Kinetics of the interaction of myo1c with phosphoinositides.
myo1c is a single-headed myosin that dynamically links membranes to the actin cytoskeleton. A putative pleckstrin homology domain has been identified in the myo1c tail that binds phosphoinositides and soluble inositol phosphates with high affinity. However, the kinetics of association and dissociation and the influence of phospholipid composition on the kinetics have not been determined. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1c(IQ-tail)) to and from 100-nm diameter large unilamellar vesicles (LUVs). We found the time course of association of myo1c(IQ-tail) with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) followed a two-exponential time course, and the rate of the predominant fast phase depended linearly upon the total lipid concentration. The apparent second-order rate constant was approximately diffusion-limited. Increasing the molar ratio of anionic phospholipid by adding phosphatidylserine, additional PtdIns(4,5)P(2), or by situating PtdIns(4,5)P(2) in a more physiologically relevant lipid background increased the apparent association rate constant less than 2-fold. myo1c(IQ-tail) dissociated from PtdIns(4,5)P(2) at a slower rate (2.0 s(-1)) than the pleckstrin homology domain of phospholipase C-delta (13 s(-1)). The presence of additional anionic phospholipid reduced the myo1c(IQ-tail) dissociation rate constant >50-fold but marginally changed the dissociation rate of phospholipase C-delta, suggesting that additional electrostatic interactions in myo1c(IQ-tail) help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1c(IQ-tail) from LUVs, suggesting that phosphoinositides are able to bind to and dissociate from myo1c(IQ-tail) as it remains bound to the membrane.
McKenna
Jennine M Dawicki
JM
Department of Physiology, Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085, USA.
Ostap
E Michael
EM
D000199:Actins / Q000737:chemistry; D000818:Animals; D000838:Anions; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008956:Models, Chemical; D024461:Myosin Type I; D009218:Myosins / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D000199:Actins; D000838:Anions; D008055:Lipids; C470422:Myo1c protein, mouse; D010716:Phosphatidylinositols; D024461:Myosin Type I; D009218:Myosins; D008274:Magnesium
Nature. 1989 Aug 17;340(6234):565-8
2770861
EMBO J. 2004 May 5;23(9):1922-33
15103325
Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10472-6
7479822
Adv Exp Med Biol. 1992;321:159-63
1449079
Dev Cell. 2007 Sep;13(3):391-404
17765682
Biochemistry. 1996 Jun 25;35(25):8193-200
8679573
Biochemistry. 2007 Oct 23;46(42):11718-26
17910470
Cell. 1995 Dec 15;83(6):1037-46
8521504
J Cell Biol. 1989 Oct;109(4 Pt 1):1519-28
2793931
J Cell Biol. 1995 Dec;131(5):1205-21
8522584
Cell. 1994 Oct 21;79(2):199-209
7954789
Mol Biol Cell. 2006 Nov;17(11):4856-65
16971510
Biochemistry. 2004 Dec 28;43(51):16161-73
15610010
Annu Rev Physiol. 2003;65:761-89
12471164
Mol Cell Biol. 2004 Jun;24(12):5447-58
15169906
J Cell Sci. 2005 Jul 1;118(Pt 13):2891-9
15976448
J Cell Sci. 1995 Dec;108 ( Pt 12):3775-86
8719884
J Cell Sci. 2008 Mar 1;121(Pt 5):551-9
18287584
J Cell Biol. 2000 Jun 26;149(7):1455-72
10871285
Adv Exp Med Biol. 1994;358:205-13
7801806
J Cell Biol. 2004 May 10;165(3):395-405
15138292
Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3118-23
16492791
Nature. 2002 Dec 19-26;420(6917):821-4
12490950
Biochemistry. 2000 May 16;39(19):5893-901
10801341
Mol Cell Biol. 2004 Oct;24(20):9102-23
15456883
J Cell Biol. 1990 Aug;111(2):443-51
2143194
J Biol Chem. 2002 Nov 8;277(45):42763-8
12221091
Dev Cell. 2001 Dec;1(6):743-7
11740936
Biophys J. 2004 Apr;86(4):2188-207
15041659
Cell. 2002 Feb 8;108(3):371-81
11853671
Dev Cell. 2006 Nov;11(5):629-40
17084356
Biochemistry. 1988 Sep 20;27(19):7435-43
2849993
Nature. 2005 Dec 1;438(7068):605-11
16319880
Curr Diabetes Rev. 2007 May;3(2):111-22
18220662
Biochem Soc Trans. 2001 Aug;29(Pt 4):377-84
11497993
Science. 2008 Jul 4;321(5885):133-6
18599791
J Muscle Res Cell Motil. 2007;28(6):285-91
17994197
J Cell Biol. 2006 Jun 5;173(5):665-71
16754954
Biochem J. 2007 Jul 15;405(2):199-221
17590154
Neuron. 2004 Oct 14;44(2):309-20
15473969