title: Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling.
abstract: Phosphoinositides are essential lipid regulators of trafficking and signaling pathways of all eukaryotic cells. Phosphatidylinositol 4-phosphate (PtdIns4P) is an intermediate in the synthesis of several important phosphoinositide species but also serves as a regulatory molecule in its own right. Phosphatidylinositol 4-kinases are most abundant in the Golgi but are also found in the plasma membrane and in endocytic compartments. To investigate the role of Golgi PtdIns4P in orchestrating trafficking events, we used a unique drug-inducible molecular approach to rapidly deplete PtdIns4P from Golgi membranes by a recruitable Sac1 phosphatase enzyme. The utility of the system was shown by the rapid loss of Golgi localization of PH domains known to bind PtdIns4P after Sac1 recruitment to the Golgi. Acute PtdIns4P depletion prevented the exit of cargo from the Golgi destined to both the plasma membrane and the late endosomes and led to the loss of some but not all clathrin adaptors from the Golgi membrane. Rapid PtdIns4P depletion in the Golgi also impaired but did not eliminate the replenishment of the plasma membrane PtdIns(4,5)P(2) during phospholipase C activation revealing a hitherto unrecognized contribution of Golgi PtdIns4P to this process. This unique approach will allow further studies on the role of phosphoinositides in endocytic compartments that have evaded detection using the conventional long-term manipulations of inositide kinase and phosphatase activities.
authors.affiliation: Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Zsofia
authors.initials: Z
authors.lastname: Szentpetery
authors.forename: Peter
authors.initials: P
authors.lastname: Várnai
authors.forename: Tamas
authors.initials: T
authors.lastname: Balla
mesh_terms: D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D004789:Enzyme Activation; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D015398:Signal Transduction; D022161:Transport Vesicles / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D002966:Clathrin; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D010738:Type C Phospholipases
references.citation: Science. 2006 Dec 1;314(5804):1454-7
references.pmid: 16990515
references.citation: Biochim Biophys Acta. 2005 Jul 10;1744(3):364-73
references.pmid: 15979507
references.citation: Biochem Cell Biol. 2004 Feb;82(1):254-62
references.pmid: 15052341
references.citation: Biochim Biophys Acta. 2007 Jun;1771(6):677-91
references.pmid: 17490911
references.citation: Proc Natl Acad Sci U S A. 1996 May 14;93(10):4604-7
references.pmid: 8643450
references.citation: Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5317-21
references.pmid: 7777504
references.citation: Curr Biol. 2002 Apr 30;12(9):695-704
references.pmid: 12007412
references.citation: Biochim Biophys Acta. 1997 Mar 1;1355(3):209-17
references.pmid: 9060992
references.citation: Biochem J. 2000 Oct 1;351(Pt 1):19-31
references.pmid: 11001876
references.citation: J Cell Biol. 2008 Feb 25;180(4):803-12
references.pmid: 18299350
references.citation: Dev Cell. 2002 May;2(5):593-605
references.pmid: 12015967
references.citation: J Biol Chem. 1999 May 7;274(19):12990-5
references.pmid: 10224048
references.citation: Mol Biol Cell. 2008 Feb;19(2):711-21
references.pmid: 18077555
references.citation: Mol Biol Cell. 2000 Aug;11(8):2673-89
references.pmid: 10930462
references.citation: J Biol Chem. 1994 Nov 11;269(45):28130-5
references.pmid: 7961749
references.citation: Nat Cell Biol. 1999 Dec;1(8):523-5
references.pmid: 10587649
references.citation: Biochem J. 1984 Jun 1;220(2):345-60
references.pmid: 6146314
references.citation: J Biol Chem. 2002 Jan 18;277(3):2012-8
references.pmid: 11704666
references.citation: Trends Cell Biol. 2005 Oct;15(10):540-7
references.pmid: 16139503
references.citation: Mol Biol Cell. 2008 May;19(5):1991-2002
references.pmid: 18287542
references.citation: Sci STKE. 2001 Dec 04;2001(111):re19
references.pmid: 11734659
references.citation: Trends Cell Biol. 2006 Jul;16(7):351-61
references.pmid: 16793271
references.citation: Physiology (Bethesda). 2009 Aug;24:231-44
references.pmid: 19675354
references.citation: J Biol Chem. 1999 Nov 26;274(48):34294-300
references.pmid: 10567405
references.citation: Cell. 2003 Aug 8;114(3):299-310
references.pmid: 12914695
references.citation: Mol Biol Cell. 2007 Jul;18(7):2646-55
references.pmid: 17494868
references.citation: J Cell Biol. 2006 Nov 6;175(3):377-82
references.pmid: 17088424
references.citation: Mol Biol Cell. 2001 Aug;12(8):2396-411
references.pmid: 11514624
references.citation: J Biol Chem. 1994 Apr 8;269(14):10758-63
references.pmid: 8144664
references.citation: Nature. 2003 Dec 18;426(6968):803-9
references.pmid: 14685229
references.citation: Mol Biol Cell. 1998 Apr;9(4):809-16
references.pmid: 9529379
references.citation: EMBO J. 1996 May 1;15(9):2094-101
references.pmid: 8641275
references.citation: Nature. 2006 Oct 12;443(7112):651-7
references.pmid: 17035995
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 17;103(42):15473-8
references.pmid: 17030795
references.citation: J Cell Sci. 2008 May 1;121(Pt 9):1488-94
references.pmid: 18411250
references.citation: Nat Cell Biol. 1999 Sep;1(5):280-7
references.pmid: 10559940
references.citation: Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3793-8
references.pmid: 17360432
references.citation: Biochim Biophys Acta. 2005 Jul 10;1744(3):396-405
references.pmid: 15979509
references.citation: J Biol Chem. 1998 Feb 27;273(9):5037-46
references.pmid: 9478953
references.citation: Biochem J. 2000 Sep 1;350 Pt 2:337-52
references.pmid: 10947947