RESUMO
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.
Assuntos
Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Canais de Translocação SEC/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Modelos Moleculares , Mutação , Biossíntese de Proteínas , Conformação Proteica , Dobramento de Proteína , Serina Endopeptidases/genéticaRESUMO
BACKGROUND: To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector. RESULTS: By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein. CONCLUSIONS: Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Membrana/genética , Proteínas Virais/metabolismo , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/metabolismo , Vetores Genéticos/metabolismo , Proteínas de Membrana/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Virais/genéticaRESUMO
Optimizing the conditions for the production of membrane proteins in E. coli is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain or the pReX T7-based expression vector with membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the optimization of membrane protein production yields. Both Lemo21(DE3) and pReX allow precise regulation of expression intensities of genes encoding membrane proteins, which is critical to identify the optimal production condition for a membrane protein. The use of GFP-fusions allows direct monitoring and visualization of membrane proteins at any stage during the production optimization process.
Assuntos
Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana/genética , Animais , Técnicas de Cultura de Células , Clonagem Molecular/métodos , Expressão Gênica , Vetores Genéticos/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Transformação GenéticaRESUMO
Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.
Assuntos
Regulação Bacteriana da Expressão Gênica , Mannheimia/genética , Fatores de Alongamento de Peptídeos/genética , Peptídeos/química , Ribossomos/genética , Sequência de Aminoácidos , Fenômenos Biomecânicos , Escherichia coli/genética , Escherichia coli/metabolismo , Mannheimia/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Elongação Traducional da Cadeia Peptídica , Fatores de Alongamento de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Ribossomos/metabolismoRESUMO
On average, every fifth residue in secretory proteins carries either a positive or a negative charge. In a bacterium such as Escherichia coli, charged residues are exposed to an electric field as they transit through the inner membrane, and this should generate a fluctuating electric force on a translocating nascent chain. Here, we have used translational arrest peptides as in vivo force sensors to measure this electric force during cotranslational chain translocation through the SecYEG translocon. We find that charged residues experience a biphasic electric force as they move across the membrane, including an early component with a maximum when they are 47-49 residues away from the ribosomal P site, followed by a more slowly varying component. The early component is generated by the transmembrane electric potential, whereas the second may reflect interactions between charged residues and the periplasmic membrane surface.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Potenciais da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Membrana Celular/metabolismo , Canais de Translocação SECRESUMO
Transmembrane helices (TMHs) in membrane proteins can be orientated with their N-terminus towards the cytoplasm (Nin), or facing the non-cytoplasmic side (Nout). Most membrane proteins are inserted co-translationally into membranes, aided by Sec-type translocons. Since the final orientation of Nin- and Nout-orientated TMHs differs, they could also interact differently with the translocon and the surrounding membrane during insertion. We measured pulling forces exerted on Nin-orientated TMHs during co-translational insertion into the inner membrane of Escherichia coli. Our results demonstrate that Nin-orientated TMHs experience a weaker pulling force but retain the overall biphasic force profile seen previously for Nout-orientated TMHs (Ismail et al., 2012 [1]).
Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Biológicos , Mutação , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Transporte Proteico , Homologia de Sequência do Ácido NucleicoRESUMO
Lipid composition can differ widely among organelles and even between leaflets of a membrane. Lipid homeostasis is critical because disequilibrium can have disease outcomes. Despite their importance, mechanisms maintaining lipid homeostasis remain poorly understood. Here, we establish a model system to study the global effects of lipid imbalance. Quantitative lipid profiling was integral to monitor changes to lipid composition and for system validation. Applying global transcriptional and proteomic analyses, a dramatically altered biochemical landscape was revealed from adaptive cells. The resulting composite regulation we term the "membrane stress response" (MSR) confers compensation, not through restoration of lipid composition, but by remodeling the protein homeostasis network. To validate its physiological significance, we analyzed the unfolded protein response (UPR), one facet of the MSR and a key regulator of protein homeostasis. We demonstrate that the UPR maintains protein biogenesis, quality control, and membrane integrity-functions otherwise lethally compromised in lipid dysregulated cells.
Assuntos
Proteínas Ligadas a Lipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Resposta a Proteínas não Dobradas , Homeostase , Proteínas Ligadas a Lipídeos/química , Lipídeos de Membrana/química , Redes e Vias Metabólicas , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse FisiológicoRESUMO
Membrane proteins destined for insertion into the inner membrane of bacteria or the endoplasmic reticulum membrane in eukaryotic cells are synthesized by ribosomes bound to the bacterial SecYEG or the homologous eukaryotic Sec61 translocon. During co-translational membrane integration, transmembrane α-helical segments in the nascent chain exit the translocon through a lateral gate that opens toward the surrounding membrane, but the mechanism of lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when entering and exiting the translocon. Using translation-arrest peptides from bacterial SecM proteins and from the mammalian Xbp1 protein as force sensors, we show that substantial force is exerted on a transmembrane helix at two distinct points during its transit through the translocon channel, providing direct insight into the dynamics of membrane integration.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Cães , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Glicosilação , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Fatores de Transcrição/genéticaRESUMO
Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the unfolded protein response (UPR), is responsible for maintaining endoplasmic reticulum (ER) homeostasis. The highly conserved Ire1 branch regulates hundreds of gene targets by activating a UPR-specific transcription factor. To understand how the UPR manages ER stress, a unique genetic approach was applied to reveal how the system corrects disequilibria. The data show that the UPR can address a wide range of dysfunctions that are otherwise lethal if not for its intervention. Transcriptional profiling of stress-alleviated cells shows that the program can be modulated, not just in signal amplitude, but also through differential target gene expression depending on the stress. The breadth of the functions mitigated by the UPR further supports its role as a major mechanism maintaining systems robustness.
Assuntos
Proteínas Fúngicas/química , Resposta a Proteínas não Dobradas , Alelos , Retículo Endoplasmático/metabolismo , Deleção de Genes , Glicosilação , Modelos Genéticos , Conformação Molecular , Mutação , Fenótipo , Desnaturação Proteica , Dobramento de Proteína , Transdução de Sinais , Temperatura , Transcrição Gênica , beta-Galactosidase/metabolismoRESUMO
A site-specific cross-linking approach was used to study the integration of TM (transmembrane) segments 4-7 of the polytopic membrane protein, opsin, at the ER (endoplasmic reticulum). We found that although TM4 exits the ER translocon rapidly, TM segments 5, 6 and 7 are all retained at the translocon until opsin biosynthesis is terminated. Furthermore, although artificial extension of the nascent chain is not sufficient to release the C-terminal region of opsin from the translocon, substitution of the native TM segment 7 with a more hydrophobic TM segment results in its rapid lateral exit into the lipid bilayer. We conclude that the intrinsic properties of a TM segment determine the timing of its membrane integration rather than its relative location within the polypeptide chain. A pronounced and prolonged association of opsin TM5 with the translocon-associated component PAT-10 was also observed, suggesting that PAT-10 may facilitate the assembly of distinct opsin subdomains during membrane integration. The results of the present study strongly support a model in which the ER translocon co-ordinates the integration of selected TM segments in response to the specific requirements of the precursor being synthesized.
Assuntos
Retículo Endoplasmático/metabolismo , Opsinas de Bastonetes/biossíntese , Animais , Bovinos , Deleção de Genes , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Mutação/genética , Transporte Proteico , Opsinas de Bastonetes/genética , Canais de Translocação SECRESUMO
Numerous factors are involved in the eradication of misfolded proteins, yet how these factors achieve substrate specificity remains unclear. In this issue of Cell, Denic et al. (2006) and Carvalho et al. (2006) report that two distinct protein complexes at the endoplasmic reticulum membrane are responsible for the recognition and degradation of specific subsets of protein substrates.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Citosol/química , Citosol/metabolismo , Ligases/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dobramento de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteína com Valosina , Proteínas de Transporte VesicularRESUMO
We used a site-specific crosslinking approach to study the membrane integration of the polytopic protein opsin at the endoplasmic reticulum. We show that transmembrane domain 1 occupies two distinct Sec61-based environments during its integration. However, transmembrane domains 2 and 3 exit the Sec61 translocon more rapidly in a process that suggests a displacement model for their integration where the biosynthesis of one transmembrane domain would facilitate the exit of another. In order to investigate this hypothesis further, we studied the integration of the first and third transmembrane domains of opsin in the absence of any additional C-terminal transmembrane domains. In the case of transmembrane domain 1, we found that its lateral exit from the translocon is clearly dependent upon the synthesis of subsequent transmembrane domains. By contrast, the lateral exit of the third transmembrane domain occurred independently of any such requirement. Thus, even within a single polypeptide chain, distinct transmembrane domains display different requirements for their integration through the endoplasmic reticulum translocon, and the displacement of one transmembrane domain by another is not a global requirement for membrane integration.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/metabolismo , Opsinas de Bastonetes/biossíntese , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Bovinos , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , Difusão , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Elongação Traducional da Cadeia Peptídica , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Transporte Proteico , Opsinas de Bastonetes/química , Canais de Translocação SEC , Homologia de Sequência de AminoácidosRESUMO
The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide. We identified strong adducts with both the ribophorin I subunit of the oligosaccharyltransferase complex and the 25-kDa subunit of the signal peptidase complex. Focusing on the association with ribophorin I, we found that adduct formation occurred exclusively after the exit of the nascent chain from the Sec61 translocon and was unaffected by the N-glycosylation status of the associated precursor. Only a subset of newly made membrane proteins associated with ribophorin I in vitro, and we could recapitulate a specific association between the amyloid precursor protein fragment and ribophorin I in vivo. Taken together, our data suggest a model where ribophorin I may function to retain potential substrates in close proximity to the catalytic subunit of the oligosaccharyltransferase and thereby stochastically improve the efficiency of the N-glycosylation reaction in vivo. Alternatively ribophorin I may be multifunctional and facilitate additional processes, for example, ER quality control.