Reciprocal Phosphorylation of Yeast Glycerol-3-Phosphate Dehydrogenases in Adaptation to Distinct Types of Stress

Phosphorylation of Gpd2 by Snf1. (A) Snf1 phosphorylates Gpd2 in a radiolabel kinase assay. TAP-tagged Gpd2 (WT or S72A mutant) purified from yeast was incubated with the bacterially expressed Snf1 catalytic domain and radiolabeled ATP, resolved by SDS-PAGE, and analyzed by autoradiography. (B) Mobility shift analysis indicates that Snf1 phosphorylates Gpd2 to high stoichiometry. TAP-tagged Gpd2 (WT or S72A mutant) was incubated alone, with λ protein phosphatase (λPP), or with the Snf1 catalytic domain in the presence of unlabeled ATP, resolved by Phos-tag PAGE, and analyzed by immunoblotting. (C) Gpd2 phosphorylation depends on Snf1 in vivo. Cultures of WT and snf1Δ and elm1Δ tos3Δ sak1Δ mutant yeast cells expressing Gpd2-His₆-GFP from its own promoter on a low-copy-number vector were grown to mid-exponential phase in SC-Leu medium containing 2% glucose (H). The cultures were then split and either left untreated or transferred into SC-Leu containing 0.05% glucose (L). After 90 min, cells were harvested and lysed and Gpd2 was partially purified and analyzed as described in the legend to Fig. 1C. (D) Reexpression of Snf1 rescues the Gpd2 phosphorylation defect of a snf1Δ mutant strain. The WT and snf1Δ mutant strains, as indicated, were transformed with an empty vector (−) or the same plasmid expressing SNF1 and then cotransformed with a plasmid expressing either Gpd2-His₆-GFP (top) or HA-tagged Mig1 (bottom). Cells were grown, treated, and lysed as described for panel C. Partially purified Gpd2 was subjected to Phos-tag PAGE (top), and total cell lysates containing Mig1 were subjected to standard SDS-PAGE (bottom) before immunoblotting with the indicated antibodies. (E) Phosphorylation of Gpd2 at Ser72 by Snf1 primes for phosphorylation at Ser75 by Yck1. Purified, HA-tagged Gpd2 was incubated with ATP and the indicated kinases, resolved by Phos-tag SDS-PAGE, and analyzed by immunoblotting. For the far right lane, after phosphorylation, the sample was treated with λPP. The arrow indicates the slowest-migrating dually phosphorylated species. (F) Priming-dependent phosphorylation of Ser75 in vivo. Cultures of either a gpd2Δ single mutant (top) or a snf1Δ mutant (bottom) expressing WT Gpd2-(His)₆-GFP or its S72A or S75A mutant form were treated and analyzed as described for panel C.

Phosphorylation of Gpd1 by Ypk1 and Ypk2 is sensitive to osmotic conditions. (A) TAP-tagged Gpd1 purified from yeast was incubated with ATP alone; in the presence Ypk1, Ypk2, or Sch9; or in the presence of λPP, as indicated. After incubation, samples were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-RXRXXS antibody to detect Gpd1 phosphorylation and with anti-HA antibody to detect total Gpd1. (B) Purified WT Gpd1 or Gpd1-S24A was incubated with [γ-³³P]ATP in the absence (−) or presence (+) of purified Ypk1. After incubation, samples were resolved by SDS-PAGE and analyzed by autoradiography (top), by staining with Coomassie blue to confirm the presence of the substrate (middle), and with anti-phospho-RXRXXS antibody as an independent confirmation of phosphorylation (bottom). (C) Purified WT Gpd1 and Gpd1-S24A were incubated with either Ypk1 or λPP in the presence of ATP and resolved by Phos-tag PAGE and analyzed by immunoblotting with anti-HA antibody. (D) Cultures of otherwise isogenic WT and ypk1^ts ypk2Δ mutant strains expressing Gpd1-His₆-GFP from its own promoter on a low-copy-number vector were grown to mid-exponential phase at 30°C and then shifted to 37°C. At the indicated times, samples were withdrawn and the harvested cells were lysed and analyzed as described in the legend to Fig. 1C. (E) Cultures of ypk1Δ ypk2Δ mutant cells expressing Gpd1-His₆-GFP and either WT Ypk1 (ypk2Δ, left) or Ypk1-as (ypk2Δ ypk1-as, right) were grown to mid-exponential phase and then treated with 1NM-PP1 or a vehicle control. After 30 min, cultures were analyzed for Gpd1 phosphorylation as described in the legend to Fig. 1D. (F) A culture of gpd1Δ mutant cells expressing Gpd1-His₆-GFP was grown to mid-exponential phase, and then samples were treated for 30 min with 1 M NaCl with or without 1.25 μM myriocin, as indicated. The cells were then analyzed as described for panel D. (G) Cells transformed with either an empty vector or the same vector expressing TAP-tagged Ypk1 from the GAL1 promoter were grown to mid-exponential phase and either not induced or induced with galactose. Cultures were then left untreated or treated with 1 M NaCl for 15 min prior to harvesting, cell lysis, and immunoprecipitation of Ypk1. Immobilized Ypk1 was incubated with ATP and purified, bacterially expressed GST-Gpd1. After incubation, samples were resolved by SDS-PAGE and immunoblotted with anti-phospho-RXRXXS to detect GST-Gpd1 phosphorylation, with anti-HA antibodies to determine the level of Ypk1, and with anti-GST antibodies to confirm that equivalent amounts of GST-Gpd1 were present. (H) Hyperosmotic shock blocks TORC2-dependent phosphorylation of Ypk1. Cultures of WT cells (strain BY4741) and an otherwise isogenic hog1Δ mutant, each expressing Ypk1^11A-myc as a reporter for TORC2-dependent phosphorylation at Thr622, as described in detail elsewhere (57), were grown to mid-exponential phase and split, and then one-half of each culture was exposed to 1 M sorbitol for 10 min. Cells were harvested and lysed, and the resulting extracts were resolved by Phos-tag PAGE and analyzed by immunoblotting with anti-c-myc MAb 9E10 to detect all Ypk1 species and with anti-phospho-p38 antibodies to detect activated, dually phosphorylated Hog1, as described in detail elsewhere (73). (I) Cultures of WT and hog1Δ mutant cells expressing tagged Gpd1-His₆-GFP (WT or S24A mutant) were grown to mid-exponential phase and then either left untreated or treated with 1 M NaCl for 30 min. Samples were then processed as described in the legend to Fig. 1C.

Phosphorylation decreases the catalytic activity of Gpd1 and Gpd2 and curtails glycerol production in vivo. (A) Gpd1 activity following phosphorylation by Ypk1. Gpd1-TAP (WT or S24A or S24E,S27E [SSEE] mutant form) purified from yeast was preincubated with ATP in the presence or absence of Ypk1 and then catalytic activity with DHAP as the substrate was measured by monitoring NADH consumption spectrophotometrically (top). In parallel, mobility on Phos-tag PAGE was used to assess the extent of phosphorylation (bottom). (B) Gpd2 activity following phosphorylation by Snf1. The catalytic activities and electrophoretic mobilities of Gpd2, Gpd2-S72A, and Gpd2-S72E,S75E were assessed as described for panel A following phosphorylation by Snf1. (C) Multisite phosphorylation has an additive inhibitory effect on Gpd2 activity. Purified Gpd2 was incubated with ATP in the absence (−) or presence of Snf1 alone, Yck1 alone, or both enzymes, and then enzymatic activity was assessed as described for panel B. In all of the panels, bars show average initial reaction rates (n = 3) and error bars show standard deviations.

Effect of Gpd2 mutation on glycerol production in vivo. Cultures of WT and gpd1Δ, gpd2Δ, or gpd1Δ gpd2Δ mutant yeast cells were transformed with either an empty low-copy-number vector (−) or the same plasmid expressing Gpd2, Gpd2-S72A, or Gpd2-S72E,S75E, Gpd1, Gpd1-S24A, or Gpd1-S24E,S27E (SSEE) as indicated. Cells were grown to exponential phase (A and B) or early stationary phase (C, D, G, and H), and the total glycerol concentration was determined as described in Materials and Methods. (E and F) Exponential-phase cultures (in medium containing 2% glucose) were transferred to fresh medium containing 0.05% glucose, and glycerol production was assayed 1 h later. Bars show the average glycerol concentrations across three separate cultures grown in parallel; error bars show standard deviations. *, P < 0.03 (compared to the identical strain expressing the WT Gpd isozyme, by unpaired one-tailed t test).

Both Gpd1 dephosphorylation and transcriptional induction are essential mechanisms for survival and long-term growth at high osmolarity. Ten-fold serial dilutions of WT or gpd1Δ or gpd1Δ gpd2Δ mutant cells, as indicated, expressing WT Gpd1, Gpd1-S24A (GPD1 SA), or Gpd1-S24E,S27E (GPD1 EE) from either the native GPD1 promoter (top and middle panels) or the GPD2 promoter (bottom panel) were spotted onto agar plates containing SC-Leu with or without 1 M NaCl or 1 M sorbitol and incubated at 30°C for 3 days.