Cloned (Comment) | Organism |
---|---|
(PCR) amplification from the SigD open reading frame from S. enterica ser. typhimurium C53 genomic DNA. 2 types plasmids: first type based on the pEG-KG vector to express GST fusion proteins in yeast, second type based on pGEX-KG for expression of GST-fused proteins in bacteria. transformation of Escherichia coli DH5alpha cells (used for molceularbiological methods and expression). Transformation of Saccharomyces cerevisiae YPH499 (used for SigD functional analyzes as a model organism). transfection of human epithelial cell line HeLa (ATCC CCL2). yeast: endogenous SigD expression results in severe growth inhibition, allele mutant in catalytic site or deletion of whole C-terminal phosphatase domain inhibit growth by loss of actin polarization and precluding the budding process. HeLa: expression of same sigD alleles causes loss of ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, actin fibres still disappear and lose their adherence. region of 25 amino acids localized (residues 118142) essentially required for the effect of SigD on actin in HeLa cells. SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity | Salmonella enterica subsp. enterica serovar Typhimurium |
Protein Variants | Comment | Organism |
---|---|---|
C462A | recombinant protein | Salmonella enterica subsp. enterica serovar Typhimurium |
DELTA1-135-DELTA118-142 | recombinant protein, Saccharomyces cerevisiae: no effect in Saccharomyces cerevisiae, HeLa: no effect (Green Fluorescent Protein linked) in HeLa | Salmonella enterica subsp. enterica serovar Typhimurium |
DELTA1-165 | recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments | Salmonella enterica subsp. enterica serovar Typhimurium |
DELTA1-351 | recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments | Salmonella enterica subsp. enterica serovar Typhimurium |
DELTA118-142 | recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, residues 118142 crucial for toxicity of SigD in the yeast cells, affecting both phosphatase activity and actin depolarization events, no effect (Green Fluorescent Protein linked) in HeLa | Salmonella enterica subsp. enterica serovar Typhimurium |
DELTA352-563 | recombinant protein, no effect in Saccharomyces cerevisiae, no effect in HeLa (Green Fluorescent Protein linked) | Salmonella enterica subsp. enterica serovar Typhimurium |
R468A | recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments | Salmonella enterica subsp. enterica serovar Typhimurium |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
phosphatidylinositol 4,5-bisphosphate + H2O | Salmonella enterica subsp. enterica serovar Typhimurium | - |
phosphatidylinositol 5-monophosphate + phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Salmonella enterica subsp. enterica serovar Typhimurium | - |
- |
- |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.0000003 | - |
approx., C462A mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate) | Salmonella enterica subsp. enterica serovar Typhimurium |
0.0000005 | - |
approx., R468A mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate) | Salmonella enterica subsp. enterica serovar Typhimurium |
0.0000007 | - |
approx., GST, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate) | Salmonella enterica subsp. enterica serovar Typhimurium |
0.0000011 | - |
approx., DELTA1-351 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate) | Salmonella enterica subsp. enterica serovar Typhimurium |
0.0000015 | - |
approx., DELTA352-563 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate) | Salmonella enterica subsp. enterica serovar Typhimurium |
0.0000032 | - |
approx., DELTA118-142 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate) | Salmonella enterica subsp. enterica serovar Typhimurium |
0.0000063 | - |
approx., SigD, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate) | Salmonella enterica subsp. enterica serovar Typhimurium |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
phosphatidylinositol 3,4,5-trisphosphate + H2O | - |
Salmonella enterica subsp. enterica serovar Typhimurium | phosphatidylinositol 3,5-bisphosphate + phosphate | - |
? | |
phosphatidylinositol 4,5-bisphosphate + H2O | - |
Salmonella enterica subsp. enterica serovar Typhimurium | phosphatidylinositol 5-monophosphate + phosphate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
phosphoinositide phosphatase | - |
Salmonella enterica subsp. enterica serovar Typhimurium |
SigD | - |
Salmonella enterica subsp. enterica serovar Typhimurium |