Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1.
Ptd(4,5)P(2) is thought to promote and organize a wide range of cellular functions, including vesicular membrane traffic and cytoskeletal dynamics, by recruiting functional protein complexes to restricted locations in cellular membranes. However, little is known about the distribution of PtdIns(4,5)P(2) in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase delta(1) (PLCdelta(1)), narrowly specific for PtdIns(4,5)P(2), to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCdelta(1) PH domain (PLCdelta(1)PH-GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P(2) labelling in the plasma membrane, and also in intracellular membranes, including Golgi (mainly stack), endosomes and endoplasmic reticulum, as well as in electron-dense structures within the nucleus. At the plasma membrane, labelling was more concentrated over lamellipodia, but not in caveolae, which contained less than 10% of the total cell-surface labelling. A dramatic decrease in signal over labelled compartments was observed on preincubation with the cognate headgroup [Ins(1,4,5)P(3)], and plasma-membrane labelling was substantially decreased after stimulation with thrombin-receptor-activating peptide (SFLLRN in the one-letter amino acid code), a treatment which markedly diminishes PtdIns(4,5)P(2) levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P(2) distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.
Watt
Stephen A
SA
School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.
Kular
Gursant
G
Fleming
Ian N
IN
Downes
C Peter
CP
Lucocq
John M
JM
D001798:Blood Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D050356:Lipid Metabolism; D016253:Microscopy, Immunoelectron; D010446:Peptide Fragments / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D054803:Phospholipase C delta; D010750:Phosphoproteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D001798:Blood Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; C039077:platelet protein P47; C082835:thrombin receptor peptide (42-47); D010738:Type C Phospholipases; C518024:PLCD1 protein, human; D054803:Phospholipase C delta