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Abstract

Both cytochrome b₅, isolated from rabbit liver microsomes, and LacZ:HP, a recombinant protein consisting of enzymatically active Escherichia coli beta-galactosidase coupled to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b₅, were shown to spontaneously associate with the plasma membranes of erythrocytes and 3T3 cells. Association was promoted by low pH values, but proceeded satisfactorily over several hours at physiological pH and temperature. About 150,000 cytochrome b₅ molecules or 100,000 LacZ:HP molecules could be associated per erythrocyte. These proteins were not removed from the membrane by extensive washing, even at high ionic strength. After incubation with fluorescently labeled cytochrome b₅ or LacZ:HP, cells displayed fluorescent membranes. The lateral mobility of fluorescently labeled cytochrome b₅ and LacZ:HP was measured by photo-bleaching techniques. In the plasma membrane of erythrocytes and 3T3 cells, the apparent lateral diffusion coefficient D ranged from 1.0.10^-9 to 8.10^-9 cm^-2 s^-1 with a mobile fraction M between 0.4 and 0.6. The lateral mobility of these proteins closely resembled that reported for lipid-anchored proteins and was much higher than that reported for Band 3, an erythrocyte membrane-spanning protein with a large cytoplasmic domain. These results suggest that the hydrophobic domain of cytochrome b₅ could be employed as a universal, laterally mobile membrane anchor to associate a variety of diagnostically and therapeutically useful recombinant proteins with cells.