Translocation Of Proteins

Published November 1, 1981

Translocation of Proteins Across the Endoplasmic Reticulum III . Signal Recognition Protein (SRP) Causes Signal Sequence-dependent and Site-specific Arrest of Chain Elongation that is Released by Microsomal Membranes
PETER WALTER and GONTER BLOBEL Laboratory of Cell Biology, The Rockefeller University, New York 10021

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The previously observed (Walter, et al . 1981 1. Cell Biol. 91 :545-550) inhibitory effect of SRP selectively on the cell-free translation of mRNA for secretory protein (preprolactin) was shown here to be caused by a signal sequence-induced and site-specific arrest in polypeptide chain elongation . The M r of the SRP-arrested nascent preprolactin chain was estimated to be 8,000corresponding to -70 amino acid residues . Because the signal sequence of preprolactin comprises 30 residues and because ---40 residues of the nascent chain are buried (protected from protease) in the large ribosomal subunit, we conclude that it is the interaction of SRP with the amino-terminal signal peptide of the nascent chain (emerged from the large ribosomal subunit) that modulates translation andthereby causes an arrest in chain elongation . This arrest is released upon SRP-mediated binding of the elongation-arrested ribosomes to the microsomal membrane, resulting in chain completion and translocation into the microsomal vesicle.
ABSTRACT

In the previous two papers we have described several functional properties of SRP . We have shown (a) that SRP binds, presumably via the signal sequenceof the nascent chain, to in vitro assembled polysomes synthesizing secretory protein but not to those synthesizing cytoplasmic protein (1); (b) that SRP inhibits translation of mRNA coding for secretory protein but not of that coding for cytoplasmic protein (1) ; and (c) that SRP mediates binding of in vitro assembled polysomes synthesizing secretory protein (but not of those synthesizingcytoplasmic protein) to microsomal membranes (2). Polysome binding and translation-inhibitory effect were observed to be correlated (1) and the presence ofmicrosomal membranes appeared to reduce the translation-inhibitory effect of SRP (2) . In this paper we describe studies on the translation-inhibitory effect of SRP and on the reversal of this effect by microsomal membranes .
MATERIALS AND METHODS
Thepreparation of various microsomal membrane fractions (RM, K-RM), the extraction and purification of SRP, the wheat germ translation system, and the quantitation of in vitro synthesized protein were described in the first paper of this series (1). The SRP preparation used was the eluate of the aminopentylagarose column.

RESULTS

To address questions concerning the nature ofthe SRP-inducedinhibition of secretory protein synthesis (1) and the observed
THE JOURNAL OF CELL BIOLOGY " VOLUME 91 NOVEMBER 1981 557-561 ©The Rockefeller University Press - 0021-9525/81/11/0557/05 $1 .00

release of this inhibition by microsomal membranes (2), we decided to use a translation system that was synchronized with respect to polypeptide chain elongation (3, 4). Synchronization can be achieved by theaddition of inhibitors of initiation a short time after the start of the incubation. Such a system allows the subsequent addition of membranes to be precisely timed with respect to chain length . Furthermore, protein synthesis stops after the initiated chains are completed . This allows us to detect any putative SRP-arrested states of preprolactin chain elongation and to ascertain that (uponrelease of the arrested state by microsomal membranes) the appearance of completed chains is due to chain completion of a previously elongation-arrested nascent chain, but not of a newly initiated chain. To characterize our synchronized translation system, the following experiments were performed . Translation of prolactin mRNA was allowed to initiate and to proceed for 2 min after which time...