Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components.
BACKGROUND
Phosphoinositides are required for the recruitment of many proteins to both the plasma membrane and the endosome; however, their role in protein targeting to other organelles is less clear. The pleckstrin homology (PH) domains of oxysterol binding protein (OSBP) and its relatives have been shown to bind to the Golgi apparatus in yeast and mammalian cells. Previous in vitro binding studies identified phosphatidylinositol (PtdIns) (4)P and PtdIns(4,5)P(2) as candidate ligands, but it is not known which is recognized in vivo and whether phosphoinositide specificity can account for Golgi-specific targeting.


RESULTS
We have examined the distribution of GFP fusions to the PH domain of OSBP and to related PH domains in yeast strains carrying mutations in individual phosphoinositide kinases. We find that Golgi targeting requires the activity of the PtdIns 4-kinase Pik1p but not phosphorylation of PtdIns at the 3 or 5 positions and that a PH domain specific for PtdIns(4,5)P(2) is targeted exclusively to the plasma membrane. However, a mutant version of the OSBP PH domain that does not bind phosphoinositides in vitro still shows some targeting in vivo. This targeting is independent of Pik1p but dependent on the Golgi GTPase Arf1p.


CONCLUSIONS
Phosphorylation of PtdIns at the 4 position but not conversion to PtdIns(4,5)P(2) contributes to recruitment of PH domains to the Golgi apparatus. However, potential phosphoinositide ligands for these PH domains are not restricted to the Golgi, and the OSBP PH domain also recognizes a second determinant that is ARF dependent, indicating that organelle specificity reflects a combinatorial interaction.
Levine
Timothy P
TP
Division of Cell Biology, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK.
Munro
Sean
S
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism*; D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry*; D019556:COS Cells; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
D001798:Blood Proteins; D008164:Luminescent Proteins; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D000098825:Oxysterol Binding Proteins; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D019870:1-Phosphatidylinositol 4-Kinase; D020823:ADP-Ribosylation Factor 1