Endoplasmic reticulum-associated degradation of a degron-containing polytopic membrane protein

Abstract

The presence of two basic amino acids strategically located within a single spanning transmembrane region has previously been shown to act as a signal for the endoplasmic reticulum associated degradation (ERAD) of several polypeptides. In contrast, the functionality of this degron motif within the context of a polytopic membrane protein has not been established. Using opsin as a model system, we have investigated the consequences of inserting the degron motif in the first of its seven transmembrane (TM) spans. Whilst these basic residue reduce the binding of the targeting factor, signal recognition particle, to the first TM span, this has no effect on membrane integration in vitro or in vivo. This most likely reflects the presence of multiple TM spans that can act as targeting signals within in the nascent opsin chain. We find that the degron motif leads to the efficient retention of mutant opsin chains at the endoplasmic reticulum. The mutant opsin polypeptides are degraded via a proteasomal pathway that involves the actions of the E3 ubiquitin ligase HRD1. In contrast, wild-type opsin remains stable for a prolonged period even when artificially accumulated at the endoplasmic reticulum. We conclude that a single dibasic degron motif is sufficient to initiate both the ER retention and subsequent degradation of ospin via an ERAD pathway.

Keywords::

• ERAD
• glycoprotein
• opsin
• signal recognition particle

Acknowledgements

We thank Samuel Crawshaw for valuable discussions during this project and Paul Hargrave, Martin Lowe, Hans-Dieter Söling, Sjaak van Voorden and Marjolein Kikkert for providing reagents. This work was supported by a Wellcome Trust PhD award (AR-S) and funding from the BBSRC (BCSC and SH) grant number BBSRC, BB/G000948/1, and MRC (SH) grant number G0501725.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.