Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages

Karin Römisch,Frederickw Miller,Bernhard Dobberstein,Stephen High

doi:10.1186/ar1895

Abstract

The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the α-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain.

Highlights

Both secretory and membrane proteins destined for entry into the eukaryotic secretory pathway carry hydrophobic signal sequences that direct them to the endoplasmic reticulum (ER)
Once a signal sequence has been bound, further elongation is retarded until contact is made between SRP54 and the signal recognition particle (SRP) receptor complex in the ER membrane
In order to better characterize functionally important regions of SRP54 in situ, we tested the effects of various sera containing antibodies specific for the SRP54 protein upon co-translational SRP-dependent targeting to the ER membrane

Summary

Introduction

Both secretory and membrane proteins destined for entry into the eukaryotic secretory pathway carry hydrophobic signal sequences that direct them to the endoplasmic reticulum (ER). The signal sequence is often located towards the amino terminus of the protein, and in the case of presecretory proteins it is proteolytically removed after targeting is completed [1] These ER targeting signals are recognized and bound by a small ribonucleoprotein complex, the signal recognition particle (SRP), as soon as they emerge from the ribosome during protein synthesis [2,3,4]. This co-translational binding of SRP causes an arrest or retardation of translation that is relieved upon the interaction of the nascent chain/ribosome/SRP complex with the SRP receptor complex located in the ER membrane [3]. Translation of the targeted nascent chain resumes and the free SRP can enter a new cycle of targeting, while the signal sequence inserts into the ER translocon and is cleaved on the luminal side of the membrane by signal peptidase when a suitable site is available [6]

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Conclusion