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

2021 ◽  
Vol 7 (21) ◽  
pp. eabg0942
Author(s):  
Jae Ho Lee ◽  
Ahmad Jomaa ◽  
SangYoon Chung ◽  
Yu-Hsien Hwang Fu ◽  
Ruilin Qian ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Translocation ◽  
Molecular Model ◽  
Genetic Diseases ◽  
Signal Recognition Particle ◽  
Biological Regulation ◽  
Single Molecule Studies ◽  
Guanosine Triphosphatase ◽  
Gtpase Cycle

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.

scholarly journals Receptor compaction and GTPase movements drive cotranslational protein translocation

2020 ◽  
Author(s):  
Jae Ho Lee ◽  
SangYoon Chung ◽  
Yu-Hsien Hwang Fu ◽  
Ruilin Qian ◽  
Xuemeng Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Biological Regulation ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Cause Of Failure ◽  
Distal Site

AbstractSignal recognition particle (SRP) is a universally conserved targeting machine that couples the synthesis of ~30% of the proteome to their proper membrane localization1,2. In eukaryotic cells, SRP recognizes translating ribosomes bearing hydrophobic signal sequences and, through interaction with SRP receptor (SR), delivers them to the Sec61p translocase on the endoplasmic reticulum (ER) membrane1,2. How SRP ensures efficient and productive initiation of protein translocation at the ER is not well understood. Here, single molecule fluorescence spectroscopy demonstrates that cargo-loaded SRP induces a global compaction of SR, driving a >90 Å movement of the SRP•SR GTPase complex from the vicinity of the ribosome exit, where it initially assembles, to the distal site of SRP. These rearrangements bring translating ribosomes near the membrane, expose conserved Sec61p docking sites on the ribosome and weaken SRP’s interaction with the signal sequence on the nascent polypeptide, thus priming the translating ribosome for engaging the translocation machinery. Disruption of these rearrangements severely impairs cotranslational protein translocation and is the cause of failure in an SRP54 mutant linked to severe congenital neutropenia. Our results demonstrate that multiple largescale molecular motions in the SRP•SR complex are required to drive the transition from protein targeting to translocation; these post-targeting rearrangements provide potential new points for biological regulation as well as disease intervention.

scholarly journals Real-time observation of signal recognition particle binding to actively translating ribosomes

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Thomas R Noriega ◽  
Jin Chen ◽  
Peter Walter ◽  
Joseph D Puglisi
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Initial Step ◽  
Signal Recognition ◽  
Fluorescence Measurements ◽  
Nascent Chain ◽  
Time Observation

The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.

scholarly journals Translation- and SRP-independent mRNA targeting to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae

2013 ◽  
Vol 24 (19) ◽  
pp. 3069-3084 ◽  
Author(s):  
Judith Kraut-Cohen ◽  
Evgenia Afanasieva ◽  
Liora Haim-Vilmovsky ◽  
Boris Slobodin ◽  
Ido Yosef ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Single Molecule ◽  
Translational Control ◽  
Rna Binding ◽  
Protein Translocation ◽  
Rna Binding Proteins ◽  
Signal Recognition Particle ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mrna Targeting

mRNAs encoding secreted/membrane proteins (mSMPs) are believed to reach the endoplasmic reticulum (ER) in a translation-dependent manner to confer protein translocation. Evidence exists, however, for translation- and signal recognition particle (SRP)–independent mRNA localization to the ER, suggesting that there are alternate paths for RNA delivery. We localized endogenously expressed mSMPs in yeast using an aptamer-based RNA-tagging procedure and fluorescence microscopy. Unlike mRNAs encoding polarity and secretion factors that colocalize with cortical ER at the bud tip, mSMPs and mRNAs encoding soluble, nonsecreted, nonpolarized proteins localized mainly to ER peripheral to the nucleus (nER). Synthetic nontranslatable uracil-rich mRNAs were also demonstrated to colocalize with nER in yeast. This mRNA–ER association was verified by subcellular fractionation and reverse transcription-PCR, single-molecule fluorescence in situ hybridization, and was not inhibited upon SRP inactivation. To better understand mSMP targeting, we examined aptamer-tagged USE1, which encodes a tail-anchored membrane protein, and SUC2, which encodes a soluble secreted enzyme. USE1 and SUC2 mRNA targeting was not abolished by the inhibition of translation or removal of elements involved in translational control. Overall we show that mSMP targeting to the ER is both translation- and SRP-independent, and regulated by cis elements contained within the message and trans-acting RNA-binding proteins (e.g., She2, Puf2).

scholarly journals Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation

2007 ◽  
Vol 178 (4) ◽  
pp. 611-620 ◽  
Author(s):  
Shu-ou Shan ◽  
Sowmya Chandrasekar ◽  
Peter Walter
Keyword(s):  
Conformational Changes ◽  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Gtp Hydrolysis ◽  
Translocation Efficiency ◽  
Cargo Proteins ◽  
Gtpase Activation ◽  
Guanosine Triphosphatase

During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP–SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP–SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.

scholarly journals Real time observation of chaperone-modulated talin mechanics under single molecule resolution

2021 ◽  
Author(s):  
Souradeep Banerjee ◽  
Deep Chaudhuri ◽  
Soham Chakraborty ◽  
Shubhasis Haldar
Keyword(s):  
Focal Adhesion ◽  
Single Molecule ◽  
Mechanical Stability ◽  
Energy Landscape ◽  
Magnetic Tweezers ◽  
Downstream Signaling ◽  
Cellular Processes ◽  
Single Molecule Studies ◽  
Time Observation

AbstractRecent single molecule studies have recognized talin as a mechanosensitive hub in focal adhesion, where its function is strongly regulated by mechanical force. For instance, at low force (less than 5pN), folded talin binds RIAM for integrin activation; whereas at high force (more than 5pN), it unfolds to activate vinculin binding for focal adhesion stabilization. Being a cytoplasmic large protein, talin must interact with various chaperones, however the role of chaperones on talin mechanics is unknown.To address this question, we investigated the force response of a mechanically stable talin domain, with a set of well-known holdase and foldase chaperones, using a single molecule magnetic tweezers technology. Our findings demonstrate a novel mechanical role of chaperones. We found holdase chaperones reduce the mechanical stability of the protein to ~6 pN, while the foldase chaperone increases it up to ~15 pN. The alteration in mechanical stability ascribes to the underlying molecular mechanism where the chaperones directly reshape the energy landscape of talin. For example, unfoldase chaperone (DnaK) decreases the unfolding barrier height from 26.8 to 21.69 kBT and increases the refolding barrier from 3.49 to 11.31 kBT. In contrast, foldase chaperone (DsbA) increases the unfolding barrier to 33.46 kBT and decreases the refolding barrier to 0.44 kBT. The quantitative mapping of the chaperone-induced free energy landscape of talin directly shows that chaperones could perturb the focal adhesion dynamics, which in turn can influence downstream signaling cascades in diverse cellular processes.

scholarly journals Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum

2003 ◽  
Vol 162 (4) ◽  
pp. 575-585 ◽  
Author(s):  
Elisabet C. Mandon ◽  
Ying Jiang ◽  
Reid Gilmore
Keyword(s):  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Chain Complex ◽  
Signal Recognition ◽  
Binding Assays ◽  
Ribosomal Subunits ◽  
Nascent Chain ◽  
Gtpase Cycle ◽  
Necessary And Sufficient

We have analyzed the interactions between the signal recognition particle (SRP), the SRP receptor (SR), and the ribosome using GTPase assays, biosensor experiments, and ribosome binding assays. Possible mechanisms that could contribute to an enhanced affinity between the SR and the SRP–ribosome nascent chain complex to promote protein translocation under physiological ionic strength conditions have been explored. Ribosomes or 60S large ribosomal subunits activate the GTPase cycle of SRP54 and SRα by providing a platform for assembly of the SRP–SR complex. Biosensor experiments revealed high-affinity, saturable binding of ribosomes or large ribosomal subunits to the SR. Remarkably, the SR has a 100-fold higher affinity for the ribosome than for SRP. Proteoliposomes that contain the SR bind nontranslating ribosomes with an affinity comparable to that shown by the Sec61 complex. An NH2-terminal 319-residue segment of SRα is necessary and sufficient for binding of SR to the ribosome. We propose that the ribosome–SR interaction accelerates targeting of the ribosome nascent chain complex to the RER, while the SRP–SR interaction is crucial for maintaining the fidelity of the targeting reaction.

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

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hao-Hsuan Hsieh ◽  
Jae Ho Lee ◽  
Sowmya Chandrasekar ◽  
Shu-ou Shan
Keyword(s):  
Protein Targeting ◽  
Molecular Model ◽  
Signal Recognition Particle ◽  
Substrate Selection ◽  
Nascent Polypeptide ◽  
Protein Biogenesis ◽  
Interaction Kinetics ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractProtein 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.

scholarly journals Interaction of theory and experiment: examples from single molecule studies of nanoparticles

2010 ◽  
Vol 368 (1914) ◽  
pp. 1109-1124 ◽  
Author(s):  
Rudolph A. Marcus
Keyword(s):  
Single Molecule ◽  
Large Body ◽  
Surface States ◽  
Biological Species ◽  
Theoretical Chemistry ◽  
Past Half Century ◽  
Single Molecule Studies ◽  
Computer Calculations ◽  
Complementary Nature

This article is in part the author's perspective on the revolution that has occurred in theoretical chemistry during the past half-century. In this period much of theoretical chemistry has moved from its initial emphasis on analytic treatments, resulting in equations for physical chemical and chemical phenomena, to the detailed computation of many different systems and processes. In the best sense the old and the new are complementary and their coexistence can benefit both. Experiment too has seen major developments. One of the newer types of experiment is that of single molecule studies. They range from those on small inorganic and organic nanoparticles to large biological species. We illustrate some of the issues that arise, using the topic of ‘quantum dots’ (QDs), and choosing a particular inorganic nanoparticle, CdSe, the most studied of these systems. Its study reflects the problems that arise in experiment and in theories in this field. The complementary nature of the conventional ensemble experiments and the new single molecule experiments is described and is illustrated by trajectories for the two types of experiments. The research in the QD field is both experimentally and theoretically a currently ongoing process, for which the answers are not fully known in spite of the large body of research. The detailed role of surface states is part of the problem. The field continues to yield new and unexpected results. In a sense this part of the article is an interim report that illustrates one analytic approach to the topic and where computer calculations and simulations can be expected to provide added insight.

scholarly journals Single-Molecule Studies on the Protein Translocon

2019 ◽  
Vol 48 (1) ◽  
pp. 185-207 ◽  
Author(s):  
Anne-Bart Seinen ◽  
Arnold J.M. Driessen
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Protein Translocation ◽  
Molecular Motor ◽  
Membrane Pore ◽  
Protein Insertion ◽  
System A ◽  
Biochemical Assays ◽  
Membrane Protein Insertion ◽  
Single Molecule Studies

Single-molecule studies provide unprecedented details about processes that are difficult to grasp by bulk biochemical assays that yield ensemble-averaged results. One of these processes is the translocation and insertion of proteins across and into the bacterial cytoplasmic membrane. This process is facilitated by the universally conserved secretion (Sec) system, a multi-subunit membrane protein complex that consists of dissociable cytoplasmic targeting components, a molecular motor, a protein-conducting membrane pore, and accessory membrane proteins. Here, we review recent insights into the mechanisms of protein translocation and membrane protein insertion from single-molecule studies.

Single-Molecule Studies of Bacterial Protein Translocation

Biochemistry ◽  
2013 ◽  
Vol 52 (39) ◽  
pp. 6740-6754 ◽  
Author(s):  
Alexej Kedrov ◽  
Ilja Kusters ◽  
Arnold J. M. Driessen
Keyword(s):  
Single Molecule ◽  
Protein Translocation ◽  
Bacterial Protein ◽  
Single Molecule Studies
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