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Biochem. J. (2002) 363 (657–666) (Printed in Great Britain)

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Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C d₁

Stephen A. WATT , Gursant KULAR , Ian N. FLEMING , C. Peter DOWNES and John M. LUCOCQ¹

School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.

Ptd(4,5)P₂ 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)P₂ in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase d₁ (PLCd₁), narrowly specific for PtdIns(4,5)P₂, to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCd₁ PH domain (PLCd₁PH–GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P₂ 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)P₃], 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)P₂ levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P₂ distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.

Key words: immunoelectron microscopy, immunogold, lipid domains, localization, phosphoinositide.

Abbreviations used: PH, pleckstrin homology; GST, glutathione S-transferase; GFP, green fluorescent protein; PLCd₁, phospholipase C d₁; MVB, multivesicular bodies; EM, electron microscopy; C.E., coefficient of error; ARF, ADP ribosylation factor; SFLLRN, one-letter amino acid code for thrombin-receptor-activating peptide.

¹To whom correspondence should be addressed (e-mail j.m.lucocq@dundee.ac.uk).

Received 3 October 2001/24 January 2002; accepted 28 February 2002

The Biochemical Society, London © 2002