Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p.
Phosphatidylinositol (PtdIns) phosphate (PtdInsP) lipids are used as intracellular signposts for the recruitment and activation of peripheral membrane proteins. Whereas the distribution of most PtdInsPs is restricted to a single organelle, PtdIns(4)P is unique in that it exists in several discrete pools, and so proteins that bind PtdIns(4)P must use extra receptors to achieve a restricted localization. Here we compare the two highly related pleckstrin homology (PH) domains from Osh1p and Osh2p, yeast homologues of oxysterol-binding protein (OSBP), that target membranes using PtdIns(4)P, and in vitro bind both PtdIns(4)P and PtdIns(4,5)P2. We show that Golgi targeting is specified by an additional site on PH(Osh1), which lies on a face of the domain not previously known to interact with receptors. In contrast, PH(Osh2) does not have a demonstrable second site, and targets multiple pools of PtdInsPs, each dependent on a different PtdIns 4-kinase. This lack of a second site in PH(Osh2) allows it to be used as an unbiased reporter for altered distribution of 4-phosphorylated PtdIns. For example, in cells with excess PtdIns(4)P caused by inactivation of the phosphatase Sac1p, PH(Osh2) indicates that PtdIns(4)P accumulates on the plasma membrane, whereas other Golgi-targeted PH domains fail to detect this change.
Roy
Anjana
A
Division of Cell Biology, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, United Kingdom.
Levine
Timothy P
TP
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism; D000483:Alleles; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008024:Ligands; D008081:Liposomes / Q000378:metabolism; D008954:Models, Biological; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D000098825:Oxysterol Binding Proteins
D001798:Blood Proteins; D002352:Carrier Proteins; D008024:Ligands; D008081:Liposomes; C432935:OSH2 protein, S cerevisiae; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D011987:Receptors, Steroid; D029701:Saccharomyces cerevisiae Proteins; D000098825:Oxysterol Binding Proteins; C037178:phosphatidylinositol 4-phosphate; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D019870:1-Phosphatidylinositol 4-Kinase