No abstract

Mechanisms that regulate the movement of a membrane spanning protein band 3 in erythrocyte ghosts were investigated at the level of a single or small groups of molecules using single particle tracking with an enhanced time resolution (0.22 ms). Two-thirds of band 3 undergo macroscopic diffusion: a band 3 molecule is temporarily corralled in a mesh of 110 nm in diameter, and hops to an adjacent mesh an average of every 350 ms. The rest (one-third) of band 3 exhibited oscillatory motion similar to that of spectrin, suggesting that these band 3 molecules are bound to spectrin. When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton. Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral. These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.

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 equilibrium, and the fractional ratios are strongly temperature dependent. At 26 degrees 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 translational 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Rotational diffusion of rhodopsin in reconstituted membranes of phosphatidylcholines of various alkyl chain lengths has been measured by using saturation-transfer electron spin resonance spectroscopy as a function of temperature and lipid/rhodopsin mole ratio. For dipalmitoyl-phosphatidylcholine, the rotational correlation time is 20 microseconds at physiological concentration, the same as in rod outer segment (ros) membranes. Dilution reduces the time to 10 microseconds, a value that is ascribed to well-dispersed monomeric rhodopsin. Use of phospholipids with longer or shorter chains results in sharply increased rotational correlation times. It is concluded that rhodopsin molecules are transiently associated in both reconstituted and ros membranes and that the nature of the association is determined by lipid type and composition.