Pop2 peptide kinase activity (Kinase activity, CPM), K-phosphate concentration (K-phosphate, mS/mL), and protein concentration (Protein, A₂₈₀) of each fraction are shown. (C) Silver staining image of the SDS-PAGE-separated elution fractions of the column indicated in panel B. The molecular weights indicated on the left side were estimated by SDS-PAGE standards (Bench-Mark protein ladder, GIBCO BRL). (D) The purified Pop2 peptide kinase was Yak1p. Amino acid sequences of three peptides derived from purified 45-kD Pop2p peptide kinase were determined and analyzed as described in the Materials and Methods. The positions of the peptides determined are shown as lines under the structure of Yak1p.

Kinase activities in the yak1 null mutant strain. (A) In vitro peptide kinase activities of peptide kinase activity in the phosphocellulose (P11) column fractions in wild-type (YAK1; H213) and yak1Δ (H214) cell lysates. The stationary-phase cell lysate of each strain was loaded on a P11 column and then eluted with a 0 to 0.5 M NaCl gradient. Peptide kinase activity of each elution fraction was determined as described in the Materials and Methods. (B) In vivo phosphorylation of the Pop2p in wild type (YAK1; H213), yak1Δ (H214), H214 harboring pRS314, and H214 harboring YCpYAK1(pHIS012). Exponentially growing cells of each strain were washed by YP medium and resuspended in YPD medium (Glucose +) or YP medium (Glucose −). After 5 min, protein samples were prepared from the cells and Pop2p was detected by Western blotting.

Analysis of Yak1p using GST-Yak1p fusion protein. (A) GST-Yak1p phosphorylates Thr 97 of full-length Pop2p. Phosphorylation assays were performed as described in the Materials and Methods. (WT) Wild-type Yak1p; (KD, kinase dead) K398R mutant of Yak1p; (Thr) wild-type Pop2p; (Ala) alanine substitution mutant for the 97th threonine of Pop2p; (GST-Yak1p*) autophosphorylation product of GST-Yak1p; (MBP-Pop2p*) phosphorylated MBP-Pop2p; (MBP-Pop2p [CBB]) CBB staining of MBP-Pop2p. (B) In vitro Pop2 peptide kinase activity of GST-Yak1p was not regulated by glucose. The Pop2 peptide phosphorylation activities of GST fusion proteins purified from yeast after treatment with the glucose conditions indicated were determined. (C) Pop2p associates with Yak1p. GST fusion proteins were purified from yeast cells after 10 min incubation in YPD medium (Glc +) or YP (Glc −) and separated by SDS-PAGE, and the proteins were stained with CBB. The Pop2p copurified with the GST fusion proteins was detected using anti-Pop2p specific antibody (Pop2p [co-purified]). The endogenous Pop2p in each yeast cell was detected by Western blotting (Pop2p [endogenous]). (D) Yak1p associates with Bmh1/2p in a glucose-dependent manner. SDS-PAGE image of GST and GST-Yak1p purified in panel C. The amino acid sequences of peptides derived from Yak1p-associated proteins were determined and analyzed as described in the Materials and Methods.

Intracellular localization of GFP-Yak1p. (A) Localization of GFP-Yak1p is regulated by glucose. The yak1Δ (H214) cells harboring the single-copy and GFP-Yak1p-expressing plasmid (pHIS063) grown in SCM-Ura were collected and resuspended in SCM-Ura (+Glc) or SCM-Ura minus glucose (−Glc). After 3 min, fluorescent images were taken. (B) Effect of the glucose analogs on the nuclear localization of GFP-Yak1p. The glucose analogs shown were added to pHIS063/H214 cells precultured in SCM-Ura minus glucose. (Glc) D-glucose; (L-Glc) L-glucose; (6-Deo-Glc) 6-deoxy-D-glucose; (2-Deo-Glc) 2-deoxy-D-glucose. (C) Time course of the translocation of GFP-Yak1p. Images were taken for the time periods indicated after the pHIS063/H214 cells were transferred to SCM-Ura from SCM-Ura minus glucose (+Glc), or SCM-Ura minus glucose from SCM-Ura (−Glc).

Physiological roles of Pop2p phosphorylation by Yak1p. (A) Growth curves of the POP2-T97A cells (open circles) and wild-type cells (closed circles) and glucose concentration of each culture. Two independent experiments were performed, and the standard deviations of each duplicated experiment are indicated as error bars. (B) The phosphorylation of Pop2p by Yak1p is required for proper cell cycle arrest upon glucose deprivation. Exponentially growing wild-type (H213), POP2-T97A (A2079), and yak1Δ (H214) cells were transferred to YPD medium and YP medium. After 2 h culture time, cell cycle profiles of each sample were analyzed by FACS analysis.

Models of the Pop2p phosphorylation and Yak1p.