RAPID COMMUNICATIONS Lateral Diffusion of Wild-type and Mutant L d Antigens in L Cells

We have compared the lateral diffusion of intact transmembrane proteins, wild-type H2L d antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type L d antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules. The lateral diffusion coefficients measured for membrane proteins range from ca. l0 -ll cm2s -~ to 5 x l0 -9 cm2s -l with values for many proteins ~2 x 10 -t° cm2s-L This value is l0 to 20 times slower than the fastest diffusion observed, for rhodopsin in disc membranes, (14, 20, 29) and is much lower than expected on the basis of any theory for diffusion in membranes (5, 9, 22). Several lines of evidence suggest that the slow lateral diffusion of many membrane proteins is due to interactions between these proteins and the cytoskeleton: (a) Diffusion of band 3 is 100 times faster in spectrin-deficient mouse erythrocytes than in normal erythrocytes (13, 23). (b) Diffusion of several membrane proteins is increased 100-fold or more in membrane blebs produced on the surface of intact cells. Such blebs appear to lack actin (26, 28, 32). (c) Diffusion of membrane proteins reconstituted into synthetic lipid vesicles is orders of magnitude faster than diffusion of the same proteins in native membranes (17, 27). Antigens specified by the mammalian major histocompatibility complex (MHC), class I MHC antigens, are expressed on most cells. Lateral diffusion of these antigens, H2 of mouse and HLA of humans, has been studied in normal and transformed cells, (4, 6, 7, 18, 25) and in liposomes reconstituted with purified antigens (1). MHC antigens are, like other membrane integral proteins, somehow constrained in their lateral diffusion. Diffusion coefficients range from <<2 to 10 x 10 j° cm2s -l in lymphocytes and in cultured fibroblasts, while they are 50-100 x l0 -l° cm2s -l for MHC antigens in liposomes (1). A good deal is known about the structure of MHC class I antigens, and complete sequences are available for several different antigetis (reviews in 8, I l). All class I antigens consist of a heavy chain of -44,000 d associated with a light chain, beta-2 microglobulin, of 12,000 d. The bulk of the complex lies outside of the cell membrane. A segment of 24 amino acids spans the membrane, and a segment of from 31 to 46 amino acids lies within the cell. This cytoplasmic domain interacts with isolated proteins of the cytoskeleton (19) and other work suggests that the MHC antigens react with the cytoskeleton in intact membranes as well (12, 30). If this is the case, then mutant MHC molecules with modified or truncated cytoplasmic domains ought to diffuse more rapidly than wild-type antigens. Truncated MHC genes have been produced by two laboratories. Zuniga et al. (33) transfected L cells with mutant H2 L d genes which lacked as many as 24 of the 31 C terminal residues of wild-type L d. They showed that these mutant antigens were integrated in the membrane and that they effectively presented viral antigens to H-2Ld-restricted cytotoxic T cells. Murre et al. (15) constructed a mutant L d in which all but six cytoplasmic residues were deleted and the deleted residues replaced by three amino acids specified by an MHC class II gene. These mutant genes were also expressed in transfected L cells and presented viral antigen to H-2 restricted killer cells. The truncated gene products also capped normally when reacted with anti-H-2 antibodies followed by antiglobulins. This implies that metabolically-driven lateral mobility of the truncated antigens is normal, but does not show if their diffusion differs from antigens with full-length cytoplasmic tails. We have used fluorescence photobleaching to measure the lateral diffusion of wild-type and three mutant H-2 L d antigens expressed in L cells transfected with the appropriate genes. Three of these genes, the wild-type L d (27.5), a mutant (BAL907) in which 25 of the 31 residues of the cytoplasmic domain are replaced by 19 residues, not derived from the L d gene, and a mutant (BAL911) truncated to a seven residue cytoplasmic domain, have been described previously (33). A third mutant, C48, is further truncated to four residues of the wild-type cytoplasmic domain (lys-arg-ser-glu). The cytoplasmic sequences predicted for all 4 L d antigens are shown in Fig. 1. Here we show that the diffusion coefficients of the four THE JOURNAL OF CELL BIOLOGY • VOLUME 99 DECEMBER 1984 2333-2335 © The Rockefeller University Press 0021-9525/84112/2333/03 $1.0