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Sul1 and Sul2 sulfate transceptors signal to protein kinase A upon exit of sulfur starvation.

Kankipati HN, Rubio-Texeira M, Castermans D, Diallinas G, Thevelein JM - J. Biol. Chem. (2015)

Bottom Line: Overall, our data suggest that transceptors can undergo independent conformational changes, each responsible for triggering different downstream processes.The Sul1 and Sul2 transceptors are the first identified plasma membrane sensors for extracellular sulfate.High affinity transporters induced upon starvation for their substrate may generally act as transceptors during exit from starvation.

View Article: PubMed Central - PubMed

Affiliation: From the Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium, the Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium, and.

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The addition of sulfate to sulfate-starved cells activates PKA targets through Sul1 and Sul2.A, activation of trehalase. B, phosphorylation of Nth1-HA detected by Western blot with phospho-specific antibodies in wild type cells and mutant sul1Δ sul2Δ cells. The phospho-specific antibody raised against phosphorylated Ser-21 of Nth1 was used to monitor the phosphorylation status of Nth1-HA. Anti-HA antibody was used to monitor the level of Nth1-HA. C, mobilization of trehalose. D, mobilization of glycogen. E, loss of heat stress tolerance. Cells were subjected to a heat shock at 52 °C for 20 min before or 6 h after the addition of sulfate, and serial dilutions of the cultures were then spotted on plates with rich medium and allowed to grow at 30 °C. Untreated cells were analyzed in parallel as a control. Shown are expression levels relative to the expression at time 0 of heat shock protein gene HSP12 (F) and ribosomal protein gene RPL25 (G), upon the addition of 3 mm sulfate to sulfur-starved cells. Strains are shown as follows: wild type (●), sul1Δ (○), sul2Δ (▴), and sul1Δ sul2Δ (▵). H, intracellular level of cAMP in wild type (●) and sul1Δ sul2Δ (○) cells at the indicated times before and after the addition of 3 mm sulfate to sulfur-starved cells.
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Figure 1: The addition of sulfate to sulfate-starved cells activates PKA targets through Sul1 and Sul2.A, activation of trehalase. B, phosphorylation of Nth1-HA detected by Western blot with phospho-specific antibodies in wild type cells and mutant sul1Δ sul2Δ cells. The phospho-specific antibody raised against phosphorylated Ser-21 of Nth1 was used to monitor the phosphorylation status of Nth1-HA. Anti-HA antibody was used to monitor the level of Nth1-HA. C, mobilization of trehalose. D, mobilization of glycogen. E, loss of heat stress tolerance. Cells were subjected to a heat shock at 52 °C for 20 min before or 6 h after the addition of sulfate, and serial dilutions of the cultures were then spotted on plates with rich medium and allowed to grow at 30 °C. Untreated cells were analyzed in parallel as a control. Shown are expression levels relative to the expression at time 0 of heat shock protein gene HSP12 (F) and ribosomal protein gene RPL25 (G), upon the addition of 3 mm sulfate to sulfur-starved cells. Strains are shown as follows: wild type (●), sul1Δ (○), sul2Δ (▴), and sul1Δ sul2Δ (▵). H, intracellular level of cAMP in wild type (●) and sul1Δ sul2Δ (○) cells at the indicated times before and after the addition of 3 mm sulfate to sulfur-starved cells.

Mentions: Previous work has shown that sulfate addition to sulfur-starved cells on a glucose-containing medium triggers activation of trehalase, a classical read-out for rapid PKA activation in vivo in yeast (27). We now show that this sulfate-induced activation of PKA requires one of the two sulfate transporters, either Sul1 or Sul2 (Fig. 1A). Using site- and phospho-specific antibodies, we show that activation of trehalase is correlated with phosphorylation on the PKA consensus site, Ser-21 (Fig. 1B), similar to trehalase activation by glucose and nitrogen sources in appropriately starved cells (28). We next confirmed the requirement of Sul1 or Sul2 for sulfate-induced activation of PKA using several other in vivo read-outs for activation of the PKA pathway. After the addition of sulfate to sulfur-starved cells, the carbohydrates trehalose (Fig. 1C) and glycogen (Fig. 1D) were mobilized, heat stress tolerance dropped (Fig. 1E), expression of the heat shock gene HSP12 was down-regulated (Fig. 1F), and expression of the ribosomal gene RPL25 was up-regulated (Fig. 1G). All of these sulfate-induced processes required the presence of either Sul1 or Sul2 (Fig. 1, A–G). Consistent with previous results (27), sulfate activation of the PKA targets in sulfur-starved cells with BY genetic background was not associated with an increase in the cAMP level (Fig. 1H). In the sul1Δ sul2Δ strain, there was a residual phosphorylation signal for trehalase (Fig. 1B) that correlated with a residual increase in its activity (Fig. 1A). Also for the other PKA targets, trehalose mobilization (Fig. 1C), glycogen mobilization (Fig. 1D), loss of heat stress tolerance (Fig. 1E), reduced expression of HSP12 (Fig. 1F), and enhanced expression of RPL25 (Fig. 1G), there was a residual effect in the sul1Δ sul2Δ strain. This residual effect is probably due to uptake of sulfate through a third sulfate carrier with much lower affinity for sulfate than Sul1 and Sul2 because the sul1Δ sul2Δ strain can grow with a very high sulfate concentration (>20 mm) as the sole source of sulfur in the medium.2


Sul1 and Sul2 sulfate transceptors signal to protein kinase A upon exit of sulfur starvation.

Kankipati HN, Rubio-Texeira M, Castermans D, Diallinas G, Thevelein JM - J. Biol. Chem. (2015)

The addition of sulfate to sulfate-starved cells activates PKA targets through Sul1 and Sul2.A, activation of trehalase. B, phosphorylation of Nth1-HA detected by Western blot with phospho-specific antibodies in wild type cells and mutant sul1Δ sul2Δ cells. The phospho-specific antibody raised against phosphorylated Ser-21 of Nth1 was used to monitor the phosphorylation status of Nth1-HA. Anti-HA antibody was used to monitor the level of Nth1-HA. C, mobilization of trehalose. D, mobilization of glycogen. E, loss of heat stress tolerance. Cells were subjected to a heat shock at 52 °C for 20 min before or 6 h after the addition of sulfate, and serial dilutions of the cultures were then spotted on plates with rich medium and allowed to grow at 30 °C. Untreated cells were analyzed in parallel as a control. Shown are expression levels relative to the expression at time 0 of heat shock protein gene HSP12 (F) and ribosomal protein gene RPL25 (G), upon the addition of 3 mm sulfate to sulfur-starved cells. Strains are shown as follows: wild type (●), sul1Δ (○), sul2Δ (▴), and sul1Δ sul2Δ (▵). H, intracellular level of cAMP in wild type (●) and sul1Δ sul2Δ (○) cells at the indicated times before and after the addition of 3 mm sulfate to sulfur-starved cells.
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Figure 1: The addition of sulfate to sulfate-starved cells activates PKA targets through Sul1 and Sul2.A, activation of trehalase. B, phosphorylation of Nth1-HA detected by Western blot with phospho-specific antibodies in wild type cells and mutant sul1Δ sul2Δ cells. The phospho-specific antibody raised against phosphorylated Ser-21 of Nth1 was used to monitor the phosphorylation status of Nth1-HA. Anti-HA antibody was used to monitor the level of Nth1-HA. C, mobilization of trehalose. D, mobilization of glycogen. E, loss of heat stress tolerance. Cells were subjected to a heat shock at 52 °C for 20 min before or 6 h after the addition of sulfate, and serial dilutions of the cultures were then spotted on plates with rich medium and allowed to grow at 30 °C. Untreated cells were analyzed in parallel as a control. Shown are expression levels relative to the expression at time 0 of heat shock protein gene HSP12 (F) and ribosomal protein gene RPL25 (G), upon the addition of 3 mm sulfate to sulfur-starved cells. Strains are shown as follows: wild type (●), sul1Δ (○), sul2Δ (▴), and sul1Δ sul2Δ (▵). H, intracellular level of cAMP in wild type (●) and sul1Δ sul2Δ (○) cells at the indicated times before and after the addition of 3 mm sulfate to sulfur-starved cells.
Mentions: Previous work has shown that sulfate addition to sulfur-starved cells on a glucose-containing medium triggers activation of trehalase, a classical read-out for rapid PKA activation in vivo in yeast (27). We now show that this sulfate-induced activation of PKA requires one of the two sulfate transporters, either Sul1 or Sul2 (Fig. 1A). Using site- and phospho-specific antibodies, we show that activation of trehalase is correlated with phosphorylation on the PKA consensus site, Ser-21 (Fig. 1B), similar to trehalase activation by glucose and nitrogen sources in appropriately starved cells (28). We next confirmed the requirement of Sul1 or Sul2 for sulfate-induced activation of PKA using several other in vivo read-outs for activation of the PKA pathway. After the addition of sulfate to sulfur-starved cells, the carbohydrates trehalose (Fig. 1C) and glycogen (Fig. 1D) were mobilized, heat stress tolerance dropped (Fig. 1E), expression of the heat shock gene HSP12 was down-regulated (Fig. 1F), and expression of the ribosomal gene RPL25 was up-regulated (Fig. 1G). All of these sulfate-induced processes required the presence of either Sul1 or Sul2 (Fig. 1, A–G). Consistent with previous results (27), sulfate activation of the PKA targets in sulfur-starved cells with BY genetic background was not associated with an increase in the cAMP level (Fig. 1H). In the sul1Δ sul2Δ strain, there was a residual phosphorylation signal for trehalase (Fig. 1B) that correlated with a residual increase in its activity (Fig. 1A). Also for the other PKA targets, trehalose mobilization (Fig. 1C), glycogen mobilization (Fig. 1D), loss of heat stress tolerance (Fig. 1E), reduced expression of HSP12 (Fig. 1F), and enhanced expression of RPL25 (Fig. 1G), there was a residual effect in the sul1Δ sul2Δ strain. This residual effect is probably due to uptake of sulfate through a third sulfate carrier with much lower affinity for sulfate than Sul1 and Sul2 because the sul1Δ sul2Δ strain can grow with a very high sulfate concentration (>20 mm) as the sole source of sulfur in the medium.2

Bottom Line: Overall, our data suggest that transceptors can undergo independent conformational changes, each responsible for triggering different downstream processes.The Sul1 and Sul2 transceptors are the first identified plasma membrane sensors for extracellular sulfate.High affinity transporters induced upon starvation for their substrate may generally act as transceptors during exit from starvation.

View Article: PubMed Central - PubMed

Affiliation: From the Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium, the Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium, and.

Show MeSH
Related in: MedlinePlus