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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Scientific Reports Open Access 07 June 2023
BIOspektrum Open Access 30 August 2022
HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins
Nature Communications Open Access 18 May 2022
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Gene Expression Omnibus
Rapoport, T. A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450, 663–669 (2007)
Walter, P. & Johnson, A. E. Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 10, 87–119 (1994)
Favaloro, V., Spasic, M., Schwappach, B. & Dobberstein, B. Distinct targeting pathways for the membrane insertion of tail-anchored (TA) proteins. J. Cell Sci. 121, 1832–1840 (2008)
Stefanovic, S. & Hegde, R. S. Identification of a targeting factor for posttranslational membrane protein insertion into the ER. Cell 128, 1147–1159 (2007)
Jonikas, M. C. et al. Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 323, 1693–1697 (2009)
Schuldiner, M. et al. The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell 134, 634–645 (2008)
Aviram, N. & Schuldiner, M. Embracing the void—how much do we really know about targeting and translocation to the endoplasmic reticulum? Curr. Opin. Cell Biol. 29, 8–17 (2014)
Ast, T. & Schuldiner, M. All roads lead to Rome (but some may be harder to travel): SRP-independent translocation into the endoplasmic reticulum. Crit. Rev. Biochem. Mol. Biol. 48, 273–288 (2013)
Ast, T., Cohen, G. & Schuldiner, M. A network of cytosolic factors targets SRP-independent proteins to the endoplasmic reticulum. Cell 152, 1134–1145 (2013)
Ng, D. T., Brown, J. D. & Walter, P. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J. Cell Biol. 134, 269–278 (1996)
Cohen, Y. & Schuldiner, M. Advanced methods for high-throughput microscopy screening of genetically modified yeast libraries. Methods Mol. Biol. 781, 127–159 (2011)
Tong, A. H. Y. & Boone, C. Synthetic genetic array analysis in Saccharomyces cerevisiae. Methods Mol. Biol. 313, 171–192 (2006)
Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002)
Breslow, D. K. et al. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat. Methods 5, 711–718 (2008)
Breker, M., Gymrek, M. & Schuldiner, M. A novel single-cell screening platform reveals proteome plasticity during yeast stress responses. J. Cell Biol. 200, 839–850 (2013)
Mandon, E. C., Trueman, S. F. & Gilmore, R. Protein translocation across the rough endoplasmic reticulum. Cold Spring Harb. Perspect. Biol. 5, 1–14 (2013)
Harada, Y., Li, H., Wall, J. S., Li, H. & Lennarz, W. J. Structural studies and the assembly of the heptameric post-translational translocon complex. J. Biol. Chem. 286, 2956–2965 (2011)
Kaganovich, D., Kopito, R. & Frydman, J. Misfolded proteins partition between two distinct quality control compartments. Nature 454, 1088–1095 (2008)
Huh, W.-K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003)
Fleischer, T. C., Weaver, C. M., McAfee, K. J., Jennings, J. L. & Link, A. J. Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes. Genes Dev. 20, 1294–1307 (2006)
Ricarte, F. et al. A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology. PLoS One 6, e23696 (2011)
Zhao, Y. et al. Transmembrane protein 208: a novel ER-localized protein that regulates autophagy and ER stress. PLoS One 8, e64228 (2013)
Yompakdee, C., Ogawa, N., Harashima, S. & Oshima, Y. A putative membrane protein, Pho88p, involved in inorganic phosphate transport in Saccharomyces cerevisiae. Mol. Gen. Genet. 251, 580–590 (1996)
Kulak, N. A., Pichler, G., Paron, I., Nagaraj, N. & Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319–324 (2014)
Jan, C. H., Williams, C. C. & Weissman, J. S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling. Science 80, 1257521 (2014). doi: 10.1126/science.1257521
Noriega, T. R. et al. Signal recognition particle-ribosome binding is sensitive to nascent chain length. J. Biol. Chem. 289, 19294–19305 (2014)
Pan, X. et al. A DNA integrity network in the yeast Saccharomyces cerevisiae. Cell 124, 1069–1081 (2006)
Brachmann, C. B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132 (1998)
Gietz, R. D. & Woods, R. A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96 (2002)
Yofe, I. & Schuldiner, M. Primers-4-Yeast: a comprehensive web tool for planning primers for Saccharomyces cerevisiae. Yeast 31, 77–80 (2014)
Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998)
Kitada, K., Yamaguchi, E. & Arisawa, M. Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation. Gene 165, 203–206 (1995)
Goldstein, A. L. & McCusker, J. H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553 (1999)
Stirling, C. J. & Hewitt, E. W. The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19. Nature 356, 534–537 (1992)
Kushnirov, V. V. Rapid and reliable protein extraction from yeast. Yeast 16, 857–860 (2000)
Graham, T. R. Metabolic labeling and immunoprecipitation of yeast proteins. Curr. Protoc. Cell Biol. 7, 7.6 (2001)
Wuestehube, L. J. & Schekman, R. W. Reconstitution of transport from endoplasmic reticulum to Golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast. Methods Enzymol. 219, 124–136 (1992)
Schwenk, J. et al. Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors. Science 323, 1313–1319 (2009)
Wittig, I., Braun, H.-P. & Schägger, H. Blue native PAGE. Nat. Protocols 1, 418–428 (2006)
Pfeffer, S. et al. Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon. Nat. Commun. 5, 3072 (2014)
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.
The authors declare no competing financial interests.
Extended data figures and tables
a, Fluorescent micrographs of RFP–Gas1 confirm that it is not mislocalized when components of SRP, SRP receptor or NAC are compromised (control image can be found in Fig. 1b). Scale bars throughout figure, 5 μm. Images throughout figure are representative of about 300 cells captured per strain. b, SND mutants accumulate RFP–Gas1 in inclusions. Fluorescent micrographs confirm that accumulations of RFP–Gas1 in Δsnd strains colocalize with the cytosolic inclusion marker VHL–GFP. c, SND deletions do not have a non-specific effect on translation, targeting or translocation. A fluorescently tagged SRP substrate (Hxt2–GFP) was mislocalized only in the temperature-sensitive strain sec65-1 when grown at the restrictive temperature of 37 °C (under these conditions the cells lack functional SRP). SND-deleted strains display normal cell surface localization of Hxt2. d, Schematic representation of the structural elements and topology predictions of Snd1 (top), Snd2 (middle) and Snd3 (bottom). Numbers indicate the number of amino acids in the proteins. e, RFP–Gas1 is correctly localized in all GFP-tagged SND proteins, indicating that the tag does not disrupt their function and endogenous localization. f, A mammalian orthologue of Snd2 (hSnd2) is present in canine pancreatic rough microsomes, which are routinely used as a source of mammalian ER proteins, as seen by immunoblotting with an antibody (data not shown) against hSnd2 that was shown to be specific by siRNA-mediated gene silencing. g, Endogenous hSnd2 is localized to human rough ER. HEK293 cells were homogenized and subfractionated into various pellet (P) and supernatant (S) fractions. Fractions were analysed by SDS–PAGE and immunoblotting. hSnd2 co-fractionated with the rough ER markers Grp170 and Sec62 and the ribosomal protein uS3 but not with the nuclear and cytosolic proteins p68 and GAPDH. The areas of interest of luminescence images from a single western blot are shown. For gel source images see Supplementary Fig. 1.
a, Snd2 and Snd3 form a complex with the Sec61 translocon, as shown by BN-PAGE followed by second-dimension SDS–PAGE. Densitometry quantification revealed that Sec61 migrates in four distinct complexes as well as a monomer. Snd2 and Snd3 reside together in two of these complexes, one approximately 669 kDa and a second with a higher molecular mass. We postulate that the two Sec61–SND complexes may differ in size depending on the presence of additional auxiliary components. For gel source images see Supplementary Fig. 1. b, Fluorescent micrographs showing that Snd2 is mislocalized upon deletion of SND3 and Snd3 is mislocalized upon deletion of SND1, suggesting functional dependence among the three proteins. Scale bars throughout figure, 5 μm. Images throughout figure are representative of around 300 cells captured per strain. c, Growth rates reveal genetic interactions among the SND genes. Heterozygous diploids of Δsnd were sporulated and tetrad-dissected to retrieve haploids. Tetrads obtained demonstrate an epistatic interaction between SND1 and SND2 mutations, and a synthetic sick interaction between SND3 and the SND1/2 mutations. As SND3 is more than an order of magnitude more abundant than SND1/2, it is possible that this interaction is due to an independent cellular function. d, Fluorescent micrographs of RFP–Gas1 in single and double SND mutants show that the mutations are epistatic to each other in terms of their effect on targeting. e, Quantification of the RFP–Gas1 mislocalization phenotype in SND single and double mutants (Extended Data Fig. 2d) reveals a buffering epistatic interaction between SND genes (100 cells were counted per strain).
Extended Data Figure 3 Substrate affinity to a targeting pathway depends on the position of its transmembrane domain.
Quantification of the mislocalization phenotype in Fig. 2f, g confirms that re-positioning of a substrate’s TMD can alter its dependence on the different targeting pathways.
Extended Data Figure 4 Compensation for loss of SRP by the SNDs is not due to alteration in protein levels.
a, Overexpression of SND genes does not affect SRP levels. SND genes were overexpressed by growth on galactose in 30 °C, and levels of Sec65 protein were measured by western blotting and normalized to a histone H3 loading control. No apparent change in sec65-1 levels was detected, implying that the rescue observed in Fig. 3b–d is not due to increased SRP levels (data shown as mean ± s.e.m., n = 3, no statistically significant difference was seen between the samples, biological replicates). b, Levels of SND proteins do not change in SRP-depleted cells. SND proteins were C-terminally tagged on the sec65-1 background, and their levels were measured by western blotting when grown at either permissive or restrictive temperatures (30 °C and 37 °C, respectively), and normalized to an actin loading control. No apparent change in Snd1 or Snd3 levels was observed. Snd2 levels were below detection threshold (data not shown). c, Pulse radioactive metabolic labelling followed by DHCαF immunoprecipitation was used to measure the translocation rate of DHCαF. SND2 overexpression induced significantly higher translocation when compared to its repression by glucose, regardless of the functional state of sec65-1. Data shown as mean ± s.e.m. **P < 0.01, ***P < 0.001, by two-tailed Student’s t-test, n = 3, biological replicates. For all gel source images see Supplementary Fig. 1.
a, Repression of SND genes is epistatic with SEC72 deletion and synthetic sick with GET3 deletion. Growth rates of strains with the SND genes expressed under the regulation of a repressible Tet-promoter when grown on tetracycline. The growth rate of Δsec72 Tetp-SNDs conditional double mutants is identical to that of the control strain, indicating that these mutations are epistatic. The Δget3 Tetp-SNDs conditional double mutants are sick, but viable. b, Double deletion of SND2 and GET3 is lethal. Heterozygous diploids of Δsnd2 and Δget3 were sporulated and tetrad-dissected to retrieve haploids. Tetrads obtained demonstrate a synthetic lethal interaction between SND2 and GET3. c, RFP–Gas1 translocation is moderately affected by SND single deletions. Pulse radioactive metabolic labelling followed by RFP–Gas1 immunoprecipitation was used to measure RFP–Gas1 translocation rates. Percentage of glycosylated ER and Golgi forms (indicated by two black lines) was reduced to 5% in Δsec72 mutants, while in Δsnd1, Δsnd2 and Δsnd3 strains it was reduced to 85%, 88% and 79%, respectively (data shown as mean (s.e.m.), n = 3, biological replicates). All strains in this assay were attenuated for degradation with the scl1-DAmP proteasome hypomorphic allele. d, Pulse radioactive metabolic labelling followed by RFP–Gas1 immunoprecipitation was performed in the presence and absence of the glycosylation inhibitor tunicamycin, allowing the identification of three forms of RFP–Gas1: cytosolic, ER and Golgi (mature). e, CPY targeting is not affected by double mutants of the SND and GET pathways. The same methodology as in c was used to follow the signal sequence (SS)-containing protein CPY in the conditional SND2/GET3 double mutant. A mild decrease in the glycosylated forms was observed in the SND2 single mutant, but there was no translocation defect in the GET3 single mutant or in the conditional double mutant. This result was repeated in three independent biological replicates. f, CPY was metabolically labelled in a control strain and a partially translocated pool was visualized with a ladder to provide a size reference to e. g, The same methodology as in c was used to measure the translocation rate of the SRP-dependent substrate DHCαF. In the temperature-sensitive strain sec65-1, at the restrictive temperature (37 °C), there was no translocated substrate. The translocation efficiency of the Δsnd1 strain was comparable to that of the wild-type control; in the Δsnd2 and Δsnd3 strains it was significantly higher (~160% glycosylated protein compared to the wild-type control). Data shown as mean (s.e.m.), n = 3, biological replicates. For all gel source images see Supplementary Fig. 1.
This file contains Supplementary Figure 1, the uncropped scans with size marker indications. (PDF 3722 kb)
This table contains a full list of genes whose deletion caused mislocalization of RFP-Gas1. (XLSX 66 kb)
This table shows physical interactors of Snd2 and Snd3. (XLSX 64 kb)
This table shows proximity specific ribosome profiling in SND deleted strains. (XLSX 910 kb)
This table contains a list of plasmids, strains and primers used in the study. (XLS 71 kb)
About this article
Cite this article
Aviram, N., Ast, T., Costa, E. et al. The SND proteins constitute an alternative targeting route to the endoplasmic reticulum. Nature 540, 134–138 (2016). https://doi.org/10.1038/nature20169
This article is cited by
Scientific Reports (2023)
Nature Reviews Molecular Cell Biology (2022)
HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins
Nature Communications (2022)
Communications Biology (2021)