Structure, dynamics and interactions of large SRP variants

Abstract Co-translational protein targeting to membranes relies on the signal recognition particle (SRP) system consisting of a cytosolic ribonucleoprotein complex and its membrane-associated receptor. SRP recognizes N-terminal cleavable signals or signal anchor sequences, retards translation, and delivers ribosome-nascent chain complexes (RNCs) to vacant translocation channels in the target membrane. While our mechanistic understanding is well advanced for the small bacterial systems it lags behind for the large bacterial, archaeal and eukaryotic SRP variants including an Alu and an S domain. Here we describe recent advances on structural and functional insights in domain architecture, particle dynamics and interplay with RNCs and translocon and GTP-dependent regulation of co-translational protein targeting stimulated by SRP RNA.

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Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1424453112 ◽

2015 ◽

Vol 112 (13) ◽

pp. 3943-3948 ◽

Cited By ~ 19

Author(s):

Ottilie von Loeffelholz ◽

Qiyang Jiang ◽

Aileen Ariosa ◽

Manikandan Karuppasamy ◽

Karine Huard ◽

...

Keyword(s):

Conformational Changes ◽

Protein Targeting ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Binding Pocket ◽

Van Der Waals Interactions ◽

Closed State ◽

Chain Complexes ◽

Nascent Chain ◽

Srp Rna

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome–nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP–RNC complex results in GTP-independent binding of the SRP–FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP–FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP–FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.

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NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e12-02-0112 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3027-3040 ◽

Cited By ~ 41

Author(s):

Ying Zhang ◽

Uta Berndt ◽

Hanna Gölz ◽

Arlette Tais ◽

Stefan Oellerer ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Targeting ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Signal Sequences ◽

Chain Complexes ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Combined Data

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.

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The many faces of SRP RNA

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314081868 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1814-C1814

Author(s):

Klemens Wild ◽

Georg Kempf ◽

Jan Grotwinkel ◽

Irmgard Sinning

Keyword(s):

Ternary Complex ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Chain Complexes ◽

Composition And Structure ◽

Nascent Chain ◽

Domains Of Life ◽

The Many ◽

Srp Rna ◽

High Diversity

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.

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SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Science ◽

10.1126/science.1249094 ◽

2014 ◽

Vol 344 (6179) ◽

pp. 101-104 ◽

Cited By ~ 26

Author(s):

Jan Timo Grotwinkel ◽

Klemens Wild ◽

Bernd Segnitz ◽

Irmgard Sinning

Keyword(s):

Ribosomal Rna ◽

Protein Targeting ◽

Rna Binding ◽

Protein Translocation ◽

Signal Recognition Particle ◽

Structural Basis ◽

Signal Recognition ◽

Rna Remodeling ◽

Srp Rna ◽

Arginine Rich Motif

The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

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Mechanisms of membrane protein biogenesis

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331408838x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1161-C1161

Author(s):

Irmgard Sinning

Keyword(s):

Membrane Proteins ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Protein Biogenesis ◽

Cargo Proteins ◽

Target Membrane ◽

Hydrophobic Nature ◽

Correct Target ◽

Srp Rna

More than 25% of the cellular proteome comprise membrane proteins that have to be inserted into the correct target membrane. Most membrane proteins are delivered to the membrane by the signal recognition particle (SRP) pathway which relies on the recognition of an N-terminal signal sequence. In contrast to this co-translational mechanism, which avoids problems due to the hydrophobic nature of the cargo proteins, tail-anchored (TA) membrane proteins utilize a post-translational mechanism for membrane insertion – the GET pathway (guided entry of tail-anchored membrane proteins). The SRP and GET pathways are both regulated by GTP and ATP binding proteins of the SIMIBI family. However, in the SRP pathway the SRP RNA plays a unique regulatory role. Recent insights into eukaryotic SRP will be discussed.

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

The Journal of Cell Biology ◽

10.1083/jcb.201502103 ◽

2015 ◽

Vol 211 (1) ◽

pp. 91-104 ◽

Cited By ~ 24

Author(s):

Patrick Kuhn ◽

Albena Draycheva ◽

Andreas Vogt ◽

Narcis-Adrian Petriman ◽

Lukas Sturm ◽

...

Keyword(s):

Protein Targeting ◽

Signal Recognition Particle ◽

Resonance Energy Transfer ◽

Chain Complex ◽

Resonance Energy ◽

Endoplasmic Reticulum Membrane ◽

Signal Recognition ◽

Ribosomal Tunnel ◽

Nascent Chain

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.

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A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e08-10-0989 ◽

2009 ◽

Vol 20 (17) ◽

pp. 3965-3973 ◽

Cited By ~ 15

Author(s):

Peera Jaru-Ampornpan ◽

Thang X. Nguyen ◽

Shu-ou Shan

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Protein Targeting ◽

Signal Recognition Particle ◽

Receptor Interaction ◽

Signal Recognition ◽

Gtpase Domain ◽

Distinct Mechanism ◽

Molecular Origin ◽

Srp Rna

Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5- to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication.

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Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

The Journal of Cell Biology ◽

10.1083/jcb.200302130 ◽

2003 ◽

Vol 161 (4) ◽

pp. 679-684 ◽

Cited By ~ 93

Author(s):

Ronald S. Ullers ◽

Edith N.G. Houben ◽

Amanda Raine ◽

Corinne M. ten Hagen-Jongman ◽

Måns Ehrenberg ◽

...

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Signal Recognition Particle ◽

Attachment Site ◽

Trigger Factor ◽

Signal Recognition ◽

Exit Site ◽

Nascent Chain ◽

Signal Anchor

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.

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A pair of non-optimal codons are necessary for the correct biosynthesis of the Aspergillus nidulans urea transporter, UreA

Royal Society Open Science ◽

10.1098/rsos.190773 ◽

2019 ◽

Vol 6 (11) ◽

pp. 190773 ◽

Cited By ~ 1

Author(s):

Manuel Sanguinetti ◽

Andrés Iriarte ◽

Sotiris Amillis ◽

Mónica Marín ◽

Héctor Musto ◽

...

Keyword(s):

Aspergillus Nidulans ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Urea Transporter ◽

Translation Rate ◽

Synonymous Codons ◽

Optimal Codons ◽

Chain Complexes ◽

Ribosomal Tunnel ◽

Nascent Chain

In both prokaryotic and eukaryotic genomes, synonymous codons are unevenly used. Such differential usage of optimal or non-optimal codons has been suggested to play a role in the control of translation initiation and elongation, as well as at the level of transcription and mRNA stability. In the case of membrane proteins, codon usage has been proposed to assist in the establishment of a pause necessary for the correct targeting of the nascent chains to the translocon. By using as a model UreA, the Aspergillus nidulans urea transporter, we revealed that a pair of non-optimal codons encoding amino acids situated at the boundary between the N -terminus and the first transmembrane segment are necessary for proper biogenesis of the protein at 37°C. These codons presumably regulate the translation rate in a previously undescribed fashion, possibly contributing to the correct interaction of ureA -translating ribosome-nascent chain complexes with the signal recognition particle and/or other factors, while the polypeptide has not yet emerged from the ribosomal tunnel. Our results suggest that the presence of the pair of non-optimal codons would not be functionally important in all cellular conditions. Whether this mechanism would affect other proteins remains to be determined.

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Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA

Molecular Biology of the Cell ◽

10.1091/mbc.e07-01-0037 ◽

2007 ◽

Vol 18 (7) ◽

pp. 2636-2645 ◽

Cited By ~ 27

Author(s):

Peera Jaru-Ampornpan ◽

Sowmya Chandrasekar ◽

Shu-ou Shan

Keyword(s):

Complex Formation ◽

Conformational Changes ◽

Protein Targeting ◽

Signal Recognition Particle ◽

The Other ◽

Signal Recognition ◽

Binding Partner ◽

Biochemical Analyses ◽

Kinetics Of ◽

Srp Rna

Cotranslational protein targeting to membranes is regulated by two GTPases in the signal recognition particle (SRP) and the SRP receptor; association between the two GTPases is slow and is accelerated 400-fold by the SRP RNA. Intriguingly, the otherwise universally conserved SRP RNA is missing in a novel chloroplast SRP pathway. We found that even in the absence of an SRP RNA, the chloroplast SRP and receptor GTPases can interact efficiently with one another; the kinetics of interaction between the chloroplast GTPases is 400-fold faster than their bacterial homologues, and matches the rate at which the bacterial SRP and receptor interact with the help of SRP RNA. Biochemical analyses further suggest that the chloroplast SRP receptor is pre-organized in a conformation that allows optimal interaction with its binding partner, so that conformational changes during complex formation are minimized. Our results highlight intriguing differences between the classical and chloroplast SRP and SRP receptor GTPases, and help explain how the chloroplast SRP pathway can mediate efficient targeting of proteins to the thylakoid membrane in the absence of the SRP RNA, which plays an indispensable role in all the other SRP pathways.

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