Structural basis of l-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.

In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.

The ribosome modulates folding inside the ribosomal exit tunnel.

Proteins commonly fold co-translationally at the ribosome, while the nascent chain emerges from the ribosomal exit tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not explain the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli.

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB's two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.

Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite.

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.

Force-Profile Analysis of the Cotranslational Folding of HemK and Filamin Domains: Comparison of Biochemical and Biophysical Folding Assays.

We have characterized the cotranslational folding of two small protein domains of different folds-the α-helical N-terminal domain of HemK and the ß-rich FLN5 filamin domain-by measuring the force that the folding protein exerts on the nascent chain when located in different parts of the ribosome exit tunnel (force-profile analysis, or FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: real-time FRET, photoinduced electron transfer, and NMR. We find that FPA identifies the same cotranslational folding transitions as do the other methods, and that these techniques therefore reflect the same basic process of cotranslational folding in similar ways.

The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding.

The E. coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Δloop ribosomes, while two ~ 100 residue proteins normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Δloop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

Folding pathway of an Ig domain is conserved on and off the ribosome.

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-ß Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.

Dynamic interaction of the sec translocon with the chaperone PpiD.

The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages of protein transport. During targeting, SecYEG interacts with SecA, the SRP receptor, or the ribosome. Protein transport into or across the membrane is then facilitated by the interaction of SecYEG with YidC and the SecDFYajC complex. During protein transport, SecYEG is likely to interact also with the protein quality control machinery, but details about this interaction are missing. By in vivo and in vitro site-directed cross-linking, we show here that the periplasmic chaperone PpiD is located in front of the lateral gate of SecY, through which transmembrane domains exit the SecY channel. The strongest contacts were found to helix 2b of SecY. Blue native PAGE analyses verify the presence of a SecYEG-PpiD complex in native Escherichia coli membranes. The PpiD-SecY interaction was not influenced by the addition of SecA and only weakly influenced by binding of nontranslating ribosomes to SecYEG. In contrast, PpiD lost contact to the lateral gate of SecY during membrane protein insertion. These data identify PpiD as an additional and transient subunit of the bacterial SecYEG translocon. The data furthermore demonstrate the highly modular and versatile composition of the Sec translocon, which is probably essential for its ability to transport a wide range of substrates across membranes in bacteria and eukaryotes.

YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.

Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.

The Sec translocon mediated protein transport in prokaryotes and eukaryotes.

Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically-synthesized proteins to extra-cytosolic compartments. The Sec translocon has a three-subunit core, termed Sec61 in Eukaryotes and SecYEG in Bacteria. It is located in the endoplasmic reticulum of Eukaryotes and in the cytoplasmic membrane of Bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins determine the mechanism of polypeptide movement across the channel. During SRP-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway is in Bacteria mainly dedicated for membrane proteins but in Eukaryotes also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by the motor protein SecA in Bacteria and a ratcheting mechanism by the lumenal chaperone BiP in Eukaryotes. Protein transport and biogenesis is also assisted by additional proteins at the lateral gate of SecY/Sec61α and in the lumen of the endoplasmic reticulum or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. In this review we summarize recent biochemical and structural information on the prokaryotic and eukaryotic Sec translocons and we describe the remarkably complex interaction network of the Sec complexes.

YidC occupies the lateral gate of the SecYEG translocon and is sequentially displaced by a nascent membrane protein.

Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements.

Protein translocation across the inner membrane of Gram-negative bacteria: the Sec and Tat dependent protein transport pathways.

Gram negative bacteria possess a large variety of protein transport systems, by which proteins that are synthesised in the cytosol are exported to destinations in the cell envelope or entirely secreted into the extracellular environment. The inner membrane (IM) contains three major transport systems for the translocation and insertion of signal sequence containing proteins: the Sec translocon, the YidC insertase, and the Tat system. The heterotrimeric SecYEG translocon forms a narrow channel in the membrane that serves a dual function; it allows the translocation of unfolded proteins across the pore and the integration of α-helical proteins into the IM. The YidC insertase is a multi-spanning membrane protein that cooperates with the SecYEG translocon during the integration of membrane proteins but also functions as an independent insertase. Depending upon the type of protein cargo that needs to be transported, the Signal Recognition Particle (SRP), the SRP receptor, SecA and chaperones are required to coordinate translation with transport and to target and energise the different transport systems. The Tat system consists of three membrane proteins (TatA, TatB and TatC) which in a still unknown manner accomplish the transmembrane passage of completely folded proteins and protein complexes.

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle.

Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.