In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments1. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway2 or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40)3,4 and homologous yeast guided entry of tail-anchored proteins (GET)5,6 pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay7,8. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae9,10, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.
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Gene Expression Omnibus
We thank Schuldiner laboratory members for discussions and comments on the manuscript; D. Kaganovich, T. Ravid, J. Gerst, S. High and H. Riezman for plasmids; P. Walter and M. Seedorf for antibodies; and I. Yofe and U. Weill for the N-terminal tagging plasmid and primers. T.A. was supported by the Adams Fellowship Program of the Israel Academy of Sciences and Humanities. The work on human cells was supported by a DFG grant (IRTG 1830 and ZI 234/13-1) to R.Z. and the generation of anti-hSnd2 antibodies was funded by HOMFOR (HOMFOR2015). Supercomplex analysis by E.C.A. and B.S. was funded by the Deutsche Forschungsgemeinschaft (SFB 1190 P04). J.S.W. is supported by the NIH/NGMS (Center for RNA Systems Biology P50 GM102706 (Cate)). E.A.C. is supported by the National Science Foundation under grant 1144247. This work was funded by the Minerva foundation and Israel Science Foundation grant number 791/14 support to M.S. M.S is an incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics.
Extended data figures
This table contains a full list of genes whose deletion caused mislocalization of RFP-Gas1.
This table shows physical interactors of Snd2 and Snd3.
This table shows proximity specific ribosome profiling in SND deleted strains.
This table contains a list of plasmids, strains and primers used in the study.
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