Microsomal membranes (RM) l lose their ability to translocate nascent secretory, 2,3 lysosomal, 4 and membrane s proteins upon salt extraction if they are assayed in the wheat germ cell-free translation system. From the salt extract, a ribonucleoprotein [termed signal recognition particle (SRP)] was purified to homogeneity and shown to be composed of six different polypeptide chains 3 and one 7 S RNA molecule.6 Addition of the crude salt extract or purified SRP to salt-extracted RM (K-RM) restored their ability not only to catalyze translocation of secretory 2,3 and lysosomal 4 proteins across the microsomal membrane, but also to catalyze the asymmetric integration of transmembrane proteins s into the membrane. The function of SRP in the translocation process was shown to involve the recognition of the signal sequence of these proteins in their nascent state, 7 the specific binding of polysomes synthesizing these proteins to RM vesicles, 8 and the initiation of the translocation event. In the absence of RM vesicles, SRP modulates translation by reversibly arresting the elongation of nascent secretory proteins after their signal peptide and about 40 additional amino acids have been polymerized. 9 This translation arrest can be released if K-RM vesicles are added, resulting in completed and efficiently translocated secretory protein. The arrest-releasing activity of K-RM vesicles has been localized to a 72,000-dalton integral membrane protein, termed SRP-receptor, l°,H or docking protein. ~2 Here we describe the purification of SRP which, as a component of the translocation machinery, has proved to be a valuable tool for studying the mechanism of protein translocation across the membrane of the endoplasmic reticulum. 7-9 Purified SRP has also been used as an affinity probe to purify SRP-receptor. ~1 Finally, it has been helpful as an additional supplement in in vitro translation systems containing RM, where it can boost the efficiency of in vitro protein translocation to virtually 100%.
Blobel G. Signal recognition particle: a ribonucleoprotein required for cotranslational translocation of proteins, isolation and properties. Meth Enzymol 96:682-91, 1983
(PMID : 6197610) (PDF)
(PMID : 6197610) (PDF)