Lateral Mobility of Erythrocyte Membrane Proteins Studied by the Fluorescence Photobleaching Recovery Technique

Abstract

Erythrocyte membrane peripheral and integral proteins have been isolated and purified, and the lateral diffusion of these proteins in a well-defined phospholipid bilayer matrix (dimyristoylphosphatidylcholine) has been studied by fluorescence photobleaching recovery measurements. Our own instrument for the recovery measurements is described and some data for lipid diffusions are compared with those previously reported by other investigators. The peripheral proteins (spectrin and band 4.1) diffuse rapidly on the lipid membrane in its fluid phase. The diffusion constant of ∼5× 10⁻⁸ cm² s⁻¹ (30°C) was only a little smaller than that for lipid diffusion. The diffusion was greatly slowed down when the host lipid matrix became solid. The integral protein band 3 also diffuses rapidly in the fluid membrane. The diffusion constant of 1.6′10-8 cm²∣s⁻ (30°C) was smaller than those for lipids and for the peripheral proteins. The lateral motion is compatible with diffusion of a cylinder with radius 3 nm in a two dimensional matrix with an inner viscosity of 2 poises and an inner thickness of 4 nm. The band 3 lateral motion was restricted by binding of the cytoskeletal component proteins (ankyrin, spectrin, actin, and band 4.1) to the reconstituted membranes. The diffusion constant decreased to half. The results provide a basis for the elucidation of transmembrane control mechanisms in more complex cellular systems.