Ribosome-bound Get4/5 facilitate the capture of tail anchored proteins by Sgt2 in yeast
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PROVIDER: PRJNA636793 | ENA |
REPOSITORIES: ENA
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Project description:The guided entry of tail-anchored proteins (GET) pathway assists in the proper delivery of tail-anchored (TA) proteins to the ER. Here we uncover how the yeast GET pathway components Get4/5 mediate capture of TA proteins by Sgt2, which interacts with TA sequences and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of TA proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting of proteins containing internal TM domains. Exposure of nascent, internal TM domains at the tunnel exit induces high-affinity ribosome-binding of SRP, which in turn prevents ribosome-binding of Get4/5. In this way, the position of TM domains within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.
2020-12-18 | GSE151664 | GEO
Project description:The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.
| S-EPMC7862611 | biostudies-literature
Project description:Hundreds of proteins are inserted post-translationally into the endoplasmic reticulum (ER) membrane by a single carboxy-terminal transmembrane domain (TMD). During targeting through the cytosol, the hydrophobic TMD of these tail-anchored (TA) proteins requires constant chaperoning to prevent aggregation or inappropriate interactions. A central component of this targeting system is TRC40, a conserved cytosolic factor that recognizes the TMD of TA proteins and delivers them to the ER for insertion. The mechanism that permits TRC40 to find and capture its TA protein cargos effectively in a highly crowded cytosol is unknown. Here we identify a conserved three-protein complex composed of Bat3, TRC35 and Ubl4A that facilitates TA protein capture by TRC40. This Bat3 complex is recruited to ribosomes synthesizing membrane proteins, interacts with the TMDs of newly released TA proteins, and transfers them to TRC40 for targeting. Depletion of the Bat3 complex allows non-TRC40 factors to compete for TA proteins, explaining their mislocalization in the analogous yeast deletion strains. Thus, the Bat3 complex acts as a TMD-selective chaperone that effectively channels TA proteins to the TRC40 insertion pathway.
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Project description:Tail-anchored (TA) proteins are transmembrane proteins with a single C-terminal transmembrane domain, which functions as both their subcellular targeting signal and membrane anchor. We show that knockout of TRC40 in cultured human cells has a relatively minor effect on endogenous TA proteins, despite their apparent reliance on this pathway in vitro These findings support recent evidence that the canonical TRC40 pathway is not essential for TA protein biogenesis in vivo We therefore investigated the possibility that other ER-targeting routes can complement the TRC40 pathway and identified roles for both the SRP pathway and the recently described mammalian SND pathway in TA protein biogenesis. We conclude that, although TRC40 normally plays an important role in TA protein biogenesis, it is not essential, and speculate that alternative pathways for TA protein biogenesis, including those identified in this study, contribute to the redundancy of the TRC40 pathway.
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Project description:Enhanced ascorbate peroxidase 2 (APEX2) can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein, ideally within a confined compartment. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. Here we used rapamycin-dependent targeting of APEX2 to tail-anchored proteins of the endoplasmic reticulum and of the inner nuclear membrane (INM). In combination with stable isotope labeling with amino acids in cell culture (SILAC), our novel approach (rapamycin- and APEX-dependent identification of proteins by SILAC, or RAPIDS) allowed the identification of proteins that are in close proximity to the prototypic INM-protein emerin and the vesicle-trafficking protein VAPB. In addition to well-known interaction partners, several new potential binding partners were identified. In RAPIDS, the comparison of plus/minus-rapamycin conditions allows a clear distinction between specific and non-specific hits.
2019-09-09 | PXD012157 | Pride
Project description:Genome-Wide Translation Profiling by Ribosome-Bound tRNA Capture
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Project description:Misfolded or damaged proteins are typically targeted for destruction by proteasome-mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome-mediated degradation of the soluble misfolded reporter, mCherry-CL1, involves two ER-resident E3 ligases, MARCH6 and TRC8. mCherry-CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail-anchored protein heme oxygenase-1 (HO-1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO-1 following intramembrane proteolysis. Our results highlight how ER-resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail-anchored proteins.
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Project description:The loss of the tail is among the notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the “anthropomorphous apes”, with a hypothesized role in contributing to human bipedalism. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here, we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element – inserted into an intron of the TBXT gene – pairs with a neighboring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of mouse Tbxt, mimicking the expression pattern of its hominoid ortholog TBXT. We found that mice expressing both Tbxt isoforms can exhibit a complete absence of the tail or a shortened tail, depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud, supporting the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. We further noted that mice expressing the exon-skipped Tbxt isoform may develop neural tube defects, a condition that affects ~1/1,000 neonates in human. Thus tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.
2024-01-01 | GSE252278 | GEO
Project description:The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when MED5/15 or MED15/16 are inactivated together.
2013-06-07 | GSE47712 | GEO
Project description:The differential ribosome-bound lncRNAs between SW620 and SW480 were investigated by RNA-seq.
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