Abstract
The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs-non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. Previously, we showed that the functional specialization lies in Ire1's RNase activity, which is either stringently splice-site specific or promiscuous (Li et al., 2018). Here, we developed an assay that reports on Ire1's RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain's dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.
Highlights
IntroductionAbout one third of all proteins are folded in the endoplasmic reticulum (ER)
In eukaryotes, about one third of all proteins are folded in the endoplasmic reticulum (ER)
Growth curves revealed that E. coli cells bearing a plasmid containing the S. pombe (Sp) IRE1-kinase/ RNase (KR) barely grew within the monitored 5 hr time window, even in the absence of the isopropyl b-d-1-thiogalactopyranoside (IPTG) inducer (Figure 1A, blue filled triangles)
Summary
About one third of all proteins are folded in the endoplasmic reticulum (ER). The protein folding homeostasis of the ER is monitored and tightly regulated by a collective of signaling pathways, known as the unfolded protein response (UPR) (Hetz et al, 2020; Walter and Ron, 2011). Ire initiates non-conventional splicing of HAC1 (in S. cerevisiae) or XBP1 (in metazoans) mRNA (Cox et al, 1993; Mori et al, 1993; Sidrauski and Walter, 1997; Yoshida et al, 2001). The cleaved mRNAs are subsequently degraded by the cellular RNA decay machinery (Guydosh et al, 2017). As a result, this process, known as regulated Ire1-dependent
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