Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad.

Membrane protein biogenesis poses acute challenges to protein homeostasis, and how they are selectively escorted to the target membrane is not well understood. Here we address this question in the guided-entry-of-tail-anchored protein (GET) pathway, in which tail-anchored membrane proteins (TAs) are relayed through an Hsp70-Sgt2-Get3 chaperone triad for targeting to the endoplasmic reticulum. We show that the Hsp70 ATPase cycle and TA substrate drive dimeric Sgt2 from a wide-open conformation to a closed state, in which TAs are protected by both substrate binding domains of Sgt2. Get3 is privileged to receive TA from closed Sgt2, whereas off-pathway chaperones remove TAs from open Sgt2. Sgt2 closing is less favorable with suboptimal GET substrates, which are rejected during or after the Hsp70-to-Sgt2 handover. Our results demonstrate how fine-tuned conformational dynamics in Sgt2 enable hydrophobic TAs to be effectively funneled onto their dedicated targeting factor while also providing a mechanism for substrate selection.

An ankyrin repeat chaperone targets toxic oligomers during amyloidogenesis.

Numerous age-linked diseases are rooted in protein misfolding; this has motivated the development of small molecules and therapeutic antibodies that target the aggregation of disease-linked proteins. Here we explore another approach: molecular chaperones with engineerable protein scaffolds such as the ankyrin repeat domain (ARD). We tested the ability of cpSRP43, a small, robust, ATP- and cofactor-independent plant chaperone built from an ARD, to antagonize disease-linked protein aggregation. cpSRP43 delays the aggregation of multiple proteins including the amyloid beta peptide (Aß) associated with Alzheimer's disease and α-synuclein associated with Parkinson's disease. Kinetic modeling and biochemical analyses show that cpSRP43 targets early oligomers during Aß aggregation, preventing their transition to a self-propagating nucleus on the fibril surface. Accordingly, cpSRP43 rescued neuronal cells from the toxicity of extracellular Aß42 aggregates. The substrate-binding domain of cpSRP43, composed primarily of the ARD, is necessary and sufficient to prevent Aß42 aggregation and protect cells against Aß42 toxicity. This work provides an example in which an ARD chaperone non-native to mammalian cells harbors anti-amyloidal activity, which may be exploited for bioengineering.

Dodecamer assembly of a metazoan AAA+ chaperone couples substrate extraction to refolding.

Ring-forming AAA+ chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA+ chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.

Role of Hsp70 in Post-Translational Protein Targeting: Tail-Anchored Membrane Proteins and Beyond.

The Hsp70 family of molecular chaperones acts as a central 'hub' in the cell that interacts with numerous newly synthesized proteins to assist in their biogenesis. Apart from its central and well-established role in facilitating protein folding, Hsp70s also act as key decision points in the cellular chaperone network that direct client proteins to distinct biogenesis and quality control pathways. In this paper, we review accumulating data that illustrate a new branch in the Hsp70 network: the post-translational targeting of nascent membrane and organellar proteins to diverse cellular organelles. Work in multiple pathways suggests that Hsp70, via its ability to interact with components of protein targeting and translocation machineries, can initiate elaborate substrate relays in a sophisticated cascade of chaperones, cochaperones, and receptor proteins, and thus provide a mechanism to safeguard and deliver nascent membrane proteins to the correct cellular membrane. We discuss the mechanistic principles gleaned from better-studied Hsp70-dependent targeting pathways and outline the observations and outstanding questions in less well-studied systems.

System-wide analyses reveal essential roles of N-terminal protein modification in bacterial membrane integrity.

The removal of the N-terminal formyl group on nascent proteins by peptide deformylase (PDF) is the most prevalent protein modification in bacteria. PDF is a critical target of antibiotic development; however, its role in bacterial physiology remains a long-standing question. This work used the time-resolved analyses of the Escherichia coli translatome and proteome to investigate the consequences of PDF inhibition. Loss of PDF activity rapidly induces cellular stress responses, especially those associated with protein misfolding and membrane defects, followed by a global down-regulation of metabolic pathways. Rapid membrane hyperpolarization and impaired membrane integrity were observed shortly after PDF inhibition, suggesting that the plasma membrane disruption is the most immediate and primary consequence of formyl group retention on nascent proteins. This work resolves the physiological function of a ubiquitous protein modification and uncovers its crucial role in maintaining the structure and function of the bacterial membrane.

Ribosome profiling reveals multiple roles of SecA in cotranslational protein export.

SecA, an ATPase known to posttranslationally translocate secretory proteins across the bacterial plasma membrane, also binds ribosomes, but the role of SecA's ribosome interaction has been unclear. Here, we used a combination of ribosome profiling methods to investigate the cotranslational actions of SecA. Our data reveal the widespread accumulation of large periplasmic loops of inner membrane proteins in the cytoplasm during their cotranslational translocation, which are specifically recognized and resolved by SecA in coordination with the proton motive force (PMF). Furthermore, SecA associates with 25% of secretory proteins with highly hydrophobic signal sequences at an early stage of translation and mediates their cotranslational transport. In contrast, the chaperone trigger factor (TF) delays SecA engagement on secretory proteins with weakly hydrophobic signal sequences, thus enforcing a posttranslational mode of their translocation. Our results elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria.

Ribosome-nascent Chain Interaction Regulates N-terminal Protein Modification.

Numerous proteins initiate their folding, localization, and modifications early during translation, and emerging data show that the ribosome actively participates in diverse protein biogenesis pathways. Here we show that the ribosome imposes an additional layer of substrate selection during N-terminal methionine excision (NME), an essential protein modification in bacteria. Biochemical analyses show that cotranslational NME is exquisitely sensitive to a hydrophobic signal sequence or transmembrane domain near the N terminus of the nascent polypeptide. The ability of the nascent chain to access the active site of NME enzymes dictates NME efficiency, which is inhibited by confinement of the nascent chain on the ribosome surface and exacerbated by signal recognition particle. In vivo measurements corroborate the inhibition of NME by an N-terminal hydrophobic sequence, suggesting the retention of formylmethionine on a substantial fraction of the secretory and membrane proteome. Our work demonstrates how molecular features of a protein regulate its cotranslational modification and highlights the active participation of the ribosome in protein biogenesis pathways via interactions of the ribosome surface with the nascent protein.

Mechanism of signal sequence handover from NAC to SRP on ribosomes during ER-protein targeting.

The nascent polypeptide-associated complex (NAC) interacts with newly synthesized proteins at the ribosomal tunnel exit and competes with the signal recognition particle (SRP) to prevent mistargeting of cytosolic and mitochondrial polypeptides to the endoplasmic reticulum (ER). How NAC antagonizes SRP and how this is overcome by ER targeting signals are unknown. Here, we found that NAC uses two domains with opposing effects to control SRP access. The core globular domain prevented SRP from binding to signal-less ribosomes, whereas a flexibly attached domain transiently captured SRP to permit scanning of nascent chains. The emergence of an ER-targeting signal destabilized NAC's globular domain and facilitated SRP access to the nascent chain. These findings elucidate how NAC hands over the signal sequence to SRP and imparts specificity of protein localization.

Subunit cooperation in the Get1/2 receptor promotes tail-anchored membrane protein insertion.

The guided entry of tail-anchored protein (GET) pathway, in which the Get3 ATPase delivers an essential class of tail-anchored membrane proteins (TAs) to the Get1/2 receptor at the endoplasmic reticulum, provides a conserved mechanism for TA biogenesis in eukaryotic cells. The membrane-associated events of this pathway remain poorly understood. Here we show that complex assembly between the cytosolic domains (CDs) of Get1 and Get2 strongly enhances the affinity of the individual subunits for Get3•TA, thus enabling efficient capture of the targeting complex. In addition to the known role of Get1CD in remodeling Get3 conformation, two molecular recognition features (MoRFs) in Get2CD induce Get3 opening, and both subunits are required for optimal TA release from Get3. Mutation of the MoRFs attenuates TA insertion into the ER in vivo. Our results demonstrate extensive cooperation between the Get1/2 receptor subunits in the capture and remodeling of the targeting complex, and emphasize the role of MoRFs in receptor function during membrane protein biogenesis.

Chloroplast SRP43 autonomously protects chlorophyll biosynthesis proteins against heat shock.

The assembly of light-harvesting chlorophyll-binding proteins (LHCPs) is coordinated with chlorophyll biosynthesis during chloroplast development. The ATP-independent chaperone known as chloroplast signal recognition particle 43 (cpSRP43) mediates post-translational LHCP targeting to the thylakoid membrane and also participates in tetrapyrrole biosynthesis (TBS). How these distinct actions of cpSRP43 are controlled has remained unclear. Here, we demonstrate that cpSRP43 effectively protects several TBS proteins from heat-induced aggregation and enhances their stability during leaf greening and heat shock. While the substrate-binding domain of cpSRP43 is sufficient for chaperoning LHCPs, the stabilization of TBS clients requires the chromodomain 2 of the protein. Strikingly, cpSRP54-which activates cpSRP43's LHCP-targeted function-inhibits the chaperone activity of cpSRP43 towards TBS proteins. High temperature weakens the interaction of cpSRP54 with cpSRP43, thus freeing cpSRP43 to interact with and protect the integrity of TBS proteins. Our data indicate that the temperature sensitivity of the cpSRP43-cpSRP54 complex enables cpSRP43 to serve as an autonomous chaperone for the thermoprotection of TBS proteins.

Molecular mechanism of cargo recognition and handover by the mammalian signal recognition particle.

Co-translational protein targeting to membranes by the signal recognition particle (SRP) is a universally conserved pathway from bacteria to humans. In mammals, SRP and its receptor (SR) have many additional RNA features and protein components compared to the bacterial system, which were recently shown to play regulatory roles. Due to its complexity, the mammalian SRP targeting process is mechanistically not well understood. In particular, it is not clear how SRP recognizes translating ribosomes with exposed signal sequences and how the GTPase activity of SRP and SR is regulated. Here, we present electron cryo-microscopy structures of SRP and SRP·SR in complex with the translating ribosome. The structures reveal the specific molecular interactions between SRP and the emerging signal sequence and the elements that regulate GTPase activity of SRP·SR. Our results suggest the molecular mechanism of how eukaryote-specific elements regulate the early and late stages of SRP-dependent protein targeting.

Receptor compaction and GTPase rearrangement drive SRP-mediated cotranslational protein translocation into the ER.

The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)-driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases.

J-domain proteins promote client relay from Hsp70 during tail-anchored membrane protein targeting.

J-domain proteins (JDPs) play essential roles in Hsp70 function by assisting Hsp70 in client trapping and regulating the Hsp70 ATPase cycle. Here, we report that JDPs can further enhance the targeting competence of Hsp70-bound client proteins during tail-anchored protein (TA) biogenesis. In the guided-entry-of-tail-anchored protein pathway in yeast, nascent TAs are captured by cytosolic Hsp70 and sequentially relayed to downstream chaperones, Sgt2 and Get3, for delivery to the ER. We found that two JDPs, Ydj1 and Sis1, function in parallel to support TA targeting to the ER in vivo. Biochemical analyses showed that, while Ydj1 and Sis1 differ in their ability to assist Hsp70 in TA trapping, both JDPs enhance the transfer of Hsp70-bound TAs to Sgt2. The ability of the JDPs to regulate the ATPase cycle of Hsp70 is essential for enhancing the transfer competence of Hsp70-bound TAs in vitro and for supporting TA insertion in vivo. These results demonstrate a role of JDPs in regulating the conformation of Hsp70-bound clients during membrane protein biogenesis.

Fidelity of Cotranslational Protein Targeting to the Endoplasmic Reticulum.

Fidelity of protein targeting is essential for the proper biogenesis and functioning of organelles. Unlike replication, transcription and translation processes, in which multiple mechanisms to recognize and reject noncognate substrates are established in energetic and molecular detail, the mechanisms by which cells achieve a high fidelity in protein localization remain incompletely understood. Signal recognition particle (SRP), a conserved pathway to mediate the localization of membrane and secretory proteins to the appropriate cellular membrane, provides a paradigm to understand the molecular basis of protein localization in the cell. In this chapter, we review recent progress in deciphering the molecular mechanisms and substrate selection of the mammalian SRP pathway, with an emphasis on the key role of the cotranslational chaperone NAC in preventing protein mistargeting to the ER and in ensuring the organelle specificity of protein localization.

A ribosome-associated chaperone enables substrate triage in a cotranslational protein targeting complex.

Protein biogenesis is essential in all cells and initiates when a nascent polypeptide emerges from the ribosome exit tunnel, where multiple ribosome-associated protein biogenesis factors (RPBs) direct nascent proteins to distinct fates. How distinct RPBs spatiotemporally coordinate with one another to affect accurate protein biogenesis is an emerging question. Here, we address this question by studying the role of a cotranslational chaperone, nascent polypeptide-associated complex (NAC), in regulating substrate selection by signal recognition particle (SRP), a universally conserved protein targeting machine. We show that mammalian SRP and SRP receptors (SR) are insufficient to generate the biologically required specificity for protein targeting to the endoplasmic reticulum. NAC co-binds with and remodels the conformational landscape of SRP on the ribosome to regulate its interaction kinetics with SR, thereby reducing the nonspecific targeting of signalless ribosomes and pre-emptive targeting of ribosomes with short nascent chains. Mathematical modeling demonstrates that the NAC-induced regulations of SRP activity are essential for the fidelity of cotranslational protein targeting. Our work establishes a molecular model for how NAC acts as a triage factor to prevent protein mislocalization, and demonstrates how the macromolecular crowding of RPBs at the ribosome exit site enhances the fidelity of substrate selection into individual protein biogenesis pathways.

A Disorder-to-Order Transition Activates an ATP-Independent Membrane Protein Chaperone.

The 43 kDa subunit of the chloroplast signal recognition particle, cpSRP43, is an ATP-independent chaperone essential for the biogenesis of the light harvesting chlorophyll-binding proteins (LHCP), the most abundant membrane protein family on earth. cpSRP43 is activated by a stromal factor, cpSRP54, to more effectively capture and solubilize LHCPs. The molecular mechanism underlying this chaperone activation is unclear. Here, a combination of hydrogen-deuterium exchange, electron paramagnetic resonance, and NMR spectroscopy experiments reveal that a disorder-to-order transition of the ankyrin repeat motifs in the substrate binding domain of cpSRP43 drives its activation. An analogous coil-to-helix transition in the bridging helix, which connects the ankyrin repeat motifs to the cpSRP54 binding site in the second chromodomain, mediates long-range allosteric communication of cpSRP43 with its activating binding partner. Our results provide a molecular model to explain how the conformational dynamics of cpSRP43 enables regulation of its chaperone activity and suggest a general mechanism by which ATP-independent chaperones with cooperatively folding domains can be regulated.

Timing and specificity of cotranslational nascent protein modification in bacteria.

The nascent polypeptide exit site of the ribosome is a crowded environment where multiple ribosome-associated protein biogenesis factors (RPBs) compete for the nascent polypeptide to influence their localization, folding, or quality control. Here we address how N-terminal methionine excision (NME), a ubiquitous process crucial for the maturation of over 50% of the bacterial proteome, occurs in a timely and selective manner in this crowded environment. In bacteria, NME is mediated by 2 essential enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). We show that the reaction of MAP on ribosome-bound nascent chains approaches diffusion-limited rates, allowing immediate methionine excision of optimal substrates after deformylation. Specificity is achieved by kinetic competition of NME with translation elongation and by regulation from other RPBs, which selectively narrow the processing time window for suboptimal substrates. A mathematical model derived from the data accurately predicts cotranslational NME efficiency in the cytosol. Our results demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity, and provides a platform to study other cotranslational protein biogenesis pathways.

Guiding tail-anchored membrane proteins to the endoplasmic reticulum in a chaperone cascade.

Newly synthesized integral membrane proteins must traverse the aqueous cytosolic environment before arrival at their membrane destination and are prone to aggregation, misfolding, and mislocalization during this process. The biogenesis of integral membrane proteins therefore poses acute challenges to protein homeostasis within a cell and requires the action of effective molecular chaperones. Chaperones that mediate membrane protein targeting not only need to protect the nascent transmembrane domains from improper exposure in the cytosol, but also need to accurately select client proteins and actively guide their clients to the appropriate target membrane. The mechanisms by which cellular chaperones work together to coordinate this complex process are only beginning to be delineated. Here, we summarize recent advances in studies of the tail-anchored membrane protein targeting pathway, which revealed a network of chaperones, cochaperones, and targeting factors that together drive and regulate this essential process. This pathway is emerging as an excellent model system to decipher the mechanism by which molecular chaperones overcome the multiple challenges during post-translational membrane protein biogenesis and to gain insights into the functional organization of multicomponent chaperone networks.

The molecular mechanism of cotranslational membrane protein recognition and targeting by SecA.

Cotranslational protein targeting is a conserved process for membrane protein biogenesis. In Escherichia coli, the essential ATPase SecA was found to cotranslationally target a subset of nascent membrane proteins to the SecYEG translocase at the plasma membrane. The molecular mechanism of this pathway remains unclear. Here we use biochemical and cryoelectron microscopy analyses to show that the amino-terminal amphipathic helix of SecA and the ribosomal protein uL23 form a composite binding site for the transmembrane domain (TMD) on the nascent protein. This binding mode further enables recognition of charged residues flanking the nascent TMD and thus explains the specificity of SecA recognition. Finally, we show that membrane-embedded SecYEG promotes handover of the translating ribosome from SecA to the translocase via a concerted mechanism. Our work provides a molecular description of the SecA-mediated cotranslational targeting pathway and demonstrates an unprecedented role of the ribosome in shielding nascent TMDs.

A molecular recognition feature mediates ribosome-induced SRP-receptor assembly during protein targeting.

Molecular recognition features (MoRFs) provide interaction motifs in intrinsically disordered protein regions to mediate diverse cellular functions. Here we report that a MoRF element, located in the disordered linker domain of the mammalian signal recognition particle (SRP) receptor and conserved among eukaryotes, plays an essential role in sensing the ribosome during cotranslational protein targeting to the endoplasmic reticulum. Loss of the MoRF in the SRP receptor (SR) largely abolishes the ability of the ribosome to activate SRP-SR assembly and impairs cotranslational protein targeting. These results demonstrate a novel role for MoRF elements and provide a mechanism for the ribosome-induced activation of the mammalian SRP pathway. Kinetic analyses and comparison with the bacterial SRP further suggest that the SR MoRF functionally replaces the essential GNRA tetraloop in the bacterial SRP RNA, providing an example for the replacement of RNA function by proteins during the evolution of ancient ribonucleoprotein particles.