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 Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

1998 ◽  
Vol 9 (1) ◽  
pp. 103-115 ◽  
Author(s):  
Andrea Neuhof ◽  
Melissa M. Rolls ◽  
Berit Jungnickel ◽  
Kai-Uwe Kalies ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Membrane Binding ◽  
Membrane Targeting ◽  
Signal Recognition ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Cytosolic Factors ◽  
Er Membrane

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

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 The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

1992 ◽  
Vol 117 (1) ◽  
pp. 15-25 ◽  
Author(s):  
G Migliaccio ◽  
CV Nicchitta ◽  
G Blobel
Keyword(s):  
Membrane Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Chain Complex ◽  
Secretory Protein ◽  
Signal Recognition ◽  
Ribosome Binding ◽  
Signal Recognition Particle Receptor ◽  
Nascent Chain

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.

scholarly journals Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.

1987 ◽  
Vol 104 (2) ◽  
pp. 201-208 ◽  
Author(s):  
M Wiedmann ◽  
T V Kurzchalia ◽  
H Bielka ◽  
T A Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Cross Linking ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Group 4 ◽  
Lysine Residues ◽  
Nascent Chain

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.

scholarly journals Integration of membrane proteins into the endoplasmic reticulum requires GTP.

1988 ◽  
Vol 107 (1) ◽  
pp. 69-77 ◽  
Author(s):  
C Wilson ◽  
T Connolly ◽  
T Morrison ◽  
R Gilmore
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
G Protein ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Membrane Insertion ◽  
Signal Recognition ◽  
Free System ◽  
Amino Terminal ◽  
Nascent Chain

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.

scholarly journals Nascent secretory chain binding and translocation are distinct processes: differentiation by chemical alkylation.

1989 ◽  
Vol 108 (3) ◽  
pp. 789-795 ◽  
Author(s):  
C V Nicchitta ◽  
G Blobel
Keyword(s):  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Chain Complex ◽  
Membrane System ◽  
Signal Recognition ◽  
Signal Recognition Particle Receptor ◽  
Nascent Chain ◽  
Microsomal Membranes ◽  
Dependent Signal ◽  
A Site

We have investigated the effects of chemical alkylation of microsomal membranes on nascent chain binding and translocation. Assays were conducted using either full-length or truncated preprolactin transcripts in combination with a reconstituted membrane system consisting of proteolyzed rough microsomes and the cytoplasmic domain of the signal recognition particle receptor. Treatment of rough microsomes with N-ethylmaleimide was observed to inhibit preprolactin processing at a site other than the signal recognition particle or the signal recognition particle receptor. As formation of a translocation competent junction between the ribosome/nascent chain complex and the membrane has recently been demonstrated to require GTP (Connolly, T., and R. Gilmore. J. Cell Biol. 1986. 103:2253-2261), the effects of membrane alkylation on this parameter were assessed. N-ethylmaleimide treatment did not inhibit nascent chain targeting or GTP-dependent signal sequence insertion. Translocation of the targeted and inserted nascent chain was, however, blocked. These data indicate (a) that the process of nascent chain translocation is distinct from targeting and signal sequence insertion, and (b) translocation of the peptide chain across the membrane is mediated by an N-ethylmaleimide-sensitive membrane protein component(s). To further substantiate the observation that nascent chain targeting and signal sequence insertion can be distinguished from translocation, the temperature dependencies of the two phenomena were compared. Signal sequence insertion occurred at low temperatures (4 degrees C) and was maximal between 10 and 15 degrees C. Translocation was only observed at higher temperatures and was maximal between 25 and 30 degrees C.

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 Early encounters of a nascent membrane protein

2005 ◽  
Vol 170 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Edith N.G. Houben ◽  
Raz Zarivach ◽  
Bauke Oudega ◽  
Joen Luirink
Keyword(s):  
Membrane Protein ◽  
Ribosomal Proteins ◽  
Transmembrane Domain ◽  
Signal Recognition Particle ◽  
Chain Complex ◽  
Signal Recognition ◽  
Inner Membrane ◽  
Ribosomal Tunnel ◽  
Nascent Chain ◽  
Inner Membrane Protein

An unbiased photo–cross-linking approach was used to probe the “molecular path” of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome–nascent chain complex to the Sec–YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

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.

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