Ras diffusion is sensitive to plasma membrane viscosity.
The cell surface contains a variety of barriers and obstacles that slow the lateral diffusion of glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins below the theoretical limit imposed by membrane viscosity. How the diffusion of proteins residing exclusively on the inner leaflet of the plasma membrane is regulated has been largely unexplored. We show here that the diffusion of the small GTPase Ras is sensitive to the viscosity of the plasma membrane. Using confocal fluorescence recovery after photobleaching, we examined the diffusion of green fluorescent protein (GFP)-tagged HRas, NRas, and KRas in COS-7 cells loaded with or depleted of cholesterol, a well-known modulator of membrane bilayer viscosity. In cells loaded with excess cholesterol, the diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas were significantly reduced, paralleling the behavior of the viscosity-sensitive lipid probes DiIC(16) and DiIC(18). However, the effects of cholesterol depletion on protein and lipid diffusion in cell membranes were highly dependent on the depletion method used. Cholesterol depletion with methyl-beta-cyclodextrin slowed Ras diffusion by a viscosity-independent mechanism, whereas overnight cholesterol depletion slightly increased both protein and lipid diffusion. The ability of Ras to sense membrane viscosity may represent a general feature of proteins residing on the cytoplasmic face of the plasma membrane.
Goodwin
J Shawn
JS
Department of Molecular Physiology & Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Drake
Kimberly R
KR
Remmert
Catha L
CL
Kenworthy
Anne K
AK
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry*; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000737:chemistry*; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D009038:Motion; D014783:Viscosity; D018631:ras Proteins / Q000737:chemistry*
D002784:Cholesterol; D018631:ras Proteins