Project description:Cotranslational targeting into the endoplasmic reticulum (ER) by the Signal Recognition Particle (SRP) is a key event determining polypeptide fate in eukaryotic cells. Here, we globally define the principles and mechanisms of SRP binding and ER targeting in vivo. Cotranslational targeting through SRP is the dominant route into the ER for all secretory proteins, regardless of targeting signal characteristics. Cytosolic SRP functions in a pioneer translation round that builds a membrane-resident mRNAs pool, explaining how low SRP levels suffice for the secretory load. SRP does not induce an elongation arrest; consequently, kinetic competition between targeting and translation elongation dictates which substrates are translocated post-translationally. Unexpectedly, SRP binds most secretory ribosomal complexes before targeting signals are synthesized. We show non-coding mRNA elements can promote 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 in vivo. Ribosome profiling (RiboSeq) and RNA-seq of subcellular fractions of ribosomes. Soluble and membrane bound ribosomes are separated by centrifugation, and SRP-bound ribosomes are immunoprecipitated from the soluble fraction. Polysomes and monosomes are separated by sucrose gradient ultracentrifugation.
Project description:Bacillus subtilis is a Gram-positive bacterium considered as a “cell-factory” for industrial enzymes and biopharmaceuticals. Given the commercial advantage of this organism, researchers have been working towards the improvement of this organism as a producer of secreted proteins. Nonetheless, there is the need to obtain more knowledge regarding the absolute composition of the gram positive cell membrane in order to increase the secretion capability of B. subtilis and gain insight into potential bottlenecks in protein production and/or secretion. However, membrane proteins are one of the most challenging classes of proteins, mainly due to their high hydrophobicity and low abundance compared to their soluble counterparts. To this regard, we established a method for global characterization of absolute protein abundances within the membrane of B. subtilis and verified the effects of large scale protein export on a super-secreting strain. We did shotgun proteomics on three different fractions (extracellular, cytosol and membrane) and spiked in UPS2 proteins in each of these to calculate absolute protein abundances. The absolute values obtained from the shotgun experiment were validated by targeted proteomics. Furthermore, we performed western blot analysis on the three subcellular fractions using antibodies against proteins localized in these three different compartments in order to visually validate the enrichment of membrane proteins in the corresponding fraction.
Project description:A proteomic analysis of a redox active membrane fraction (doublet at >200 kDa) of Mariprofundus ferrooxydans, a chemolithoautotrophic, neutrophilic, iron-oxidizing Zetaproteobacterium.
Project description:We performed shotgun proteomics on the bacteria Prevotella brevis GA33 and Prevotella ruminicola 23. We did this for two types of samples (cell extract and cell membrane) and using two methods (data-dependent and data-independent acquisition).
Project description:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library. Ribosome profiling in cultured mammalian cells under three different footprinting conditions
Project description:Genome engineering offers the possibility to create completely novel cell factories with enhanced properties for biotechnological application. In recent years, the possibilities for genome engineering have been extensively explored in the Gram-positive bacterial cell factory Bacillus subtilis, where up to 42% of the genome, encoding dispensable functions has been removed. Such studies have shown that some strains with minimized genomes gained beneficial features, for instance in protein production. However, strains with the most minimal genomes also showed particular growth defects. This has focused our attention on strains with less extensive genome deletions that show close-to-wild-type growth properties, while retaining the acquired beneficial traits in secretory protein production of strains lacking larger genomic segments. A strain of the latter category is B. subtilis IIG-Bs27-47-24, here referred to as midiBacillus II, which lacks 30.95% of the parental genome. To date, it was unknown how the altered genomic configuration of midiBacillus II impacts on cell physiology at large, and protein secretion in particular. Therefore, the present study was aimed at bridging this knowledge gap through an in-depth proteomics analysis with special focus on protein secretion stress responses. Interestingly, the results show that the secretion stress response of midiBacillus II as elicited by high-level expression of a staphylococcal antigen is completely different from the secretion stress responses that occur in the parental strain 168. This implies that high-level protein secretion has different implications for wild-type and genome-engineered Bacillus strains, dictated by the altered genomic and proteomic configurations.
Project description:Electron transport, or oxidative phosphorylation, is one of the hallmarks of life. To this end, prokaryotes evolved a vast variety of protein complexes, only a small part of which have been discovered and studied. These protein complexes allow them to occupy virtually every ecological niche on Earth. Here, we applied the advanced method of proteomics-based complexome profiling to get a better understanding of the electron transport systems of the anaerobic ammonium-oxidizing (anammox) bacteria, the N2-producing key players of the global nitrogen cycle. By this method nearly all respiratory complexes that were previously predicted from genome analysis to be involved in energy and cell carbon fixation were validated. More importantly, new and unexpected ones were discovered. We believe that complexome profiling in concert with (meta)genomics offers great opportunities to expand our knowledge on bacterial respiratory processes at a rapid and massive pace, in particular in new and thus far poorly investigated non-model and environmentally-relevant species.
Project description:Desulfovibrio vulgaris Hildenborough is a gram-negative anaerobic bacterium belonging to the sulfate-reducing bacteria, a group of microbes that can perform dissimilatory sulfate reduction coupled to the oxidation of various substrates as carbon and energy sources. In the absence of sulfate, they can also ferment organic acids in syntrophy with methanogens. They exhibit high metabolic diversity switching from one energy mode to another depending on nutrients availability in the environments. Hence, they play a central role in shaping ecosystems. Despite, intensive efforts to study D. vulgaris energy metabolism at the genomic, biochemical and ecological level, bioenergetics in this microorganism remains far from being fully understood. Alternatively, few metabolic models were also proposed to explain D. vulgaris bioenergetics. However, they appeared to be not easily adaptable to various environmental conditions. To lift off these limitations, here we constructed a new transparent and robust metabolic model of D. vulgaris bioenergetics by combining whole-cell proteomic analysis with modeling approaches (Flux Balance Analysis). The iDvu71 model showed over 0.95 correlation with experimental data. Further simulations allowed a detailed description of D. vulgaris metabolism in various conditions of growth. Altogether, the simulations run in this study highlighted the sulfate-to-lactate consumption ratio as a pivotal factor in D. vulgaris energy metabolism. In particular, the impact on the hydrogen/formate balance and biomass synthesis is discussed. Overall, this study provides a novel insight into D. vulgaris metabolic flexibility.