Mechanisms of integration of de novo-synthesized polypeptides into membranes: Signal-recognition particle is required for integration into microsomal membranes of calcium ATPase and of lens MP26 but not of cytochrome b_5

摘要

We have investigated the in vitro integration into dog pancreas microsomal membranes of three integral membrane proteins that were synthesized de novo in a wheat germ cell-free translation system: calcium ATPase of rabbit sarcoplasmic reticulum, MP26 of bovine lens fiber plasma membrane, and rat liver cytochrome b_5. Biosynthetically these proteins show a common feature in that they are synthesized without a transient NH_2-terminal signal sequence. Two of these proteins, ATPase and MP26, were shown to require the recently discovered signal-recognition particle (SRP) [Walter, P. & Blobel, G. (1982) Nature (London) 299, 691-698] for integration. By this criterion, therefore, they each contain at least one uncleaved signal sequence. Surprisingly, however, the uncleaved signal sequence(s) of these two proteins did not induce the characteristic SRP-mediated translation arrest that was previously shown for a cleaved signal sequence. Unlike ATPase and MP26, cytochrome b_5 did not require SRP for integration into microsomal membrane. Thus, the distinction between an "insertion" sequence (specifying unassisted and opportunistic integration into any exposed membrane) and a "signal" sequence (directing integration into a specific membrane by a receptor-mediated mechanism) is a valid one. By assaying for SRP dependence, the two mechanisms of integration can now be experimentally distinguished.