Regulation of Rac1 translocation and activation by membrane domains and their boundaries.
Journal Article (Journal Article)
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.
Full Text
Duke Authors
Cited Authors
- Moissoglu, K; Kiessling, V; Wan, C; Hoffman, BD; Norambuena, A; Tamm, LK; Schwartz, MA
Published Date
- June 2014
Published In
Volume / Issue
- 127 / Pt 11
Start / End Page
- 2565 - 2576
PubMed ID
- 24695858
Pubmed Central ID
- PMC4038948
Electronic International Standard Serial Number (EISSN)
- 1477-9137
International Standard Serial Number (ISSN)
- 0021-9533
Digital Object Identifier (DOI)
- 10.1242/jcs.149088
Language
- eng