The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N‐terminal signal ...peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro‐alpha‐factor. The unbinding parameters including off‐rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko–Hummer–Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off‐rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.
Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA ...transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved “core” subunits that respectively elaborate “minimized” systems in Gram‐positive or ‐negative bacteria. However, many “expanded” T4SSs are built from “core” subunits plus numerous others that are system‐specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure–function relationships for model Gram‐negative bacterial T4SSs, including “minimized” systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and “expanded” systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid‐encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long‐standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.
Type IV secretion systems (T4SSs) are a functionally diverse superfamily of translocation nanomachines deployed by most species of bacteria. Collectively, T4SSs mediate the transfer of mobile genetic elements or protein toxins to other bacteria, or effector proteins or other macromolecules to eukaryotic cells to aid in infection. Recent structural and fluorescence approaches are generating exciting new insights into how T4SSs assemble in the cell envelope and recruit and translocate substrates to other cells.
Conservation translocations are becoming common conservation practice, so there is an increasing need to understand the drivers of plant translocation performance through reviews of cases at global ...and regional levels. The establishment of the Italian Database of Plant Translocation (IDPlanT) provides the opportunity to review the techniques used in 186 plant translocation cases performed in the last 50 years in the heart of the Mediterranean Biodiversity Hotspot. We described techniques and information available in IDPlanT and used these data to identify drivers of translocation outcomes. We tested the effect of 15 variables on survival of translocated propagules as of the last monitoring date with binomial logistic mixed-effect models. Eleven variables significantly affected survival of transplants: life form, site protection, material source, number of source populations, propagation methods, propagule life stage, planting methods, habitat suitability assessment, site preparation, aftercare, and costs. The integration of vegetation studies in the selection of suitable planting sites significantly increased the success of translocation efforts. Although posttranslocation watering had a generally positive effect on translocation outcome, other aftercare techniques did not always increase transplant survival. Finally, we found that how funds were spent appeared to be more important than the actual amount spent. Plant translocations in Italy and in the Mediterranean area should account for the complexity of speciation, gene flow, and plant migrations that has led to local adaptations and has important implications for the choice and constitution of source material.
Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia ...patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.
Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still ...unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8
T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.
The Sec61 complex is the major protein translocation channel of the endoplasmic reticulum (ER), where it plays a central role in the biogenesis of membrane and secretory proteins. Whilst ...Sec61‐mediated protein translocation is typically coupled to polypeptide synthesis, suggestive of significant complexity, an obvious characteristic of this core translocation machinery is its surprising simplicity. Over thirty years after its initial discovery, we now understand that the Sec61 complex is in fact the central piece of an elaborate jigsaw puzzle, which can be partly solved using new research findings. We propose that the Sec61 complex acts as a dynamic hub for co‐translational protein translocation at the ER, proactively recruiting a range of accessory complexes that enhance and regulate its function in response to different protein clients. It is now clear that the Sec61 complex does not have a monopoly on co‐translational insertion, with some transmembrane proteins preferentially utilising the ER membrane complex instead. We also have a better understanding of post‐insertion events, where at least one membrane‐embedded chaperone complex can capture the newly inserted transmembrane domains of multi‐span proteins and co‐ordinate their assembly into a native structure. Having discovered this array of Sec61‐associated components and competitors, our next challenge is to understand how they act together in order to expand the range and complexity of the membrane proteins that can be synthesised at the ER. Furthermore, this diversity of components and pathways may open up new opportunities for targeted therapeutic interventions designed to selectively modulate protein biogenesis at the ER.
In eukaryotes, one third of cellular proteins are co‐translationally inserted into or translocated across the membrane of the endoplasmic reticulum during their biogenesis. Here, we distil recent advances in our understanding of this process; highlighting the Sec61 translocon as a dynamic hub that co‐ordinates the roles of key partners to provide a system that accommodates a diverse array of membrane protein clients and provides opportunities for therapeutic interventions.
Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic ...compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
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•The inhibited Sec translocon adopts a conformation optimal for substrate engagement•The inhibitor mycolactone wedges open the lateral gate of Sec61α•Mycolactone blocks the path taken by the signal peptide during engagement•Resistance mutations are likely to operate by modulating translocon dynamics
Gérard et al. determine the structure of the mammalian Sec translocon in an inhibited state. Mycolactone holds the translocon in a conformation optimal for substrate engagement, wedging open the cytosolic side of the lateral gate, while also blocking the path taken by the signal peptide during engagement.
A balanced constitutional reciprocal translocation (RT) is a mutual exchange of terminal segments of two non-homologous chromosomes without any loss or gain of DNA in germline cells. Carriers of ...balanced RTs are viable individuals with no apparent phenotypical consequences. These animals produce, however, unbalanced gametes and show therefore reduced fertility and offspring with congenital abnormalities. This cytogenetic abnormality is usually detected using chromosome staining techniques. The aim of this study was to test the possibilities of using paired end short read sequencing for detection of balanced RTs in boars and investigate their breakpoints and junctions.
Balanced RTs were recovered in a blinded analysis, using structural variant calling software DELLY, in 6 of the 7 carriers with 30 fold short read paired end sequencing. In 15 non-carriers we did not detect any RTs. Reducing the coverage to 20 fold, 15 fold and 10 fold showed that at least 20 fold coverage is required to obtain good results. One RT was not detected using the blind screening, however, a highly likely RT was discovered after unblinding. This RT was located in a repetitive region, showing the limitations of short read sequence data. The detailed analysis of the breakpoints and junctions suggested three junctions showing microhomology, three junctions with blunt-end ligation, and three micro-insertions at the breakpoint junctions. The RTs detected also showed to disrupt genes.
We conclude that paired end short read sequence data can be used to detect and characterize balanced reciprocal translocations, if sequencing depth is at least 20 fold coverage. However, translocations in repetitive areas may require large fragments or even long read sequence data.
The wheat – Thinopyrum intermedium derived line Z4 has displayed novel and effective stripe rust resistance for over 40 years. This study aimed to precisely identify the chromosome constitution of ...line Z4 and determine the stripe rust resistance contribution using multicolor fluorescent in situ hybridization (FISH) and molecular marker analysis. The results indicated that the Z4 line (2n = 44) contained two pairs of non-Robertsonian translocations without the 3A chromosomes of wheat. FISH karyotypes of F
3
progenies derived from crosses between Z4 and MY11 indicated that the transmission of the translocated chromosomes appeared normal and the number of wheat chromosomes 3A and 3D frequently varied. The FISH signal distribution of a new repetitive probe, named Oligo-3A1, confirmed the physical breakage points on chromosome 3AL incorporated into translocated chromosomes. PLUG markers revealed the breakage points on chromosomes 3A, 7J
S
, and 3D invloved in the translocated chromosomes, and they were designated as T3DS-3AS.3AL-7J
S
S and T3AL-7J
S
S.7J
S
L. Stripe rust resistances surveys indicated that the proximal region of 7J
S
S or 7J
S
L may confer the resistance at the adult plant stage. The precise characterization of the chromosome complements of wheat – Th. intermedium Z4 and derived progenies has demonstrated the importance of combining cytogenetic and molecular approaches in the genomics era for further wheat genetic manipulation and breeding purposes.
SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA ...binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery.
SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as "posttranslational." Here, we show that SecA interacts with nascent Sec substrates. This interaction is not dependent on SecB or trigger factor, which also interact with nascent Sec substrates. Moreover, the interaction of SecB with nascent polypeptides is dependent on the interaction of SecA with the ribosome, suggesting that interaction of the nascent chain with SecA precedes interaction with SecB. Our results suggest that SecA could recognize substrate proteins cotranslationally in order to efficiently target them for uncoupled protein translocation.
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