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1.
Figure 3

Figure 3. From: Yeast PAS kinase coordinates glucose partitioning in response to metabolic and cell integrity signaling.

Differential roles for Psk1 and Psk2 in Ugp1 phosphorylation activated by cell integrity stress and nonfermentative carbon source. (A) Cells of the indicated genotype were grown in triplicate in YPAD to an OD600 of 0.6, treated for 2 h with 0.05% SDS and assayed for Ugp1 phosphorylation. Percent Ugp1 phosphorylation (±s.d.) is displayed. (B) Cells of the indicated genotype were grown in triplicate in YPA-Raffinose to an OD600 of 0.6 and assayed for Ugp1 phosphorylation. Percent Ugp1 phosphorylation (±s.d.) is displayed. The strains used were JRY245 (PSK1 PSK2), JRY276 (psk1 PSK2), JRY277 (PSK1 psk2) and JRY 278 (psk1 psk2).

Julianne H Grose, et al. EMBO J. 2007 November 28;26(23):4824-4830.
2.
Figure 4

Figure 4. From: Yeast PAS kinase coordinates glucose partitioning in response to metabolic and cell integrity signaling.

Activation of Psk1 and Psk2 by cell integrity stress and growth in nonfermentative carbon source. Kinase assay and western blot of immunoprecipitated Psk1 (A) or Psk2 (B). Cells expressing the Psk1-TAP and Psk2-TAP fusion proteins (or controls) were grown in either YPAD or YPA-Raffinose (YPARaff) to an OD600 of 0.6 and then either the harvested (YPAD and YPARaff) or the YPAD grown cells were subjected to 0.05% SDS for 2 h (+SDS). PAS kinase was immunoprecipitated and assayed for kinase activity using ϒ[32P]ATP and Ugp1 as a substrate (upper panels). PAS kinase protein was visualized by western blotting (lower panels). Identically treated duplicates are shown for each condition. The strains used were JRY405 (PSK1-TAP∷kanMX4) and JRY406 (PSK2-TAP∷kanMX4).

Julianne H Grose, et al. EMBO J. 2007 November 28;26(23):4824-4830.
3.
Figure 6

Figure 6. From: Yeast PAS kinase coordinates glucose partitioning in response to metabolic and cell integrity signaling.

A model for PAS kinase activation by nonfermentative carbon source and cell integrity stress in S. cerevisiae. Autoinhibited PAS kinase can be activated by either of two signals. Nonfermentative carbon sources, acting via the Snf1 kinase complex, activate PAS kinase perhaps through the production of a PAS domain-binding metabolite (star). PAS kinase can also be activated by cell integrity stress acting through the Wsc protein family. Activated PAS kinase phosphorylates Ugp1, causing a conformational change in the protein, which leads to increased glucan synthesis (structural carbohydrate) at the expense of glycogen synthesis (storage carbohydrate). A full color version of this figure is available at the EMBO Journal Online.

Julianne H Grose, et al. EMBO J. 2007 November 28;26(23):4824-4830.
4.
Figure 2

Figure 2. From: Yeast PAS kinase coordinates glucose partitioning in response to metabolic and cell integrity signaling.

The glucose derepression pathway stimulates phosphorylation of Ugp1. (A) Nonfermentative carbon sources stimulate Ugp1 phosphorylation. Wild-type cells (JRY 245) were grown in triplicate to an OD600 of 0.6 in YPA with the carbon source indicated, were assayed for Ugp1-phosphorylation and the percent of phosphorylated Ugp1 (±s.d.) is shown. (B) Snf1 kinase activity is necessary and sufficient for PAS kinase-dependent Ugp1 phosphorylation in response to the metabolic stimulus, but not cell integrity stress. Cells of the indicated genotype were grown in triplicate in either YPAD or YPA-Raffinose (Raff) to an OD600 of 0.6 and then either harvested (YPAD and Raff) or subjected to 0.05% SDS for 2 h (YPAD+SDS). Cells were assayed for Ugp1 phosphorylation and percent Ugp1 phosphorylation (±s.d.) is displayed. The strains used were JRY245 (wild type), JRY456 (REG1 snf1) JRY506 (reg1 SNF1) and JRY514 (reg1 snf1).

Julianne H Grose, et al. EMBO J. 2007 November 28;26(23):4824-4830.
5.
Figure 5

Figure 5. From: Yeast PAS kinase coordinates glucose partitioning in response to metabolic and cell integrity signaling.

Activation of constitutively expressed Psk1 and Psk2 by cell integrity stress and growth in nonfermentative carbon source. (A) Strains wherein PSK2 was expressed under the control of each of four different promoters were grown to an OD600 of 0.6 in YPAD or YPA-Raffinose as indicated. They were then either harvested (YPAD and YPA-Raffinose) or subjected to 0.05% SDS for 2 h (YPAD+SDS). Cells were assayed for Ugp1 phosphorylation and percent Ugp1 phosphorylation (±s.d.) is displayed. (B, C) Kinase assay and western blot of immunoprecipitated constitutively expressed Psk1 (B) or Psk2ΔN (C), which lacks the N-terminal 819 residues of Psk2. The strain expressing a Psk1-TAP fusion (JRY684) (B) or Psk2ΔN-TAP fusion (JRY674) (C) from the STE20 promoter was grown to an OD600 of 0.6 and then either harvested (YPAD and YPARaff) or the YPAD samples were subjected to 0.05% SDS for 2 h (+SDS). PAS kinase was immunoprecipitated and assayed for kinase activity using ϒ[32P]ATP and Ugp1 as a substrate (upper panel). PAS kinase protein was visualized by western blotting (lower panel). Identically treated duplicates are shown for each condition. The promoters and strains used are described in Materials and Methods.

Julianne H Grose, et al. EMBO J. 2007 November 28;26(23):4824-4830.
6.
Figure 1

Figure 1. From: Yeast PAS kinase coordinates glucose partitioning in response to metabolic and cell integrity signaling.

Cell integrity stress activates PAS kinase-dependent phosphorylation of Ugp1. (A) Ugp1 activity in fractions of crude yeast extracts separated by MonoQ. Wild-type cells (JRY 245) were treated with the concentration of SDS indicated for 2 h. (B) Quantification of the percent phosphorylation of Ugp1 from cells treated as indicated. Cells were grown in triplicate to an OD600 of 0.6 before treatment with SDS, calcofluor white (CW), high temperature (37°C) or chlorpromazine (CPZ) for 2 h. Cells were assayed for Ugp1-phosphorylation and the percent of phosphorylated Ugp1 (±s.d.) is shown. (C) Wsc1 overexpression is sufficient to activate PAS kinase. The wild-type strain (JRY245) containing either pRS426 (vector) or pRS426-WSC1 (WSC1) was grown to saturation in SD-uracil medium and then diluted and grown in YPAD in triplicate to an OD600 of 0.6 before harvest (YPAD) or treatment with SDS for 2 h (YPAD+SDS). Cells were assayed for Ugp1-phosphorylation and the percent of phosphorylated Ugp1 (±s.d.) is shown. (D) Deletion of PSK2 exacerbates the temperature sensitive growth phenotype of a rom2 deletion mutant. Strains of the indicated genotype were grown to saturation and serially diluted in water. These diluted samples were spotted onto minimal glucose medium lacking uracil, and incubated at 37°C for 3 days. The strains used were JRY245 (wild type+pRS416), JRY853 (psk2 ROM2+pRS416), JRY854 (PSK2 rom2+pRS416), JRY858 (rom2 psk2+pRS416) and JRY858 (rom2 psk2+pRS416-PSK2).

Julianne H Grose, et al. EMBO J. 2007 November 28;26(23):4824-4830.

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