The SND proteins constitute an alternative targeting route to the endoplasmic reticulum
TL;DR: Three uncharacterized proteins are uncovered that can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system, putting in place an essential piece of the endoplasmic reticulum targeting puzzle.
Abstract: 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 compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) 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 relay. 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 cerevisiae, 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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TL;DR: It is found that known membrane insertion pathways fail to effectively engage tail-anchored membrane proteins with moderately hydrophobic transmembrane domains, and these proteins are instead shielded in the cytosol by calmodulin.
Abstract: Insertion of proteins into membranes is an essential cellular process. The extensive biophysical and topological diversity of membrane proteins necessitates multiple insertion pathways that remain incompletely defined. Here we found that known membrane insertion pathways fail to effectively engage tail-anchored membrane proteins with moderately hydrophobic transmembrane domains. These proteins are instead shielded in the cytosol by calmodulin. Dynamic release from calmodulin allowed sampling of the endoplasmic reticulum (ER), where the conserved ER membrane protein complex (EMC) was shown to be essential for efficient insertion in vitro and in cells. Purified EMC in synthetic liposomes catalyzed the insertion of its substrates in a reconstituted system. Thus, EMC is a transmembrane domain insertase, a function that may explain its widely pleiotropic membrane-associated phenotypes across organisms.
204 citations
TL;DR: It is demonstrated that differences in attention to genes can be explained, to a large extent, exclusively from a small set of identifiable chemical, physical, and biological properties of genes.
Abstract: Biomedical research has been previously reported to primarily focus on a minority of all known genes Here, we demonstrate that these differences in attention can be explained, to a large extent, exclusively from a small set of identifiable chemical, physical, and biological properties of genes Together with knowledge about homologous genes from model organisms, these features allow us to accurately predict the number of publications on individual human genes, the year of their first report, the levels of funding awarded by the National Institutes of Health (NIH), and the development of drugs against disease-associated genes By explicitly identifying the reasons for gene-specific bias and performing a meta-analysis of existing computational and experimental knowledge bases, we describe gene-specific strategies for the identification of important but hitherto ignored genes that can open novel directions for future investigation
178 citations
TL;DR: The systematic proteomic approaches revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters.
Abstract: The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.
145 citations
Cites methods from "The SND proteins constitute an alte..."
...We also performed the same analysis on data from a BirA fusion to Sec63, a 195 component of the Sec translocon, produced for an earlier study (31)....
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TL;DR: It is shown that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino terminus, and an unanticipated consequence of SRP loss was revealed: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects.
Abstract: The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP’s in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino terminus. By contrast, many proteins containing cleavable amino-terminal signal peptides were efficiently cotranslationally targeted in SRP’s absence. We also reveal an unanticipated consequence of SRP loss: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects. These results elucidate SRP’s essential roles in maintaining the efficiency and specificity of protein targeting.
143 citations
TL;DR: The yeast Hsp70 chaperone Ssb employs substrate-tailored dynamic nascent chain associations to coordinate co-translational protein folding, facilitate accelerated translation, and support membrane targeting of organellar proteins.
139 citations
Cites background or methods from "The SND proteins constitute an alte..."
...Tail-anchored proteins (Jan et al., 2014) (D), unclassified proteins (E), SND-pathway proteins (Aviram et al., 2016) (F) and SRP substrates with distinct SRP binding peaks (Chartron et al., 2016) (G)....
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...We therefore individually analyzed Ssb binding to substrates of the SRP and the newly discovered SND (SRP independent) pathway (Aviram et al., 2016), tail-anchored proteins, and unclassified substrates....
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References
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TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.
Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd
5,301 citations
Stanford University1, Yale University2, Johns Hopkins University School of Medicine3, Washington University in St. Louis4, McGill University5, Merck & Co.6, Université catholique de Louvain7, Université libre de Bruxelles8, University of California, Berkeley9, University of Salamanca10, University of Basel11, University of Padua12, Goethe University Frankfurt13, National Institutes of Health14, Aberystwyth University15, Concordia University16, Katholieke Universiteit Leuven17
TL;DR: It is shown that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment, and less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal Growth in four of the tested conditions.
Abstract: Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.
4,328 citations
TL;DR: The construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.
Abstract: A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories, and provide localization information for 70% of previously unlocalized proteins. Analysis of this high-resolution, high-coverage localization data set in the context of transcriptional, genetic, and protein-protein interaction data helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.
4,310 citations
TL;DR: A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed and will reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications.
Abstract: A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.
3,448 citations
TL;DR: In this chapter the authors have provided instructions for transforming yeast by a number of variations of the LiAc/SS-DNA/PEG method to generate large numbers of transformants or deliver DNA constructs or oligonucleotides into the yeast cell.
Abstract: In this chapter we have provided instructions for transforming yeast by a number of variations of the LiAc/SS-DNA/PEG method for a number of different applications. The rapid transformation protocol is used when small numbers of transformants are required. The high efficiency transformation protocol is used to generate large numbers of transformants or to deliver DNA constructs or oligonucleotides into the yeast cell. The large-scale transformation protocol is primarily applicable to the analysis of complex plasmid DNA libraries, such as those required for the yeast two-hybrid system. The microtiter plate versions of the rapid and high efficiency transformation protocols can be applied to high-throughput screening technologies.
2,712 citations