Defining the physiological role of SRP in protein-targeting efficiency and specificity

Boydston, Elizabeth A.; Subramanian, Kelly; Nunnari, Jodi; Weissman, Jonathan S.

doi:10.1126/science.aar3607

Cited by 183 publications

(198 citation statements)

References 33 publications

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“…The latter observations led to the conclusion that yeast SRP is responsible for ER targeting of virtually all ribosomes synthesizing secretome proteins except for the tailanchored (TA) membrane proteins that are targeted by the GET pathway (17). However, rapid inactivation of yeast SRP combined with ER-specific ribosome profiling indicates that ribosomes synthesizing SRP-independent substrates remain ER-localized in SRPdeficient cells (18). In contrast, SRPdependent secretome proteins including integral membrane proteins are mistargeted to the mitochondria in SRP deficient cells (18).…”

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confidence: 99%

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Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations

Mandon

Butova²,

Lachapelle³

et al. 2018

Journal of Biological Chemistry

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The Sec61 complex is the primary cotranslational protein translocation channel in yeast (Saccharomyces cerevisiae). The structural transition between the closed inactive conformation of the Sec61 complex and its open and active conformation is thought to be promoted by binding of the ribosome nascent-chain complex to the cytoplasmic surface of the Sec61 complex. Here, we have analyzed new yeast Sec61 mutants that selectively interfere with cotranslational translocation across the endoplasmic reticulum. We found that a single substitution at the junction between transmembrane segment TM7 and the L6/7 loop interferes with cotranslational translocation by uncoupling ribosome binding to the L6/7 loop from the separation of the lateral gate transmembrane spans. Substitutions replacing basic residues with acidic residues in the C-terminal tail of Sec61 had an unanticipated impact upon binding of ribosomes to the Sec61 complex. We found that similar charge-reversal mutations in the N-terminal tail and in cytoplasmic loop L2/3 did not alter ribosome binding but interfered with translocation channel gating. These findings indicated that these segments are important for the structural transition between the inactive and active conformations of the Sec61 complex. In summary our results have http://www.jbc.org/cgi

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mentioning

confidence: 99%

“…However, rapid inactivation of yeast SRP combined with ER-specific ribosome profiling indicates that ribosomes synthesizing SRP-independent substrates remain ER-localized in SRPdeficient cells (18). In contrast, SRPdependent secretome proteins including integral membrane proteins are mistargeted to the mitochondria in SRP deficient cells (18).…”

mentioning

confidence: 99%

Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations

Mandon

Butova²,

Lachapelle³

et al. 2018

Journal of Biological Chemistry

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“…Allowing mecciRNA-bounded proteins to enter the mitochondria may even serve as a sorting mechanism to make sure the entry of right types and amounts of proteins and to keep unwanted cytosolic proteins and RNAs from entering the mitochondria. Interestingly, in a recent study, many proteins are found to demonstrate aberrant mitochondrial targeting in the absence of SRP that facilitates protein targeting to the endoplasmic reticulum (Aviram and Schuldiner, 2017;Costa et al, 2018;Walter and Blobel, 1982). There are also g-circRNAs encoded by the nuclear genome in the mitochondria ( Figure 1B), and it is also possible for them to play similar functions like mecciRNAs in mitochondrial protein importation.…”

Section: Discussionmentioning

confidence: 96%

The identification of mecciRNAs and their roles in mitochondrial entry of proteins

Liu

Wang

et al. 2019

Preprint

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Mammalian mitochondria have small genomes encoding very limited numbers of proteins. Over one thousand proteins and noncoding RNAs encoded by nuclear genome have to be imported from the cytosol into the mitochondria. Here we report the identification of hundreds of circular RNAs (mecciRNAs) encoded by mitochondrial genome. We provide both in vitro and in vivo evidence to show that mecciRNAs facilitate mitochondrial entry of nuclear-encoded proteins by serving as molecular chaperones in the folding of imported proteins. Known components of mitochondrial protein and RNA importation such as TOM40 and PNPASE interact with mecciRNAs and regulate protein entry. Expression of mecciRNAs is regulated, and these transcripts are critical for mitochondria in adapting to physiological conditions and diseases such as stresses and cancers by modulating mitochondrial protein importation. mecciRNAs and their associated physiological roles add categories and functions to eukaryotic circular RNAs, and shed novel lights on communication between mitochondria and nucleus.

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“…This led to the concept that availability of ribosomes, aminoacyl-tRNA syntheases and charged tRNAs might be rate limiting and influence the speed of protein translation in one condition but not in another. 15 To test this possibility, we treated HEK293 cells with RO-3306, a CDK1inhibitor, which arrests cells in the G2 state [27]. The results from a flow cytometry analysis show that treatment with 7 µM RO-3306 caused the expected arrest of cells in G2 after 48 h of incubation (Fig.…”

Section: Sorting Is Affected By State Of the Cell Cycle But Not By Enmentioning

confidence: 99%

“…It is also possible that expression of Kesv reduces the amount of a ratelimiting chaperon like the signal recognition particle (SRP) [14], which is responsible for the correct sorting of the protein to the SP. Such a process could result in miss-sorting of a nascent protein [15]. To test this possibility we examined whether sorting of small K + channel proteins is generally compromised by codon bias.…”

Section: Mitochondrial Sorting Of Channel Protein Is Modulated By Codmentioning

confidence: 99%

Codon bias determines sorting of ion channel protein

Kithil

Langhans

et al. 2020

Preprint

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The choice of codons can influence local translation kinetics during protein synthesis. The question of whether the modulation of polypeptide folding and binding to chaperons influences sorting of nascent membrane proteins remains unclear. Here, we use two similar K + channels as model systems to examine the impact of codon choice on protein sorting. By monitoring transient expression of GFP tagged proteins in mammalian cells we find that targeting of one channel to the secretory pathway is insensitive to codon optimization. In contrast, sorting of the second channel to the mitochondria is very sensitive to codon choice. The protein with an identical amino acid sequence is sorted in a codon and cell cycle dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves together with a cell-state depending decoding mechanism as a secondary code for sorting intracellular proteins. Introduction:Eukaryotic cells have developed efficient systems that guarantee specific targeting of nascent membrane proteins to their final destination. This is either the plasma membrane or the membranes of organelles such as mitochondria, chloroplasts or the nucleus. A few proteins exhibit dual targeting, which means that the same or a very similar protein is located in the plasma membrane as well as in organelle membranes [1]. Typically protein synthesis begins in the cytosol regardless of the final destination of the protein. The canonical pathway for most plasma membrane proteins involves specific motifs at the N-terminus of the nascent polypeptide that interact with the signal recognition particle (SRP). This complex then guides the ribosome together with the nascent polypeptide to the translocon in the endoplasmic reticulum (ER), which serves as the entry point for further protein synthesis.In addition to this canonical pathway, there are other SRP independent routes for targeting membrane proteins to the ER. The best-known alternative route involves tail-anchored (TA) proteins [2]. In these proteins the transmembrane 3 domains at or near their C-termini are recognized by a complex of chaperons at the exit tunnel of the ribosome where they are guided and inserted into the ER membrane [3,4]. In contrast, membrane proteins destined for mitochondria or chloroplasts avoid these targeting systems. After synthesis, these proteins are guided by chaperons to the mitochondrial or chloroplast translocation apparatus and then inserted in a post-translational manner into their target membranes [5].While many components of the co-and post-translational targeting pathways are well understood, many questions remain about how cells prevent misstargeting of ER proteins to mitochondria and how the same membrane protein is targeted to both the ER and mitochondria in the same cell. Included in the list of proteins with dual localization properties are ion channels; for example the K + channel Kv1.3 is present in both the plasma membrane and in the inner membrane of the mitocho...

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Defining the physiological role of SRP in protein-targeting efficiency and specificity

Cited by 183 publications

References 33 publications

Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations

Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations

The identification of mecciRNAs and their roles in mitochondrial entry of proteins

Codon bias determines sorting of ion channel protein

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