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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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Mutagenesis of the putative proton binding sites, Glu-427 in Sul1 and Glu-443 in Sul2, blocks the uptake of sulfate but does not affect the signaling function.A, ClustalX sequence alignment of sulfate transporters from different organisms showing the conserved glutamic acid residue (Glu-427 in Sul1 and Glu-443 in Sul2) (in green) and other conserved residues (in lavender). Bottom, the position of all mutagenized residues, including Glu-427 in Sul1 and Glu-443 in Sul2, is indicated on the predicted topology of Sul1 and Sul2. B, Sul1,2-HA expression level as detected by Western blot of immunoprecipitated Sul1-HA, Sul2-HA, and their mutant forms in membrane-enriched P13 fractions isolated from cell cultures before and after the addition of 3 mm sulfate. C, uptake rate of 0.1 mm [35S]sulfate in Sul1-HA-expressing (black bar) and Sul1E427Q-HA-expressing (gray bar) sulfur-starved cells. D, uptake rate of 0.1 mm [35S]sulfate in Sul2-HA-expressing (black bar) and Sul2E443Q-HA-expressing (gray bar) sulfur-starved cells. E, activation of trehalase in Sul1-HA (●), Sul1E427Q-HA (○), Sul2-HA (▴), and Sul2E443Q-HA (▵) upon the addition of 3 mm sulfate. F, relative expression of HSP12 in sulfur-starved sul1Δ sul2Δ cells expressing Sul1-HA (●), Sul1E427Q-HA (○), Sul2-HA (▴), and Sul2E443Q-HA (▵) upon the addition of 3 mm sulfate. Error bars, S.D.
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Figure 5: Mutagenesis of the putative proton binding sites, Glu-427 in Sul1 and Glu-443 in Sul2, blocks the uptake of sulfate but does not affect the signaling function.A, ClustalX sequence alignment of sulfate transporters from different organisms showing the conserved glutamic acid residue (Glu-427 in Sul1 and Glu-443 in Sul2) (in green) and other conserved residues (in lavender). Bottom, the position of all mutagenized residues, including Glu-427 in Sul1 and Glu-443 in Sul2, is indicated on the predicted topology of Sul1 and Sul2. B, Sul1,2-HA expression level as detected by Western blot of immunoprecipitated Sul1-HA, Sul2-HA, and their mutant forms in membrane-enriched P13 fractions isolated from cell cultures before and after the addition of 3 mm sulfate. C, uptake rate of 0.1 mm [35S]sulfate in Sul1-HA-expressing (black bar) and Sul1E427Q-HA-expressing (gray bar) sulfur-starved cells. D, uptake rate of 0.1 mm [35S]sulfate in Sul2-HA-expressing (black bar) and Sul2E443Q-HA-expressing (gray bar) sulfur-starved cells. E, activation of trehalase in Sul1-HA (●), Sul1E427Q-HA (○), Sul2-HA (▴), and Sul2E443Q-HA (▵) upon the addition of 3 mm sulfate. F, relative expression of HSP12 in sulfur-starved sul1Δ sul2Δ cells expressing Sul1-HA (●), Sul1E427Q-HA (○), Sul2-HA (▴), and Sul2E443Q-HA (▵) upon the addition of 3 mm sulfate. Error bars, S.D.

Mentions: The hidden Markov model-based topology prediction algorithm, PHOBIUS (45), predicted 12 and 11 transmembrane domains for Sul1 and Sul2, respectively. However, homology modeling (threading) using the HHpred algorithm showed that Sul1,2 are structurally most similar to the Escherichia coli uracil UraA transporter, the crystal structure of which showed 14 TMDs3 (46) (also see “Discussion”). Based on topology predictions and the similarity with UraA, we screened the 14 putative transmembrane domains of the Sul1,2 transporters for charged residues, in particular Glu and Asp residues, which are the best candidates for H+ binding and symport. We found six residues in Sul1 and five residues in Sul2, located within or very close to predicted transmembrane domains (Table 4). Alignment of sulfate transporters from different organisms revealed that Glu-406 and Glu-427 in Sul1 and Glu-422 and Glu-443 in Sul2, located in TMD8 and TMD9, respectively, were absolutely conserved in other sulfate transporters from different organisms (Fig. 5A) (47). Furthermore, residues Glu-406 and Glu-422 in Sul1 and Sul2, respectively, correspond to the His-245 residue proposed to interact with H+ in the UraA structure (46). We therefore selected to mutate these residues from Sul1 and two from Sul2 for site-directed mutagenesis (Table 4).


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)

Mutagenesis of the putative proton binding sites, Glu-427 in Sul1 and Glu-443 in Sul2, blocks the uptake of sulfate but does not affect the signaling function.A, ClustalX sequence alignment of sulfate transporters from different organisms showing the conserved glutamic acid residue (Glu-427 in Sul1 and Glu-443 in Sul2) (in green) and other conserved residues (in lavender). Bottom, the position of all mutagenized residues, including Glu-427 in Sul1 and Glu-443 in Sul2, is indicated on the predicted topology of Sul1 and Sul2. B, Sul1,2-HA expression level as detected by Western blot of immunoprecipitated Sul1-HA, Sul2-HA, and their mutant forms in membrane-enriched P13 fractions isolated from cell cultures before and after the addition of 3 mm sulfate. C, uptake rate of 0.1 mm [35S]sulfate in Sul1-HA-expressing (black bar) and Sul1E427Q-HA-expressing (gray bar) sulfur-starved cells. D, uptake rate of 0.1 mm [35S]sulfate in Sul2-HA-expressing (black bar) and Sul2E443Q-HA-expressing (gray bar) sulfur-starved cells. E, activation of trehalase in Sul1-HA (●), Sul1E427Q-HA (○), Sul2-HA (▴), and Sul2E443Q-HA (▵) upon the addition of 3 mm sulfate. F, relative expression of HSP12 in sulfur-starved sul1Δ sul2Δ cells expressing Sul1-HA (●), Sul1E427Q-HA (○), Sul2-HA (▴), and Sul2E443Q-HA (▵) upon the addition of 3 mm sulfate. Error bars, S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Mutagenesis of the putative proton binding sites, Glu-427 in Sul1 and Glu-443 in Sul2, blocks the uptake of sulfate but does not affect the signaling function.A, ClustalX sequence alignment of sulfate transporters from different organisms showing the conserved glutamic acid residue (Glu-427 in Sul1 and Glu-443 in Sul2) (in green) and other conserved residues (in lavender). Bottom, the position of all mutagenized residues, including Glu-427 in Sul1 and Glu-443 in Sul2, is indicated on the predicted topology of Sul1 and Sul2. B, Sul1,2-HA expression level as detected by Western blot of immunoprecipitated Sul1-HA, Sul2-HA, and their mutant forms in membrane-enriched P13 fractions isolated from cell cultures before and after the addition of 3 mm sulfate. C, uptake rate of 0.1 mm [35S]sulfate in Sul1-HA-expressing (black bar) and Sul1E427Q-HA-expressing (gray bar) sulfur-starved cells. D, uptake rate of 0.1 mm [35S]sulfate in Sul2-HA-expressing (black bar) and Sul2E443Q-HA-expressing (gray bar) sulfur-starved cells. E, activation of trehalase in Sul1-HA (●), Sul1E427Q-HA (○), Sul2-HA (▴), and Sul2E443Q-HA (▵) upon the addition of 3 mm sulfate. F, relative expression of HSP12 in sulfur-starved sul1Δ sul2Δ cells expressing Sul1-HA (●), Sul1E427Q-HA (○), Sul2-HA (▴), and Sul2E443Q-HA (▵) upon the addition of 3 mm sulfate. Error bars, S.D.
Mentions: The hidden Markov model-based topology prediction algorithm, PHOBIUS (45), predicted 12 and 11 transmembrane domains for Sul1 and Sul2, respectively. However, homology modeling (threading) using the HHpred algorithm showed that Sul1,2 are structurally most similar to the Escherichia coli uracil UraA transporter, the crystal structure of which showed 14 TMDs3 (46) (also see “Discussion”). Based on topology predictions and the similarity with UraA, we screened the 14 putative transmembrane domains of the Sul1,2 transporters for charged residues, in particular Glu and Asp residues, which are the best candidates for H+ binding and symport. We found six residues in Sul1 and five residues in Sul2, located within or very close to predicted transmembrane domains (Table 4). Alignment of sulfate transporters from different organisms revealed that Glu-406 and Glu-427 in Sul1 and Glu-422 and Glu-443 in Sul2, located in TMD8 and TMD9, respectively, were absolutely conserved in other sulfate transporters from different organisms (Fig. 5A) (47). Furthermore, residues Glu-406 and Glu-422 in Sul1 and Sul2, respectively, correspond to the His-245 residue proposed to interact with H+ in the UraA structure (46). We therefore selected to mutate these residues from Sul1 and two from Sul2 for site-directed mutagenesis (Table 4).

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