Signal Sequence Recognition Research Articles

Molecular basis for pH sensing in the KDEL trafficking receptor

Trafficking receptors control protein localization through the recognition of specific signal sequences that specify unique cellular locations. Differences in luminal pH are important for the vectorial trafficking of cargo receptors. The KDEL receptor is responsible for maintaining the integrity of the ER by retrieving luminally localized folding chaperones in a pH-dependent mechanism. Structural studies have revealed the end states of KDEL receptor activation and the mechanism of selective cargo binding. However, precisely how the KDEL receptor responds to changes in luminal pH remains unclear. To explain the mechanism of pH sensing, we combine analysis of X-ray crystal structures of the KDEL receptor at neutral and acidic pH with advanced computational methods and cell-based assays. We show a critical role for ordered water molecules that allows us to infer a direct connection between protonation in different cellular compartments and the consequent changes in the affinity of the receptor for cargo.

The technology of adversarial attacks in signal recognition

The wide application of contour stellar images has helped researchers transform signal classification problems into image classification problems to solve signal recognition based on deep learning. However, deep neural networks (DNN) are quite vulnerable to adversarial examples, thus simply evaluating the adversarial attack performance on the signal sequence recognition model cannot meet the current security requirements. From the perspective of an attacker, this study converts individual signals into stellar contour images, and then generates adversarial examples to evaluate the adversarial attack impacts. The results show that whether the current input sample is a signal sequence or a converted image, the DNN is vulnerable to the threat of adversarial examples. In the selected methods, whether it is under different perturbations or signal-to-noise ratio (SNRs), the momentum iteration method has the best performance among them, and under the perturbation of 0.01, the attack performance is more than 10% higher than the fast gradient sign method. Also, to measure the invisibility of adversarial examples, the contour stellar images before and after the attack were compared to maintain a balance between the attack success rate and the attack concealment.

Targeted-Capture Sequencing Is a Useful Method for MRD Markers Screening in KMT2A (MLL) Rearranged Leukemia

Immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangement-based molecular minimum residual disease (MRD) monitoring has become one of the most powerful prognostic indicators for patients with acute lymphoblastic leukemia (ALL). Infant ALL has a poor prognosis and requires therapeutic development based on accurate MRD assessment. However, there are some difficulties associated with the highly sensitive detection of MRD based on Ig/TCR gene rearrangements in infant ALL cases, compared to other B-ALL cases, because of the immature Ig/TCR gene rearrangement patterns and their oligoclonality. Since KMT2A(MLL) gene rearrangements are identified in infant ALL at a high frequency, the use of KMT2A rearranged sequences is an alternative method for MRD detection in KMT2A rearranged ALL cases. However, identifying the genomic breakpoints in KMT2A rearrangements by conventional methods is laborious and difficult in routine practice. To detect molecular MRD markers in infant ALL cases, we designed capture probes covering the coding and recognition signal sequences of the V, D, and J genes of the Ig/TCR loci and common genomic breakpoints in KMT2A, AFF1 (AF4), MLLT3 (AF9), MLLT1 (ENL), MLLT10 (AF10), and ELL. 88 infant ALL cases from the JPLSG MLL-10 study, including 73 cases with KMT2A rearrangements, were analyzed by high-throughput target-capture sequencing on the Illumina platform. The sequenced data were analyzed with the Ig/TCR recombination analysis tool Vidjil and the Genomon pipeline for detection of KMT2A rearrangements. Although 40% of cases in this study were marker-negative in the conventional marker screening analysis, clonal Ig/TCR targets for MRD measurements were identified in more than 80% of the cases by target-capture sequencing. Ig / TCR rearrangements detected in infant ALL were unique and related to age at diagnosis and type of KMT2A rearragement. KMT2A-rearranged cases showed a significantly higher frequency of incomplete rearrangements of IGH and TCRD compared to KMT2A-negative infant ALL cases. The target-capture method also showed an increased sensitivity of Ig/TCR-based MRD measurements even in cases where MRD measurement was possible. Genomic breakpoints of KMT2A rearrangements were detected in 80% of KMT2A-rearranged cases, in which KMT2A was most frequently rearranged with AFF1, followed by MLLT1, MLLT3, and MLLT10. Other rare fusion partners of the KMT2A gene were also captured by probes for KMT2A sequences. The determined patient-specific genomic breakpoint sequences could be used for monitoring the MRD of KMT2A-rearranged cases. Thus, target-capture sequencing is an easy and useful method for screening MRD markers in infant leukemia cases. Disclosures No relevant conflicts of interest to declare.

The Ways of Tails: the GET Pathway and more.

Due to their topology tail-anchored (TA) proteins must target to the membrane independently of the co-translational route defined by the signal sequence recognition particle (SRP), its receptor and the translocon Sec61. More than a decade of work has extensively characterized a highly conserved pathway, the yeast GET or mammalian TRC40 pathway, which is capable of countering the biogenetic challenge posed by the C-terminal TA anchor. In this review we briefly summarize current models of this targeting route and focus on emerging aspects such as the intricate interplay with the proteostatic network of cells and with other targeting pathways. Importantly, we consider the lessons provided by the in vivo analysis of the pathway in different model organisms and by the consideration of its full client spectrum in more recent studies. This analysis of the state of the field highlights directions in which the current models may be experimentally probed and conceptually extended.

Capture Sequencing Is a Useful Method for Comprehensive Clonality Analysis Based on Ig/TCR Gene Rearrangements in Acute Lymphoblastic Leukemia

IntroductionImmunoglobulin (Ig)/ T-cell receptor (TCR) gene rearrangements are the most widely used clonal marker to detect residual leukemic cells in patients with Acute Lymphoblastic Leukemia (ALL). Ig/TCR gene rearrangements based molecular minimum residual disease (MRD) monitoring has become one of the most powerful prognostic indicators for patients with ALL. Although the standard method of real-time quantitative PCR (RQ-PCR) provides very good sensitivity in MRD measurement, the workflow is very complicated and time-consuming, requiring expert technique and much work for operation, which limits the number of patients to be examined for MRD monitoring and the number of testable markers per patient.Material and methodsTo reveal clonal architecture and detect appropriate MRD marker, we designed capture probes covering the coding and recognition signal sequences of V, D, J genes of the Ig/TCR loci. We performed high-throughput target-capture sequencing in 208 pediatric cases with BCP-ALL and 35 pediatric cases with T-cell ALL, including 20 relapsed cases and 14 MRD marker negative cases. Extracted DNA samples were enriched with about 420 capture probes (Agilent Technology) and sequenced by HiSeq2500 platform (Illumina) in order to obtain enough sequence coverage (> 500 mean depth). Sequenced data were analyzed with Ig/TCR recombination analysis tool Vidjil (Giraud et al, 2014) and V(D)J recombination clones were listed according to a number of detected read for each clone.ResultsTotal 2379 clonal Ig/TCR gene rearrangements (median 9 per patient, range 0-82) were detected by capture sequencing among 236 (97%) cases. A clonal IGH sequence with V(D)J recombination was identified in 91% of BCP-ALL cases, followed by TRG (68 %), IGK (67 %), TRA+D (66%), TRD (59 %), TRB (49%), and IGL (15 %), respectively. On the other hand, clonal TRG V(D)J recombination was detected in 74% of T-ALL cases, followed by TRB (69%), TRD (57%), IGH (26%), and TRA+D (6%), respectively. About half of BCP-ALL cases were identified two independent IGH rearrangements. These frequencies agree with previous reports obtained by PCR based experiments.In the cases in this study with well-characterized clonal Ig/TCR gene rearrangements by PCR and Sanger sequencing, our capture sequencing was able to detect all rearrangements used in MRD measurements. Although 8 BCP-ALL cases in this study were marker-negative in standard PCR-MRD diagnostics, clonal Ig/TCR gene rearrangements were identified for 5 out of 8 cases by capture sequencing. Some of the hidden clonal rearrangements showed specific and good quantitative amplification by RQ-PCR and can be used as sensitive PCR-MRD targets. On the other hand, all the MRD marker negative 6 T-ALL cases were not detected clonal Ig/TCR gene rearrangements.Finally, we compared the clonal architecture based on Ig/TCR gene rearrangements between diagnosis and relapse in relapsed B-ALL patients. Changes in the clonal architecture were associated with remission duration. In very early relapse cases, detected Ig/TCR rearrangements and their proportion at relapse are very similar to those at diagnosis. In early to late relapse cases, some major Ig/TCR gene rearrangements were lost at relapse and other minor rearrangements expanded at relapse. Most of the identified Ig/TCR gene rearrangements were different between at diagnosis and at relapse in a case relapsed after more than 10 years. Loss of rearrangements were commonly seen in TRA, TRB, and IgL, while most of the IgK and TRD rearrangements were steady during disease course.ConclusionIntroducing target capture sequencing enables to high throughput sample preparation and automated data analysis. Capture sequencing is a useful method for comprehensive detection of Ig/TCR gene rearrangements and contributes to better understanding clonal architecture and detecting appropriate MRD markers in ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Vacuolar Sorting Determinants: Isolation and Study.

As it serves an important function in the compartmentalization of a series of components, from secondary metabolites to proteins, the vacuole is a central organelle in plant cell biology and development. One of the most important mechanisms regulating not only vacuolar biogenesis but also its luminal content, is the vacuolar sorting of proteins. This sorting mechanism is based upon the recognition of specific signal sequences, vacuolar sorting determinants, by sorting receptors, which then act by redirecting vacuolar cargo away from the default secretory pathway, and into the vacuolar lumen. One of the most direct ways of understanding if a given peptide possesses vacuolar sorting determinant capability is to isolate it, fuse it with a marker and express it in a plant system such as Nicotiana tabacum, a pipeline which will be described in more detail in this chapter.

Conformational Changes That Coordinate the Activity of BamA and BamD Allowing β-Barrel Assembly.

Most integral outer membrane proteins (OMPs) of Gram-negative bacteria, such as Escherichia coli, assume a β-barrel structure. The β-barrel assembly machine (Bam), a five-member complex composed of β-barrel OMP BamA and four associated lipoproteins, BamB, BamC, BamD, and BamE, folds and inserts OMPs into the outer membrane. The two essential proteins BamA and BamD interact to stabilize two subcomplexes, BamAB and BamCDE, and genetic and structural evidence suggests that interactions between BamA and BamD occur via an electrostatic interaction between a conserved aspartate residue in a periplasmic domain of BamA and a conserved arginine in BamD. In this work, we characterize charge-change mutations at these key BamA and BamD residues and nearby charged residues in BamA with respect to OMP assembly and Bam complex stability. We show that Bam complex stability does not correlate with function, that BamA and BamD must adopt at least two active conformational states during OMP assembly, and that these charged residues are not required for function. Rather, these charged residues are important for coordinating the activities of BamA and BamD to allow efficient OMP assembly. We present a model of OMP assembly wherein recognition and binding of unfolded OMP substrate by BamA and BamD induce a signaling interaction between the two proteins, causing conformational changes necessary for the assembly reaction to proceed. By analogy to signal sequence recognition by SecYEG, we believe these BamA-BamD interactions ensure that both substrate and complex are competent for OMP assembly before the assembly reaction commences.IMPORTANCE Conformational changes in the proteins of the β-barrel assembly machine (Bam complex) are associated with the folding and assembly of outer membrane proteins (OMPs) in Gram-negative bacteria. We show that electrostatic interactions between the two essential proteins BamA and BamD coordinate conformational changes upon binding of unfolded substrate that allow the assembly reaction to proceed. Mutations affecting this interaction are lethal not because they destabilize the Bam complex but rather because they disrupt this coordination. Our model of BamA-BamD interactions regulating conformation in response to proper substrate interaction is reminiscent of conformational changes the secretory (Sec) machinery undergoes after signal sequence recognition that ensure protein quality control.

Signal peptidase I processed secretory signal sequences: Selection for and against specific amino acids at the second position of mature protein

Signal peptides direct proteins from the cytoplasm to the periplasm. These N-terminal peptides are cleaved upon entry to the periplasm by either signal peptidase I, or signal peptidase II for lipoproteins. Signal peptidase I is a serine protease that has either a serine-lysine or serine-histidine catalytic dyad present in the active site. The recognition site for signal peptide cleavage by signal peptidase I has been defined primarily by an Ala-X-Ala motif at the C-terminal end of the signal peptide, one amino acid away from the cleavage site. We used a verified set of signal peptidase I cleaved proteins from E. coli to look for novel conserved features, focusing on the N-terminus of the mature protein. We observed a striking bias for the presence of acidic residues at second position of the mature protein (P2′), and a complete absence of aromatic amino acids at the same position. Whole genome analysis of the predicted set of all E. coli and B. subtilis secreted proteins confirmed the same strong bias for acidic residues at P2′ of the mature protein, and against aromatic amino acids at the same position. When these studies were extended to archaeal genomes (M. voltae and S. tokodaii) and the yeast genome from S. cerevisiae, this bias was not observed. E. coli and B. subtilis primarily express a signal peptidase I contains a serine-lysine catalytic dyad, whilst those of archaeal and eukaryotic origin generally have a serine-histidine catalytic dyad. This difference may explain the differential bias for acidic residues and against aromatic residues at P2′. These observations suggest additional key residues that may favor or prevent signal sequence recognition or cleavage by signal peptidase I, and thereby facilitate more accurate in silico prediction of signal peptidase I cleavage sites.

Cotranslational signal-independent SRP preloading during membrane targeting.

Ribosome-associated factors must faithfully decode the limited information available in nascent polypeptides to direct them to their correct cellular fate1. It is unclear how the low complexity information exposed by the nascent chain suffices for accurate recognition by the many factors competing for the limited surface near the ribosomal exit site2,3. Questions remain even for the well-studied cotranslational targeting cycle to the endoplasmic reticulum (ER), involving recognition of linear hydrophobic Signal Sequences (SS) or Transmembrane Domains (TMD) by the Signal Recognition Particle (SRP)4,5. Intriguingly, SRP is in low abundance relative to the large number of ribosome nascent chain complexes (RNCs), yet it accurately selects those destined to the ER6. Despite their overlapping specificities, SRP and the cotranslational Hsp70 SSB display exquisite mutually exclusive selectivity in vivo for their cognate RNCs7,8. To understand cotranslational nascent chain recognition in vivo, we interrogated the cotranslational membrane targeting cycle using ribosome profiling (herein Ribo-seq)9 coupled with biochemical fractionation of ribosome populations. Unexpectedly, SRP preferentially binds secretory RNCs before targeting signals are translated. We show non-coding mRNA elements can promote this signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting.

Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.

SummaryThe Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide1. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates2. Also hepatitis C virus (HCV) remains a major medical problem with 160 million chronically infected patients and only expensive treatment on the market3. Despite distinct differences in pathogenesis and mode of transmission, the two viruses share common replication strategies4. A detailed understanding of the host functions that determine viral infection is lacking. Here, we utilized a pooled CRISPR genetic screening strategy5,6 to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified ER-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER associated degradation. Dengue virus replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as critical step requiring the OST complex. Moreover, we showed that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects7. In contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA binding proteins and enzymes involved in metabolism. We discovered an unexpected link between intracellular FAD levels and HCV replication. This study shows remarkable divergence in host dependency factors between DENV and HCV and illuminates novel host targets for antiviral therapy.

Abstract B073: Structural basis of the 12-23 rule in V(D)J recombination

Abstract The V, D and J segment recombination (V(D)J recombination) is the key event in the development and maturation of T and B lymphocytes, which generates a diverse repertoire of T and B cell clones for the further selection of useful specificities. V(D)J recombination is also the basis for antibody diversity. The initiation step of V(D)J recombination is carried out by a pair of lymphocyte specific enzymes, which are called recombination-activating gene 1 and 2 (RAG1 and RAG2). RAG1 and RAG2 associate with each other and with two recognition signal sequences (RSS, 12RSS and 23RSS) adjacent to the coding ends of the V, D and J segments to catalyze the double stranded breaks required for joining. The reaction goes through several catalytic steps that result in formation of the single recombination complex (SC), the paired complex (PC), the cleaved signal complex (CSC) as well as the signal end complex (SEC). In order to understand the mechanisms of how RAG complex functions on DNA, we solved the structures PC and CSC by cryo-electron microscopy near the atomic resolution. Compared with the Apo-RAG complex that constitutes an open conformation, the PC and CSC reveal a closed conformation with RAG1 in one protomer interacts with RAG2 in another protomer. Furthermore, the first nucleotide of the heptamer in the non-transfer strand is flipped out to avoid the steric hindrance of the 3'-OH to attack the phosphodiester bond in the transfer strand. Upon nucleophilic attack and hairpin formation, the nucleotide adjacent to the 5'-position of the attacked phosphate is also flipped out to accommodate the tensile force in the hairpin DNA. In addition, we found that the conformation changes in the nonamer-binding domain (NBD), which is distorted toward to the 12RSS, and the distorsion requires two turns for the nonamer DNA in the 23RSS to reach the NBD. Finally, we found that two HMGB1 molecules, rather than one, are required to bind and bend both 12RSS and 23RSS DNA to adjust the conformational change of NBD on each side. In summary, our structural models not only uncover the mechanisms of how RAG protein catalyzes the hairpin formation, but also provide the structural basis of the 12-23 rule in V(D)J recombination. Citation Format: Heng Ru, Melissa G. Chambers, Maofu Liao, Hao Wu. Structural basis of the 12-23 rule in V(D)J recombination. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B073.

Crystal Structures of SecYEG in Lipidic Cubic Phase Elucidate a Precise Resting and a Peptide-Bound State

The bacterial SecYEG translocon functions as a conserved protein-conducting channel. Conformational transitions of SecYEG allow protein translocation across the membrane without perturbation of membrane permeability. Here, we report the crystal structures of intact SecYEG at 2.7-Å resolution and of peptide-bound SecYEG at 3.6-Å resolution. The higher-resolution structure revealed that the cytoplasmic loop of SecG covers the hourglass-shaped channel, which was confirmed to also occur in the membrane by disulfide bond formation analysis and molecular dynamics simulation. The cytoplasmic loop may be involved in protein translocation. In addition, the previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation. These SecYEG structures therefore provide a number of structural insights into the Sec machinery for further study.

VH replacement in primary immunoglobulin repertoire diversification

The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.

The Glove-like Structure of the Conserved Membrane Protein TatC Provides Insight into Signal Sequence Recognition in Twin-Arginine Translocation

In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0Å and 6.8Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading tostructural changes that facilitate higher order assemblies.

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.

Interplay between signal sequence recognition and N‐terminal protein modification at the ribosome exit site

Proteins destined for secretion are targeted to the endoplasmic reticulum by N‐terminal signal sequences. In contrast, the majority of cytosolic proteins are modified at their extreme N‐termini by methionine‐aminopeptidase (MetAP) and/or N‐acetylation. Both of these processes typically occur co‐translationally soon after the nascent chain emerges from the ribosome exit site.Proteins targeted by the Signal Recognition Particle‐independent targeting pathway have a strong bias against N‐terminal processing and mutations that induce such processing inhibit translocation. Detailed examination of SRP‐dependent substrates reveals that mutations that induce N‐terminal processing have no effect on ER targeting. In the context of signal anchor proteins with a long N‐terminal cytoplasmic region, methionine processing and acetylation can occur, but has no effect on translocation. In contrast, signal anchor sequences with short N‐terminal regions are inefficiently processed at their N‐termini. This is rationalized by the finding that SRP and MetAP compete for a shared binding site on the ribosome. Furthermore, we find that additional ribosome‐associated factors regulate the differential recruitment of these two factors to secretory and cytosolic ribosome nascent chains. Hence a tightly regulated interplay exists to ensure the fidelity of the early events of nascent chain processing.This work is funded by the Biotechnology and Biological Sciences Research Council.

Methionine Scanning as an NMR Tool for Detecting and Analyzing Biomolecular Interaction Surfaces

Methyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments.

Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB

The twin-arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates the transmembrane transport of folded proteins, which harbour signal sequences with a conserved twin-arginine motif. Many Tat translocases comprise the three membrane proteins TatA, TatB and TatC. TatC was previously shown to be involved in recognizing twin-arginine signal peptides. Here we show that beyond recognition, TatC mediates the transmembrane insertion of a twin-arginine signal sequence, thereby translocating the signal sequence cleavage site across the bilayer. In the absence of TatB, this can lead to the removal of the signal sequence even from a translocation-incompetent substrate. Hence interaction of twin-arginine signal peptides with TatB counteracts their premature cleavage uncoupled from translocation. This capacity of TatB is not shared by the homologous TatA protein. Collectively our results suggest that TatC is an insertase for twin-arginine signal peptides and that translocation-proficient signal sequence recognition requires the concerted action of TatC and TatB.

The recognition of endocytic signal sequences by the AP2 complex

The recognition of endocytic signal sequences by the AP2 complex

Translocation channel gating kinetics balances protein translocation efficiency with signal sequence recognition fidelity

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence-binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles, which reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity.