Regulation of Rac1 translocation and activation by membrane domains and their boundaries.
The activation of Rac1 and related Rho GTPases involves dissociation from Rho GDP-dissociation inhibitor proteins and translocation to membranes, where they bind effectors. Previous studies have suggested that the binding of Rac1 to membranes requires, and colocalizes with, cholesterol-rich liquid-ordered (lo) membrane domains (lipid rafts). Here, we have developed a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac1 membrane targeting in living cells. Surprisingly, FRET with acceptor constructs that were targeted to either raft or non-raft areas indicated that Rac1 was present in both regions. Functional studies showed that Rac1 localization to non-raft regions decreased GTP loading as a result of inactivation by GTPase-activating proteins. In vitro, Rac1 translocation to supported lipid bilayers also required lo domains, yet Rac1 was concentrated in the liquid-disordered (ld) phase. Single-molecule analysis demonstrated that translocation occurred preferentially at lo-ld boundaries. These results, therefore, suggest that Rac1 translocates to the membrane at domain boundaries, then diffuses into raft and non-raft domains, which controls interactions. These findings resolve discrepancies in our understanding of Rac biology and identify novel mechanisms by which lipid rafts modulate Rho GTPase signaling.
Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA konstadinos.moissoglu@nih.gov martin.schwartz@yale.edu.
Konstadinos
K
Moissoglu
Center for Membrane Biology, University of Virginia, Charlottesville, VA 22908, USA Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
Volker
V
Kiessling
Center for Membrane Biology, University of Virginia, Charlottesville, VA 22908, USA Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
Chen
C
Wan
Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.
Brenton D
BD
Hoffman
Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.
Andres
A
Norambuena
Center for Membrane Biology, University of Virginia, Charlottesville, VA 22908, USA Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
Lukas K
LK
Tamm
Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA konstadinos.moissoglu@nih.gov martin.schwartz@yale.edu.
Martin Alexander
MA
Schwartz
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020690:GTPase-Activating Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D011485:Protein Binding; D021381:Protein Transport; D015398:Signal Transduction; D053835:Unilamellar Liposomes / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism
D020690:GTPase-Activating Proteins; D053835:Unilamellar Liposomes; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020830:rac1 GTP-Binding Protein
Domain boundaries; FRET; Lipid rafts; Rac; Supported bilayers