In 1971, “next to nothing was known about the organization of membrane proteins,” says S. Jonathan Singer (University of California, San Diego, CA). Singer had proposed that there were two kinds of membrane proteins— integral and peripheral—but the idea was, at the time, largely speculative (Singer, 1971). It was a collaborative study between Singer, his then graduate student Garth Nicolson, and Vincent Marchesi of the National Institutes of Health that provided strong evidence for the existence of peripheral proteins (Nicolson et al., 1971a).
Earlier work by Marchesi and Steers (1968) had shown that the protein spectrin was associated with the membranes of red blood cells. It could be isolated by mild treatments and behaved like a water-soluble protein. Some researchers thought spectrin was typical of membrane proteins in general. Singer's model, however, proposed that integral membrane proteins, which passed through the membrane, would be insoluble in water. In contrast, proteins like spectrin belonged to a distinct category. “I thought that spectrin would be peripheral to the membrane and attached to specific integral proteins where they stuck out from the membrane into the cytoplasm,” recalls Singer.
To investigate this idea, Singer wanted to see where spectrin was located. He used his own ferritin-conjugated antibody technique (Singer, 1959) and got the antibodies into the red cell ghosts by fixing the ghosts while they had holes in their membranes from incubation in hypotonic medium (Seeman, 1967). Electron micrscopy (EM) analysis then localized the electron-dense ferritin-conjugated anti-spectrin antibodies specifically to the inner surface of the cell membrane (Nicolson et al., 1971a).
Several years later these predictions were borne out. It is now known that spectrin is the most abundant peripheral membrane protein in red blood cells and the principal component of a protein meshwork, or membrane skeleton, that underlies the cell membrane. This membrane skeleton, which contains other proteins, including actin (Tilney and Detmers, 1975), restricts the lateral mobility of membrane-penetrating protein molecules (Nicolson et al., 1971b; Elgsaeter and Branton, 1973). It also maintains the structural integrity and biconcave shape of the red blood cell membrane, and explains why membranes are not mechanochemically identical to lipid bilayers (Evans, 1973). Peripheral proteins also exist beneath the plasma membrane of many nucleated cells, but these proteins form a discontinuous network under the membrane, do not include spectrin, and generally allow most integral proteins to diffuse globally in the membrane (Frye and Edidin, 1970). LB