Biochem. J. (2009) 422
(23–35) (Printed in Great Britain)
Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P2
Gerald R. V. Hammond*1, Giampietro Schiavo† and Robin F. Irvine*
*Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K., and †Molecular Neuropathobiology Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.
PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P2. Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P2.
Key words: Golgi, immunofluorescence, phosphoinositides, plasma membrane, PtdIns(4,5)P2, PtdIns4P.
Abbreviations used: Arf1, ADP-ribosylation factor 1; DAPI, 4′,6-diamidino-2-phenylindole; DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanineperchlorate; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; EGF, epidermal growth factor; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; FA, formaldehyde; FAPP1, phosphatidylinositol-4-phosphate adaptor protein 1; FYVE-Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; GA, glutaraldehyde; GFP, green fluorescent protein; GM130, cis-Golgi matrix protein of 130 kDa; GST, glutathione transferase; M6PR, mannose-6-phosphate receptor; NBD-C6-ceramide, N-(ϵ-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine; NGS, normal goat serum; OSBP, oxysterol-binding protein; PAO, phenylarsine oxide; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PI4K, phosphoinositide 4-kinase; PLC, phospholipase C; POPC, 1-palmitoyl 2-oleoyl phosphatidylcholine; TBS, tris-buffered saline; WGA, wheat germ agglutinin.
1To whom correspondence should be addressed (email gruh2@cam.ac.uk).
Received 16 March 2009/2 June 2009; accepted 9 June 2009
Published as BJ Immediate Publication 9 June 2009, doi:10.1042/BJ20090428
© 2009 The Author(s)
The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
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