Cotranslational Protein Targeting Research Articles

Cotranslational protein targeting to the membrane: Nascent-chain transfer in a quaternary complex formed at the translocon

Membrane proteins in bacteria are cotranslationally inserted into the plasma membrane through the SecYEG translocon. Ribosomes exposing the signal-anchor sequence (SAS) of a membrane protein are targeted to the translocon by the signal recognition particle (SRP) pathway. SRP scans translating ribosomes and forms high-affinity targeting complexes with those exposing a SAS. Recognition of the SAS activates SRP for binding to its receptor, FtsY, which, in turn, is primed for SRP binding by complex formation with SecYEG, resulting in a quaternary targeting complex. Here we examine the effect of SecYEG docking to ribosome-nascent-chain complexes (RNCs) on SRP binding and SAS transfer, using SecYEG embedded in phospholipid-containing nanodiscs and monitoring FRET between fluorescence-labeled constituents of the targeting complex. SecYEG–FtsY binding to RNC–SRP complexes lowers the affinity of SRP to both ribosome and FtsY, indicating a general weakening of the complex due to partial binding competition near the ribosomal peptide exit. The rearrangement of the quaternary targeting complex to the pre-transfer complex requires an at least partially exposed SAS. The presence of SecYEG-bound FtsY and the length of the nascent chain strongly influence nascent-chain transfer from SRP to the translocon and repositioning of SRP in the post-transfer complex.

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates.

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

Co-translational protein targeting in bacteria.

About 30% of all bacterial proteins execute their function outside of the cytosol and have to be transported into or across the cytoplasmic membrane. Bacteria use multiple protein transport systems in parallel, but the majority of proteins engage two distinct targeting systems. One is the co-translational targeting by two universally conserved GTPases, the signal recognition particle (SRP) and its receptor FtsY, which deliver inner membrane proteins to either the SecYEG translocon or the YidC insertase for membrane insertion. The other targeting system depends on the ATPase SecA, which targets secretory proteins, i.e. periplasmic and outer membrane proteins, to SecYEG for their subsequent ATP-dependent translocation. While SRP selects its substrates already very early during their synthesis, the recognition of secretory proteins by SecA is believed to occur primarily after translation termination, i.e. post-translationally. In this review we highlight recent progress on how SRP recognizes its substrates at the ribosome and how the fidelity of the targeting reaction to SecYEG is maintained. We furthermore discuss similarities and differences in the SRP-dependent targeting to either SecYEG or YidC and summarize recent results that suggest that some membrane proteins are co-translationally targeted by SecA.

Two-Step Membrane Binding by the Bacterial SRP Receptor Enables Efficient and Accurate Co-Translational Protein Targeting

The signal recognition particle (SRP) delivers ∼30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

Characterization of FtsY, its interaction with Ffh, and proteomic identification of their potential substrates in Mycobacterium tuberculosis.

The universally conserved signal recognition particle (SRP) pathway that mediates co-translational targeting of membrane and secretory proteins is essential for eukaryotic and prokaryotic cells. The Mycobacterium tuberculosis SRP pathway consists of 2 proteins, Ffh and FtsY, and a 4.5S RNA molecule. Although the Escherichia coli SRP pathway is well studied, understanding of the M. tuberculosis SRP pathway components is very limited. In this study, we have overexpressed and characterized the M. tuberculosis SRP receptor (SR) FtsY as a GTP binding protein. Further, we established the direct protein-protein interaction between Ffh and FtsY. The Ffh-FtsY complex formation resulted in mutual stimulation of their GTP hydrolysis activity. We also attempted to biochemically characterize the SRP components by constructing the antisense gene knockdown strains of ffh and ftsY in M. tuberculosis. Loss of ffh and ftsY resulted in a decreased in vitro growth rate of the antisense ffh strain as compared with the antisense ftsY strain. Finally, 2-D gel electrophoresis of antisense depleted ffh and ftsY strains identified differential expression of 14 proteins.

A Localization Screen Reveals Translation Factories and Widespread Co-Translational Protein Targeting

Local translation allows a spatial control of gene expression. Here, we performed a dual protein/mRNA localization screen, using smFISH on 521 human cell lines expressing GFPtagged genes. A total of 32 mRNAs displayed specific cytoplasmic localizations, and we observed local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope and centrosomes, the latter being cell-cycle dependent. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational targeting. Nevertheless, the kinesin KIF1C localized mRNAs to cytoplasmic protrusions in a translation-independent manner. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For β-catenin, foci formation was regulated by Wnt, relied on APCdependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting.

The signal recognition particle (SRP) delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

Structure of the quaternary complex between SRP, SR, and translocon bound to the translating ribosome

During co-translational protein targeting, the signal recognition particle (SRP) binds to the translating ribosome displaying the signal sequence to deliver it to the SRP receptor (SR) on the membrane, where the signal peptide is transferred to the translocon. Using electron cryo-microscopy, we have determined the structure of a quaternary complex of the translating Escherichia coli ribosome, the SRP–SR in the ‘activated’ state and the translocon. Our structure, supported by biochemical experiments, reveals that the SRP RNA adopts a kinked and untwisted conformation to allow repositioning of the ‘activated’ SRP–SR complex on the ribosome. In addition, we observe the translocon positioned through interactions with the SR in the vicinity of the ribosome exit tunnel where the signal sequence is extending beyond its hydrophobic binding groove of the SRP M domain towards the translocon. Our study provides new insights into the mechanism of signal sequence transfer from the SRP to the translocon.

Human apo-SRP72 and SRP68/72 complex structures reveal the molecular basis of protein translocation.

The co-translational targeting or insertion of secretory and membrane proteins into the endoplasmic reticulum (ER) is a key biological process mediated by the signal recognition particle (SRP). In eukaryotes, the SRP68–SRP72 (SRP68/72) heterodimer plays an essential role in protein translocation. However, structural information on the two largest SRP proteins, SRP68 and SRP72, is limited, especially regarding their interaction. Herein, we report the first crystal structures of human apo-SRP72 and the SRP68/72 complex at 2.91Å and 1.7Å resolution, respectively. The SRP68-binding domain of SRP72 contains four atypical tetratricopeptide repeats (TPR) and a flexible C-terminal cap. Apo-SRP72 exists mainly as dimers in solution. To bind to SRP68, the SRP72 homodimer disassociates, and the indispensable C-terminal cap undergoes a pronounced conformational change to assist formation of the SRP68/72 heterodimer. A 23-residue polypeptide of SRP68 is sufficient for tight binding to SRP72 through its unusually hydrophobic and extended surface. Structural, biophysical, and mutagenesis analyses revealed that cancer-associated mutations disrupt the SRP68–SRP72 interaction and their co-localization with ER in mammalian cells. The results highlight the essential role of the SRP68–SRP72 interaction in SRP-mediated protein translocation and provide a structural basis for disease diagnosis, pathophysiology, and drug design.

Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction.

Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway.

The Chloroplast SRP Systems of Chaetosphaeridium globosum and Physcomitrella patens as Intermediates in the Evolution of SRP-Dependent Protein Transport in Higher Plants.

The bacterial signal recognition particle (SRP) mediates the cotranslational targeting of membrane proteins and is a high affinity complex consisting of a SRP54 protein subunit (Ffh) and an SRP RNA. The chloroplast SRP (cpSRP) pathway has adapted throughout evolution to enable the posttranslational targeting of the light harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. In spermatophytes (seed plants), the cpSRP lacks the SRP RNA and is instead formed by a high affinity interaction of the conserved 54-kD subunit (cpSRP54) with the chloroplast-specific cpSRP43 protein. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane. However, in contrast to spermatophytes, plastid SRP RNAs were identified within all streptophyte lineages and in all chlorophyte branches. Furthermore, it was shown that cpSRP43 does not interact with cpSRP54 in chlorophytes (e.g., Chlamydomonas reinhardtii). In this study, we biochemically characterized the cpSRP system of the charophyte Chaetosphaeridium globosum and the bryophyte Physcomitrella patens. Interaction studies demonstrate low affinity binding of cpSRP54 to cpSRP43 (Kd ~10 μM) in Chaetosphaeridium globosum and Physcomitrella patens as well as relatively low affinity binding of cpSRP54 to cpSRP RNA (Kd ~1 μM) in Physcomitrella patens. CpSRP54/cpSRP43 complex formation in charophytes is supported by the finding that specific alterations in the second chromodomain of cpSRP43, that are conserved within charophytes and absent in land plants, do not interfere with cpSRP54 binding. Furthermore, our data show that the elongated apical loop structure of the Physcomitrella patens cpSRP RNA contributes to the low binding affinity between cpSRP54 and the cpSRP RNA.

Analysis of Myelodysplastic Syndrome Stem Cells at Single Cell Resolution during DNA Methyltransferase Inhibitor Therapy

Abstract The myelodysplastic syndromes (MDS) arise in and are maintained by hematopoietic stem cells (HSCs). Serial sampling of patients treated with DNA methyltransferase inhibitors (DNMTIs) and lenalidomide has demonstrated that disease HSCs (MDS HSCs) persist at significant levels even in patients achieving complete clinical and cytogenetic responses. As MDS HSCs are the functional unit of clonal selection both during therapy and subsequent disease progression, we hypothesized that the molecular heterogeneity of MDS HSCs may underlie therapeutic resistance. We therefore sought to perform single cell RNA-sequencing (RNA-seq) on MDS HSCs from patients with known responses to therapy, with the intention of identifying novel therapeutic vulnerabilities. To characterize MDS HSC heterogeneity, we FACS-purified HSCs (Lin-CD34+CD38-CD90+CD45RA-) from paired bone marrow (BM) specimens taken from four MDS patients before and after two to four 28-day cycles of the DNMTI decitabine, as well as two patients who were not treated due to stable disease, and two normal age matched controls. Specimens from both responding and non-responding patients were included. We captured and sequenced a total of 869 single cells from 14 samples, sequencing to an average depth of 4.8 million reads. In a subset of samples (n=7) we also performed bulk RNA-seq (average 1500 cells) for comparison. The sequencing data was of high quality, with an average of 80% mapped reads. We confirmed our ability to accurately quantify transcript levels using ERCC spike-in controls, observing a linear correlation between expected concentration and observed FPKM (fragments per kilobase per million). Single cell RNA-seq revealed vast intratumoral heterogeneity in MDS HSCs that was otherwise missed by bulk RNA-seq, as evidenced by the presence of transcripts variably expressed among cells from the same specimen (Fig. 1A). Despite this intratumoral heterogeneity, single cell transcriptomes were able to completely separate individual MDS patients using principal components analysis and hierarchical clustering, consistent with the known heterogeneity of MDS. MDS HSCs further clustered separately from normal age-matched HSCs, with the top 10% of genes contributing to this separation enriched for Gene Ontology (GO) categories including pathways implicated in MDS biology such as "mRNA splicing," "nonsense mediated decay," and "P53 mediated DNA Damage Response" (all P<1e-9). Unsupervised hierarchical clustering of all pre-treatment MDS HSCs revealed clustering of cells from responders separately from non-responders (Fig. 1B). Differential gene expression analysis identified a cluster of genes (FDR<0.01) enriched for GO categories including "translational termination," "SRP dependent co-translational protein targeting to membrane," and "nonsense mediated decay" (all P<1e-9). Notably, this cluster included 60 ribosomal proteins, all of which were decreased in non-responders (t-test, P<1e-16), with responders demonstrating levels of expression closer to but still lower than normal controls (t-test, P<1e-3). Thus, defective ribosomal biogenesis, a hallmark of MDS pathogenesis, may also contribute to therapeutic resistance. Finally, within each sample we measured the spread of gene expression using dispersion (log[variance/mean]) within bins based on expression levels, defining variable genes as those with a dispersion >1.75 at a mean FPKM >2. The highest number of variable genes were in normal HSCs (mean=141), with the next highest in responders prior to treatment (mean=80), and the least number of variable genes in non-responders prior to treatment (mean=9.5). We speculate that the low number of variable genes in non-responders reflects a higher degree of clonal dominance. All post-treatment MDS HSCs demonstrated a relatively low number of variable genes (mean=25), suggesting that therapy induces clonal selection. In sum, our data illustrate the robustness of single cell RNA-seq to define the intrinsic variability of individual MDS HSCs, implicating perturbed ribosomal biogenesis and transcriptional variability as novel predictors of response to therapy. As we expand our data set with additional patients, we expect to identify additional pathways that mediate therapeutic response and resistance, as well as mutations and variably expressed genes that are selected for during therapy and drive disease progression. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon

Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY-SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP-RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY-FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex.

New genetic system for translocating proteins through the mitochondrial membrane of the yeast Yarrowia lipolytica

Currently, the extremophilic yeast Yarrowia lipolytica is used as a powerful system for expression of recombinant proteins. However, attempts to obtain recombinant producers based on the Y. lipolytica yeast with cytosolic location of the product almost always fail. It concerns proteins with N-terminal sequences, which are detected by proteasomes and induce the product proteolysis. For stabilization of recombinant products in the Y. lipolytica cells, they should be transported from cytoplasm into mitochondria, where they will be shielded from ubiquitination and from contact with proteosomes. In this work, a method is designed that enhances the proteolytic stability of recombinant proteins in Y. lipolytica without N-terminal modification. For the protein transport through the mitochondrial membrane of Y. lipolytica, a genetic system is proposed that employs specific non-translated sequences of mRNA and performs cotranslational targeting of proteins into mitochondria. Using a fluorescent protein 6His-EYFP that has a low stability in Y. lipolytica cytoplasm as a model, it has been shown that gene expression of this protein under the control of the promoter and transcriptional terminator of mitochondrial superoxide dismutase SOD2 leads to the accumulation in the yeast mitochondria of fluorescent product displaying a high stability in vivo. A new system of targeting recombinant proteins into mitochondria can be of great importance for pharmaceutical purposes, as it enables the creation of proteins with a native primary structure without leader sequence. This system can be used to create a genetic system for maintenance and modification of human native mitochondrial genome in vivo and for the treatment of genetic mitochondrial illnesses.

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.

Distinct Membrane Association Modes Facilitate Co-Translational Protein Targeting

Deciphering the nature of protein interactions at the membrane surface is crucial to understanding the molecular mechanism, timing, and regulation of many essential cellular processes. The signal recognition particle (SRP) and its receptor (SR), which target the translating ribosomes to translocation machineries at the target membrane, provide an ideal model system for investigating the mechanism of membrane recruitment of protein machineries. Here we show that FtsY, the E. coli SR, interacts with the membrane through two distinct modes. Using single-molecule techniques that directly observe membrane association of FtsY in a supported lipid bilayer environment, we were able to detect and differentiate between the previously identified stable association mode as well as a transient interaction mode that is also crucial for targeting. Transition between these two modes is regulated by the SRP. Functional assays show that switching between transient and stabilized modes upon SRP binding is critical for successful targeting. We propose that, in contrast to currently accepted models in which FtsY is anchored on the membrane and awaits SRP encounter, this receptor rapidly cycles between cytosol and membrane and actively searches for cargo-loaded SRP. This active search could provide a mechanism to enhance efficient and accurate membrane localization of the targeting complex.

Optimizing membrane-protein biogenesis through nonoptimal-codon usage.

Two studies provide insights into the distinct strategies used by prokaryotes and eukaryotes to pause translation in order to facilitate cotranslational targeting of membrane proteins to the translocon.

Allosteric Response and Substrate Sensitivity in Peptide Binding of the Signal Recognition Particle

We characterize the conformational dynamics and substrate selectivity of the signal recognition particle (SRP) using a thermodynamic free energy cycle approach and microsecond timescale molecular dynamics simulations. The SRP is a central component of the co-translational protein targeting machinery that binds to the N-terminal signal peptide (SP) of nascent proteins. We determined the shift in relative conformational stability of the SRP upon substrate binding to quantify allosteric coupling between SRP domains. In particular, for dipeptidyl aminopeptidase, an SP that is recognized by the SRP for co-translational targeting, it is found that substrate binding induces substantial changes in the SRP toward configurations associated with targeting of the nascent protein, and it is found that the changes are modestly enhanced by a mutation that increases the hydrophobicity of the SP. However, for alkaline phosphatase, an SP that is recognized for post-translational targeting, substrate binding induces the reverse change in the SRP conformational distribution away from targeting configurations. Microsecond timescale trajectories reveal the intrinsic flexibility of the SRP conformational landscape and provide insight into recent single molecule studies by illustrating that 10-nm lengthscale changes between FRET pairs occur via the rigid-body movement of SRP domains connected by the flexible linker region. In combination, these results provide direct evidence for the hypothesis that substrate-controlled conformational switching in the SRP provides a mechanism for discriminating between different SPs and for connecting substrate binding to downstream steps in the protein targeting pathway.

The many faces of SRP RNA

The signal recognition particle (SRP) is a ribonucleoprotein complex that plays an essential role in co-translational targeting of membrane proteins. It is found in all three domains of life and exhibits a high diversity regarding composition and structure. In most organisms, SRP can be divided into two functional domains. The S domain mediates recognition and transport of ribosome-nascent chain complexes to the translocation channel, while the Alu domain stalls elongation of the ribosome until the complex has been faithfully delivered._x000B_Here we present the crystal structures of the complete bacterial SRP Alu domain and the ternary complex of human SRP S domain RNA, SRP19, and the SRP68-RBD. Together with previous structures, our data underline the taxon-specific evolutionary adaptation of SRP RNA that has important implications in SRP-mediated targeting.

The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase.

Hundreds of tail-anchored proteins, including soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) involved in vesicle fusion, are inserted post-translationally into the endoplasmic reticulum membrane by a dedicated protein-targeting pathway. Before insertion, the carboxy-terminal transmembrane domains of tail-anchored proteins are shielded in the cytosol by the conserved targeting factor Get3 (in yeast; TRC40 in mammals). The Get3 endoplasmic-reticulum receptor comprises the cytosolic domains of the Get1/2 (WRB/CAML) transmembrane complex, which interact individually with the targeting factor to drive a conformational change that enables substrate release and, as a consequence, insertion. Because tail-anchored protein insertion is not associated with significant translocation of hydrophilic protein sequences across the membrane, it remains possible that Get1/2 cytosolic domains are sufficient to place Get3 in proximity with the endoplasmic-reticulum lipid bilayer and permit spontaneous insertion to occur. Here we use cell reporters and biochemical reconstitution to define mutations in the Get1/2 transmembrane domain that disrupt tail-anchored protein insertion without interfering with Get1/2 cytosolic domain function. These mutations reveal a novel Get1/2 insertase function, in the absence of which substrates stay bound to Get3 despite their proximity to the lipid bilayer; as a consequence, the notion of spontaneous transmembrane domain insertion is a non sequitur. Instead, the Get1/2 transmembrane domain helps to release substrates from Get3 by capturing their transmembrane domains, and these transmembrane interactions define a bona fide pre-integrated intermediate along a facilitated route for tail-anchor entry into the lipid bilayer. Our work sheds light on the fundamental point of convergence between co-translational and post-translational endoplasmic-reticulum membrane protein targeting and insertion: a mechanism for reducing the ability of a targeting factor to shield its substrates enables substrate handover to a transmembrane-domain-docking site embedded in the endoplasmic-reticulum membrane.