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[53] Signal recognition particle: A ribonucleoprotein required for cotranslational translocation of proteins, isolation and properties

1983; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(83)96057-3

1557-7988

Peter Walter, Günter Blobel,

Molecular Biology Techniques and Applications

This chapter describes the purification of signal recognition particle (SRP), which, as a component of the translocation machinery, proves to be a valuable tool for studying the mechanism of protein translocation across the membrane of the endoplasmic reticulum. The function of SRP in the translocation process involves the recognition of the signal sequence of these proteins in their nascent state, the specific binding of polysomes synthesizing the proteins to microsomal membranes (RM) vesicles, and the initiation of the translocation event. The procedure involves salt extraction of RM, fractionation of the salt extract and hydrophobic-ion exchange chromatography, further purification of SRP, and translocation assay. The SRP is composed of six different polypeptide chains (molecular weights: 72,000, 68,000, 54,000, 19,000, 14,000, and 9000) and one molecule of RNA. Signal recognition particles reconstitute the translocation activity of K-RM, which is monitored by the cotranslational conversion of preprolactin to prolactin.