scholarly journals Protein Targeting to the Bacterial Cytoplasmic Membrane

1999 ◽  
Vol 63 (1) ◽  
pp. 161-173 ◽  
Author(s):  
Peter Fekkes ◽  
Arnold J. M. Driessen
Keyword(s):  
Protein Targeting ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Specific Interaction ◽  
Signal Recognition Particle ◽  
High Fidelity ◽  
Signal Recognition ◽  
Nascent Chain ◽  
Major Route ◽  
Mature Domain

SUMMARY Proteins that perform their activity within the cytoplasmic membrane or outside this cell boundary must be targeted to the translocation site prior to their insertion and/or translocation. In bacteria, several targeting routes are known; the SecB- and the signal recognition particle-dependent pathways are the best characterized. Recently, evidence for the existence of a third major route, the twin-Arg pathway, was gathered. Proteins that use either one of these three different pathways possess special features that enable their specific interaction with the components of the targeting routes. Such targeting information is often contained in an N-terminal extension, the signal sequence, but can also be found within the mature domain of the targeted protein. Once the nascent chain starts to emerge from the ribosome, competition for the protein between different targeting factors begins. After recognition and binding, the targeting factor delivers the protein to the translocation sites at the cytoplasmic membrane. Only by means of a specific interaction between the targeting component and its receptor is the cargo released for further processing and translocation. This mechanism ensures the high-fidelity targeting of premembrane and membrane proteins to the translocation site.

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

2012 ◽  
Vol 23 (16) ◽  
pp. 3027-3040 ◽  
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.

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

2015 ◽  
Vol 112 (13) ◽  
pp. 3943-3948 ◽  
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.

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

2015 ◽  
Vol 211 (1) ◽  
pp. 91-104 ◽  
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.

scholarly journals Structure, dynamics and interactions of large SRP variants

2019 ◽  
Vol 401 (1) ◽  
pp. 63-80 ◽  
Author(s):  
Klemens Wild ◽  
Matthias M.M. Becker ◽  
Georg Kempf ◽  
Irmgard Sinning
Keyword(s):  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Domain Architecture ◽  
Particle Dynamics ◽  
Signal Recognition ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Target Membrane ◽  
Signal Anchor ◽  
Srp Rna

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.

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 The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane.

1995 ◽  
Vol 128 (3) ◽  
pp. 273-282 ◽  
Author(s):  
J D Miller ◽  
S Tajima ◽  
L Lauffer ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Targeting ◽  
Signal Sequence ◽  
Transmembrane Protein ◽  
Signal Recognition Particle ◽  
Endoplasmic Reticulum Membrane ◽  
Protein Chain ◽  
Signal Recognition ◽  
Signal Recognition Particle Receptor ◽  
Er Membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30-kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.

scholarly journals Signal Recognition Particle-dependent Targeting of Ribosomes to the Rough Endoplasmic Reticulum in the Absence and Presence of the Nascent Polypeptide-associated Complex

1998 ◽  
Vol 9 (1) ◽  
pp. 117-130 ◽  
Author(s):  
David Raden ◽  
Reid Gilmore
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Chain Complex ◽  
Dual Function ◽  
Signal Recognition ◽  
Nascent Polypeptide ◽  
Specific Attachment ◽  
Nascent Chain

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.

scholarly journals Signal sequence–independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle

2011 ◽  
Vol 22 (13) ◽  
pp. 2309-2323 ◽  
Author(s):  
David Braig ◽  
Miryana Mircheva ◽  
Ilie Sachelaru ◽  
Eli O. van der Sluis ◽  
Lukas Sturm ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Targeting ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Substrate Recognition ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Dependent Protein ◽  
Membrane Bound ◽  
Influence Of Substrate

Protein targeting by the signal recognition particle (SRP) and the bacterial SRP receptor FtsY requires a series of closely coordinated steps that monitor the presence of a substrate, the membrane, and a vacant translocon. Although the influence of substrate binding on FtsY-SRP complex formation is well documented, the contribution of the membrane is largely unknown. In the current study, we found that negatively charged phospholipids stimulate FtsY-SRP complex formation. Phospholipids act on a conserved positively charged amphipathic helix in FtsY and induce a conformational change that strongly enhances the FtsY-lipid interaction. This membrane-bound, signal sequence–independent FtsY-SRP complex is able to recruit RNCs to the membrane and to transfer them to the Sec translocon. Significantly, the same results were also observed with an artificial FtsY-SRP fusion protein, which was tethered to the membrane via a transmembrane domain. This indicates that substrate recognition by a soluble SRP is not essential for cotranslational targeting in Escherichia coli. Our findings reveal a remarkable flexibility of SRP-dependent protein targeting, as they indicate that substrate recognition can occur either in the cytosol via ribosome-bound SRP or at the membrane via a preassembled FtsY-SRP complex.

scholarly journals Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites

2006 ◽  
Vol 174 (5) ◽  
pp. 715-724 ◽  
Author(s):  
Sandra Angelini ◽  
Diana Boy ◽  
Emile Schiltz ◽  
Hans-Georg Koch
Keyword(s):  
Binding Sites ◽  
Protein Targeting ◽  
Cytoplasmic Membrane ◽  
Signal Recognition Particle ◽  
Membrane Association ◽  
Signal Recognition ◽  
Gtpase Activity ◽  
Signal Recognition Particle Receptor ◽  
Membrane Anchoring ◽  
Lipid Anchor

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRα subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRβ subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY–SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.

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