A functional interaction between the signal peptide and the translation apparatus is detected by the use of a single point mutation which blocks translocation across mammalian endoplasmic reticulum (original) (raw)
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Bacterial Signal Peptides- Navigating the Journey of Proteins
Frontiers in Physiology
In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradatio...
Proceedings of the …, 2007
Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal ␣1 helix lies outside the core structure. However, the function of the N-terminal ␣1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the ␣1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2-7 from the N terminus, including 4 aa from the ␣1 helix, were deleted by site-directed mutagenesis. The deletion resulted in complete inhibition of LTB expression in E. coli when expressed along with its signal sequence. A single amino acid deletion within the ␣1 helix also resulted in loss of expression. However, a single amino acid deletion outside the ␣1 helix did not affect LTB synthesis. Mutant proteins, whose synthesis was not detected in vivo, could be successfully translated in vitro by using the coupled transcriptiontranslation system. Immunoblot analysis, Northern blot analysis, and in vitro transcription-translation data collectively indicate that the lack of synthesis of the mutant proteins is caused by the immediate degradation of the expressed product by cellular proteases rather than by faulty translation of mutant LTB mRNA. Coexpression of the LTA could not rescue the degradation of LTB mutants.
FEMS Microbiology Letters, 2010
The plasmid-encoded toxin, Pet, a prototypical member of the serine protease autotransporters of the Enterobacteriaceae, possesses an unusually long signal peptide, which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions correspond to a conserved N-terminal extension previously designated the extended signal peptide region (ESPR), while the N2, H2 and C regions resemble typical Sec-dependent signal sequences and exhibit considerable sequence variability. We have shown previously that the ESPR directs Sec-dependent, posttranslational translocation of Pet across the bacterial inner membrane. In this study, we demonstrate that the ESPR is not essential for the secretion or the function of Pet.
Molecular …, 1997
The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.
Optimisation of signal peptide for recombinant protein secretion in bacterial hosts
Applied Microbiology and Biotechnology, 2013
Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.
Molecular Microbiology, 1996
Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, GSα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.
…, 2007
Members of the type V secretion family are among the most prevalent secreted proteins in Gram-negative bacteria. A subset of this family, including Pet, the prototypical member of the Enterobacteriaceae serine proteases, possess unusual signal peptides which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions, which the authors have named the extended signal peptide region (ESPR), demonstrate remarkable conservation. In contrast, the N2, H2 and C regions show significant variability, and are reminiscent of typical Sec-dependent signal sequences. Despite several investigations, the function of the ESPR remains obscure. Here, it is shown that proteins possessing the ESPR are translocated in a posttranslational fashion. The presence of the ESPR severely impairs inner membrane translocation. Mutational analysis suggests that the ESPR delays inner membrane translocation by adopting a particular conformation, or by interacting with a cytoplasmic or inner membrane co-factor, prior to inner membrane translocation.