Regulation of band 3 mobilities in erythrocyte ghost membranes by protein association and cytoskeletal meshwork

Abstract

Rotational diffusion of erythrocyte anion channel protein band 3 was measured in ghost membranes by observing time-resolved phosphorescence anisotropy decays of eosinyl-5-maleimide covalently attached to the protein. Experiments were carried out under conditions similar to those employed by Tsuji and Ohnishi (1986) for translational diffusion measurement of band 3 [(1986) Biochemistry 25, 6133-6139] to allow direct comparison of rotational and translational diffusion of band 3. Detailed analysis of diffusive properties of band 3 in ghost membranes was made on the basis of these rotational and translational diffusion data. Rotational diffusion measurements indicated that there are at least three populations of band 3 molecules with high, low, and no rotational mobilities in the time scale of 10-4-10-2 s. These populations are in equilbirum, and the fractional ratios are strongly temperature dependent. At 26.degree. C, 44% of band 3 molecules are mobile (16% have an average rotational correlation time of 0.19 ms, and 28% have an average correlation time of 2.4 ms), and 56% are immobile. These results correlate well with translational diffusion data which indicated 40% mobile and 60% immobile fractions of band 3. The rotational diffusion data together with the trnaslational diffusion data by Tsuji and Ohnishi (1986) and Golan and Veatch [(1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2537-2541] suggest that immobilization of band 3 is largely caused by binding of band 3 oligomers to ankyrin, which abolishes both rotational and translational diffusion of band 3. Dimer/tetramer equilibrium of spectrin, a major constituent protein of the cytoskeletal meshwork, was shifted by varying the ionic strength of the medium or by addition of polyamines. An increase in the dimer fraction of spectrin, which greatly increases the lateral diffusion constant of the translationally mobile component of band 3 without changing the fraction of the translationally mobile component, had little influence either on the mobile/immobile fraction of band 3 in terms of rotational mobility or on the rotational correlation time of the mobile fraction of band 3 in ghost membranes. These results are consistent with a model in which (1) band 3 oligomers, aggregates, and band 3 molecules that are bound to the cytoskeletal network via ankyrin (or via glycophorin and band 4.1) are in temperature-dependent equilibrium in ghost membranes, (2) oligomers and aggregates that are not bound to the cytoskeletal/peripheral protein network membranes, (2) oligomers and aggregates that are not bound to the cytoskeletal/peripheral protein network are rotationally and translationally mobile, and (3) long-range translational diffusion of mobile oligomers and aggregates is restricted by nonspecific barriers imposed by the cytoskeletal network and the rate of translational diffusion is regulated by the fraction of spectrin dimers (open gate) and tetramers (closed gate).