Isolation of human erythrocyte inside-out vesicles alters their molecular architecture

Inside-out (I.O.) vesicles isolated from human erythrocyte ghosts induced to endocytose have been used for biochemical studies to determine localization of molecules within the membrane. It was the purpose of this study to examine such vesicles by freeze-etch electron microscopy to determine the architecture of the peripheral proteins on the protoplasmic surface. Examination of the I.O. vesicles while still in the interior of the ghosts showed that a globular material was randomly distributed on the outer surface (protoplasmic surface of the original plasma membrane) of the I.O. vesicles. The random distribution of the globular material becomes altered, however, if the I.O. vesicles are isolated from the ghosts by shearing and centrifugation. Freeze-etching of these isolated I.O. vesicles revealed that the globular material was now clustered on the protoplasmic surface (PS), as were the intramembranous particles (IMPs) in the extracellular face. Thus, the lateral mobility of the IMPs is dependent upon the distribution of molecules at the protoplasmic surface of the membrane. Moreover, this change in distribution of the globular material at the surface is not due to a partial loss of major membrane proteins, because SDS-polyacrylamide gel electrophoresis revealed that equal amounts of the major proteins were present in nonisolated vesicles as compared to isolated vesicles. Although isolated I.O. vesicles have been used extensively to demonstrate that glycoproteins span the membrane, these results suggest that one should cautiously interpret data obtained from such isolated vesicles in view of the fact that there is an alteration of the distribution and perhaps configuration of molecules at the PS following isolation of I.O. vesicles.