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Is the tandem Osh2 PH domain a suitable probe for plasma membrane PtdIns(4)P?

Background and purpose: It has been reported in the literature that the tandem Osh2p pleckstrin homology domain is a suitably selective probe for plasma membrane (PM) phosphatidylinositol 4-phosphate and it is currently being used as such. In vitro data suggest that it has an equally high affinity for phosphatidylinositol 4,5-bisphosphate, and some of the original literature that is used to support the use of the probe is equivocal as to its merit. With this background, the present study sets out to gather evidence to support or refute the use of the tandem Osh2p pleckstrin homology domain as a probe for plasma membrane phosphatidylinositol 4-phosphate.

Experimental approach: A series of experiments were carried out on COS-7 cells that were transfected to express various constructs and then challenged pharmacologically and viewed using confocal microscopy. In the first experiment an inhibitor of PtdIns 4-kinase (phenylarsine oxide - PAO) was added to deplete the PtdIns(4)P and a cytosolic PtdIns(4,5)P2 5-phosphatase was then recruited to the PM to deplete the PtdIns(4,5)P2. In the second experiment either a single or a tandem Osh2p PH-domain was co-transfected with a tandem PH domain from phospholipase δ1 (PLCδ). In this case either PAO or ionomycin followed by ethylene glycol tetraacetic acid (EGTA) were added to the dishes. A PtdIns phosphatase with multiple substrates including PtdIns(4)P or a PtdIns(4,5)P2 5-phosphatase were then co-transfected with either the Osh2 tandem PH construct or the PLCδ construct in a third set of experiments. Finally, both the PtdIns(4)P and the PtdIns(4,5)P2 5-phosphatase were co-transfected along with either the Osh2 tandem PH construct or the PLCδ construct.

Key results: The findings from this work were that a PtdIns(4,5)P2 5-phosphatase alone can cause translocation of tandem Osh2p pleckstrin homology domain to the cytoplasm. The 5-phosphatase and sac combined were even more efficacious than the 5-phosphatase alone. Sac alone did not result in any translocation of the Osh2 PH domain to the cytoplasm.

Conclusions and implications: Taken together, these results imply that GFP-PH-Osh2 binds to PtdIns(4,5)P2 in vivo, contrary to the consensus view in the field. Whilst further research will be required to fully understand the interactions, it would be advisable to refrain from using the tandem Osh2 pleckstrin homology domain as a selective probe for PM PtdIns(4)P.

Keywords: phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, oxysterol-binding-protein homolog 2, probe, phenylarsine oxide, fluorescent tags, pleckstrin homology domain, confocal imaging, protein translocation

Abbreviations: PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PM, plasma membrane; PAO, phenylarsine oxide; GFP-PH-Osh2, Osh2 tandem PH domain bound to green fluorescent protein.

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Dr Pól Roibeárd Ó Catnaigh