Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling.
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.
Szentpetery
Zsofia
Z
Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Várnai
Peter
P
Balla
Tamas
T
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
D002966:Clathrin; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D010738:Type C Phospholipases
Science. 2006 Dec 1;314(5804):1454-7
16990515
Biochim Biophys Acta. 2005 Jul 10;1744(3):364-73
15979507
Biochem Cell Biol. 2004 Feb;82(1):254-62
15052341
Biochim Biophys Acta. 2007 Jun;1771(6):677-91
17490911
Proc Natl Acad Sci U S A. 1996 May 14;93(10):4604-7
8643450
Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5317-21
7777504
Curr Biol. 2002 Apr 30;12(9):695-704
12007412
Biochim Biophys Acta. 1997 Mar 1;1355(3):209-17
9060992
Biochem J. 2000 Oct 1;351(Pt 1):19-31
11001876
J Cell Biol. 2008 Feb 25;180(4):803-12
18299350
Dev Cell. 2002 May;2(5):593-605
12015967
J Biol Chem. 1999 May 7;274(19):12990-5
10224048
Mol Biol Cell. 2008 Feb;19(2):711-21
18077555
Mol Biol Cell. 2000 Aug;11(8):2673-89
10930462
J Biol Chem. 1994 Nov 11;269(45):28130-5
7961749
Nat Cell Biol. 1999 Dec;1(8):523-5
10587649
Biochem J. 1984 Jun 1;220(2):345-60
6146314
J Biol Chem. 2002 Jan 18;277(3):2012-8
11704666
Trends Cell Biol. 2005 Oct;15(10):540-7
16139503
Mol Biol Cell. 2008 May;19(5):1991-2002
18287542
Sci STKE. 2001 Dec 04;2001(111):re19
11734659
Trends Cell Biol. 2006 Jul;16(7):351-61
16793271
Physiology (Bethesda). 2009 Aug;24:231-44
19675354
J Biol Chem. 1999 Nov 26;274(48):34294-300
10567405
Cell. 2003 Aug 8;114(3):299-310
12914695
Mol Biol Cell. 2007 Jul;18(7):2646-55
17494868
J Cell Biol. 2006 Nov 6;175(3):377-82
17088424
Mol Biol Cell. 2001 Aug;12(8):2396-411
11514624
J Biol Chem. 1994 Apr 8;269(14):10758-63
8144664
Nature. 2003 Dec 18;426(6968):803-9
14685229
Mol Biol Cell. 1998 Apr;9(4):809-16
9529379
EMBO J. 1996 May 1;15(9):2094-101
8641275
Nature. 2006 Oct 12;443(7112):651-7
17035995
Proc Natl Acad Sci U S A. 2006 Oct 17;103(42):15473-8
17030795
J Cell Sci. 2008 May 1;121(Pt 9):1488-94
18411250
Nat Cell Biol. 1999 Sep;1(5):280-7
10559940
Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3793-8
17360432
Biochim Biophys Acta. 2005 Jul 10;1744(3):396-405
15979509
J Biol Chem. 1998 Feb 27;273(9):5037-46
9478953
Biochem J. 2000 Sep 1;350 Pt 2:337-52
10947947