On the asymmetry of frog retinal rod outer segment disk membranes

Abstract

Freeze-fracture studies on intact frog rod outer segments have been carried out using a device designed to minimize surface contamination and inadvertent specimen etching. When fractured and shadowed at −170°C, each double-membrane disk gives rise to two membrane fracture faces. In replicas with particularly favorable directions of heavy metal deposition, it is possible to infer the axial spacings between the membrane fracture faces by analyzing the widths of the oblique fracture jumps. The distance of each membrane fracture plane from the center of the disk can then be determined from these axial spacings and the structural centrosymmetry of the disk. Our analysis indicates that each membrane fracture plane is located at a distance of ∼47 Å from the coordinate origin at the center of the disk. This coordinate corresponds in position to the electron density minimum of the membrane. Our results therefore support the original hypothesis by Clark and Branton (1968) that disk membranes fracture along some interior hydrophobic plane, presumably the region of terminal methyl groups at the center of the lipid bilayer. Fracture face A is characterized by a rough, particulate appearance, and represents the hydrophobic face of that half of the disk membrane adjacent to the cytoplasmic matrix. Face B is smooth, and represents the hydrophobic face of that half of the membrane adjacent to the intradisk compartment. When these membrane fracture faces are compared with the characteristic textures observed in pure lipid bilayers and in rhodopsin-lipid recombinant membranes, it appears that in frozen disk membranes rhodopsin is restricted to the cytoplasmic half of the membrane. The smooth B face has the appearance of a lipid monolayer. Although the A face particles have a rather low surface relief, it is still possible to estimate their apparent size and shape. These particles are roughly circular; the range of diameters is about 120–140 Å, when measured in the projected direction of heavy metal shadowing. Optical diffraction patterns of the A face show broad intensity bands centered at ± ( ∼ 1 130 A ̊ ). When examining the published equatorial X-ray diffraction data from frog outer segments, it is apparent that there is no broad band of intensity within the interval ( 1 150 A ̊ ) to ( 1 70 A ̊ ), corresponding to the optical diffraction data. Accordingly, it must be postulated that at physiological temperatures, the distribution of matter within the plane of the disk membrane does not correspond to the distribution evident in the A fracture face. Hence, the A face particles appear to represent artifactual, derivative phenomena. An explanation in terms of lateral phase separations of disk membrane components is developed. The consequences of such an hypothesis for the etching behavior of the B face are derived in the context of those parameters which are known to affect the passage of water molecules across lipid monolayers.