Upon endoplasmic reticulum (ER) stress, the transmembrane endoribonuclease Ire1α performs mRNA cleavage reactions to increase the ER folding capacity. It is unclear how the low abundant Ire1α efficiently finds and cleaves the majority of mRNAs at the ER membrane. Here, we reveal that Ire1α forms a complex with the Sec61 translocon to cleave its mRNA substrates. We show that Ire1α's key substrate, XBP1u mRNA, is recruited to the Ire1α-Sec61 translocon complex through its nascent chain, which contains a pseudo-transmembrane domain to utilize the signal recognition particle (SRP)-mediated pathway. Depletion of SRP, the SRP receptor or the Sec61 translocon in cells leads to reduced Ire1α-mediated splicing of XBP1u mRNA. Furthermore, mutations in Ire1α that disrupt the Ire1α-Sec61 complex causes reduced Ire1α-mediated cleavage of ER-targeted mRNAs. Thus, our data suggest that the Unfolded Protein Response is coupled with the co-translational protein translocation pathway to maintain protein homeostasis in the ER during stress conditions.
Proteins are made up of long chains of smaller building blocks called amino acids. To build this chain, a molecule called mRNA is ‘translated’ into the sequence of amino acids by a molecular machine called a ribosome. In order to work, the protein chain must then be folded into a complex shape. For many proteins, this happens inside a cell compartment called the endoplasmic reticulum.
Newly made proteins are guided to the endoplasmic reticulum by ‘signal recognition particles’, and then enter the endoplasmic reticulum through a channel protein called Sec61. If too many protein chains arrive at once, or they are folded too slowly, the accumulation of unfolded proteins can stress the endoplasmic reticulum. To fix this, cells trigger a process called the unfolded protein response.
In mammals, an enzyme called Ire1α detects when the endoplasmic reticulum is becoming stressed and responds by cleaving mRNA molecules. One particular target of Ire1α is the mRNA molecule that encodes a protein called XBP1, which can activate hundreds of genes to increase the size—and hence reduce the stress—of the endoplasmic reticulum. This protein is only made if a section of the mRNA molecule is removed from it; thus, by cleaving the mRNA, Ire1α enables the protein to be made. It remains unknown, however, how Ire1α finds and cleaves its mRNA targets.
Plumb, Zhang et al. identified the proteins that bind to Ire1α in human cells, and found that the Sec61 channel is one such protein. This interaction localizes Ire1α to the Sec61 channel in the endoplasmic reticulum membrane. The XBP1 protein is then brought to this channel by a signal recognition particle while it is still being translated—that is, when it is still attached to the ribosome and its mRNA molecule. Ire1α can then cleave the XBP1 mRNA. In cells that lack the signal recognition particle or the Sec61 channel protein, Ire1α cannot efficiently cleave the XBP1 mRNA molecule. In addition, if Ire1α is unable to interact with the channel protein, it does not efficiently cleave mRNA molecules at the endoplasmic reticulum membrane.
This work establishes a new link between the unfolded protein response and the pathway that brings new proteins to the endoplasmic reticulum membrane. It provides a basis for future studies examining the details of Ire1α signaling in mammals and, in particular, work investigating the mechanism of insulin mRNA cleavage by Ire1α, which has been implicated in type 2 diabetes.