Functional ramifications of FRET-detected nascent chain folding far inside the membrane-bound ribosome

2004 ◽  
Vol 32 (5) ◽  
pp. 668-672 ◽  
Author(s):  
A.E. Johnson
Keyword(s):  
Protein Trafficking ◽  
Protein Biosynthesis ◽  
Ribosomal Subunit ◽  
Regulatory Control ◽  
Secretory Proteins ◽  
Induced Folding ◽  
Ribosomal Tunnel ◽  
Nascent Chain ◽  
Membrane Bound ◽  
Transmembrane Sequence

During protein biosynthesis, nascent protein chains are directed along a long narrow tunnel that spans the large ribosomal subunit. It has recently become clear that this structural feature has evolved to effect regulatory control over aspects of protein synthesis and protein trafficking. Since this control is nascent chain-specific, ribosomal components that form the tunnel must be involved in recognizing selected nascent proteins as they pass by. The present study focuses on one such situation in which nascent secretory proteins and membrane proteins are distinguished by the ribosome-induced folding of the latter's hydrophobic transmembrane sequence far inside the ribosomal tunnel and close to the peptidyltransferase centre.

scholarly journals RIBOSOME-MEMBRANE INTERACTION

1973 ◽  
Vol 56 (1) ◽  
pp. 206-229 ◽  
Author(s):  
M. R. Adelman ◽  
David D. Sabatini ◽  
Günter Blobel
Keyword(s):  
Ionic Strength ◽  
Room Temperature ◽  
Ribosomal Subunit ◽  
High Ionic Strength ◽  
Ribosomal Subunits ◽  
Insoluble Material ◽  
Polyuridylic Acid ◽  
Nascent Chain ◽  
Membrane Bound ◽  
Polypeptide Chains

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl2, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [3H]puromycin into hot acid-insoluble material and from the release of [3H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (∼15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.

scholarly journals Polytopic membrane protein folding at L17 in the ribosome tunnel initiates cyclical changes at the translocon

2011 ◽  
Vol 195 (1) ◽  
pp. 55-70 ◽  
Author(s):  
Pen-Jen Lin ◽  
Candice G. Jongsma ◽  
Martin R. Pool ◽  
Arthur E. Johnson
Keyword(s):  
Membrane Protein ◽  
Conformational Changes ◽  
Membrane Protein Folding ◽  
Multiple Components ◽  
Induced Folding ◽  
Ribosomal Tunnel ◽  
Nascent Chain ◽  
Polytopic Membrane Protein ◽  
Peptidyltransferase Center ◽  
Compact Conformation

Multi-spanning membrane protein loops are directed alternately into the cytosol or ER lumen during cotranslational integration. Nascent chain exposure is switched after a newly synthesized transmembrane segment (TMS) enters the ribosomal tunnel. FRET measurements revealed that each TMS is initially extended, but folds into a compact conformation after moving 6–7 residues from the peptidyltransferase center, irrespective of loop size. The ribosome-induced folding of each TMS coincided with its photocrosslinking to ribosomal protein L17 and an inversion of compartmental exposure. This correlation indicates that successive TMSs fold and bind at a specific ribosomal tunnel site that includes L17, thereby triggering structural rearrangements of multiple components in and on both sides of the ER membrane, most likely via TMS-dependent L17 and/or rRNA conformational changes transmitted to the surface. Thus, cyclical changes at the membrane during integration are initiated by TMS folding, even though nascent chain conformation and location vary dynamically in the ribosome tunnel. Nascent chains therefore control their own trafficking.

scholarly journals Molecular Mimicry of SecA and Signal Recognition Particle Binding to the Bacterial Ribosome

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Lara Knüpffer ◽  
Clara Fehrenbach ◽  
Kärt Denks ◽  
Veronika Erichsen ◽  
Narcis-Adrian Petriman ◽  
...  
Keyword(s):  
Protein Transport ◽  
Molecular Mimicry ◽  
Signal Recognition Particle ◽  
Transport Systems ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Bacterial Protein ◽  
Ribosomal Tunnel ◽  
N Terminus ◽  
Nascent Chain

ABSTRACT Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome. IMPORTANCE Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time.

scholarly journals Discrete nascent chain lengths are required for the insertion of presecretory proteins into microsomal membranes.

1993 ◽  
Vol 121 (6) ◽  
pp. 1211-1219 ◽  
Author(s):  
S L Wolin ◽  
P Walter
Keyword(s):  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Translation Arrest ◽  
Nascent Chain ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Chain Lengths ◽  
Edta Extraction ◽  
Er Membrane

Ribosomes synthesizing nascent secretory proteins are targeted to the membrane by the signal recognition particle (SRP), a small ribonucleoprotein that binds to the signal peptide as it emerges from the ribosome. SRP arrests further elongation, causing ribosomes to stack behind the arrested ribosome. Upon interaction of SRP with its receptor on the ER membrane, the translation arrest is released and the ribosome becomes bound to the ER membrane. We have examined the distribution of unattached and membrane-bound ribosomes during the translation of mRNAs encoding two secretory proteins, bovine preprolactin and rat preproinsulin I. We find that the enhancement of ribosome stacking that occurs when SRP arrests translation of these proteins is relaxed in the presence of microsomal membranes. We also demonstrate that two previously described populations of membrane-associated ribosomes, distinguished by their sensitivity to high salt or EDTA extraction, correspond to ribosomes that have synthesized differing lengths of the nascent polypeptide. This analysis has revealed that nascent chain insertion into the membrane begins at distinct points for different presecretory proteins.

scholarly journals Structural features within the nascent chain regulate alternative targeting of secretory proteins to mitochondria

2013 ◽  
Vol 32 (7) ◽  
pp. 1036-1051 ◽  
Author(s):  
Natalie V Pfeiffer ◽  
Daniela Dirndorfer ◽  
Sven Lang ◽  
Ulrike K Resenberger ◽  
Lisa M Restelli ◽  
...  
Keyword(s):  
Structural Features ◽  
Secretory Proteins ◽  
Nascent Chain

scholarly journals Negamycin Binds to the Wall of the Nascent Chain Exit Tunnel of the 50S Ribosomal Subunit

2007 ◽  
Vol 51 (12) ◽  
pp. 4462-4465 ◽  
Author(s):  
Susan J. Schroeder ◽  
Gregor Blaha ◽  
Peter B. Moore
Keyword(s):  
Protein Synthesis ◽  
Small Molecule ◽  
Ribosomal Subunit ◽  
Small Molecule Inhibitor ◽  
Single Site ◽  
Haloarcula Marismortui ◽  
Molecule Inhibitor ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Inhibitor Of Protein Synthesis

ABSTRACT Negamycin, a small-molecule inhibitor of protein synthesis, binds the Haloarcula marismortui 50S ribosomal subunit at a single site formed by highly conserved RNA nucleotides near the cytosolic end of the nascent chain exit tunnel. The mechanism of antibiotic action and the function of this unexplored tunnel region remain intriguingly elusive.

scholarly journals The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria

2018 ◽  
Vol 29 (20) ◽  
pp. 2386-2396 ◽  
Author(s):  
Braulio Vargas Möller-Hergt ◽  
Andreas Carlström ◽  
Katharina Stephan ◽  
Axel Imhof ◽  
Martin Ott
Keyword(s):  
Membrane Protein ◽  
Mitochondrial Gene ◽  
Ribosomal Subunit ◽  
Mitochondrial Translation ◽  
Protein Insertion ◽  
Mitochondrial Ribosomes ◽  
Protein Biogenesis ◽  
Membrane Bound ◽  
Highly Hydrophobic ◽  
Soluble C

Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for the production of highly hydrophobic subunits of the oxidative phosphorylation system. Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry–based approach, we discovered the previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria.

scholarly journals ORF Ι of Mycovirus SsNSRV-1 is Associated with Debilitating Symptoms of Sclerotinia sclerotiorum

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 456
Author(s):  
Zhixiao Gao ◽  
Junyan Wu ◽  
Daohong Jiang ◽  
Jiatao Xie ◽  
Jiasen Cheng ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Protein Biosynthesis ◽  
Secretory Proteins ◽  
Viral Mrna ◽  
Fungal Host ◽  
Reading Frame ◽  
Mutant Strains ◽  
Hypovirulent Strain ◽  
Viral Proliferation ◽  
Host Genes

We previously identified Sclerotinia sclerotiorum negative-stranded virus 1 (SsNSRV-1), the first (−) ssRNA mycovirus, associated with hypovirulence of its fungal host Sclerotinia sclerotiorum. In this study, functional analysis of Open Reading Frame Ι (ORF Ι) of SsNSRV-1 was performed. The integration and expression of ORF Ι led to defects in hyphal tips, vegetative growth, and virulence of the mutant strains of S. sclerotiorum. Further, differentially expressed genes (DEGs) responding to the expression of ORF Ι were identified by transcriptome analysis. In all, 686 DEGs consisted of 267 up-regulated genes and 419 down-regulated genes. DEGs reprogramed by ORF Ι were relevant to secretory proteins, pathogenicity, transcription, transmembrane transport, protein biosynthesis, modification, and metabolism. Alternative splicing was also detected in all mutant strains, but not in hypovirulent strain AH98, which was co-infected by SsNSRV-1 and Sclerotinia sclerotiorum hypovirus 1 (SsHV-1). Thus, the integrity of SsNSRV-1 genome may be necessary to protect viral mRNA from splicing and inactivation by the host. Taken together, the results suggested that protein ORF Ι could regulate the transcription, translation, and modification of host genes in order to facilitate viral proliferation and reduce the virulence of the host. Therefore, ORF Ι may be a potential gene used for the prevention of S. sclerotiorum.

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.

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