Biomedical subjects

Harris D Bernstein

Publications and source records attributed to Harris D Bernstein.

10 recordsLinked to original sources

PubMed2006

The surprising complexity of signal sequences.

Most secreted and many membrane proteins contain cleavable N-terminal signal sequences that mediate their targeting to and translocation across the endoplasmic reticulum or bacterial cytoplasmic membrane. Recent studies have identified many exceptions to the widely held view that signal sequences are simple, degenerate and interchangeable. Growing evidence indicates that signal sequences contain information that specifies the choice of targeting pathway, the efficiency of translocation, the timing of cleavage and even postcleavage functions. As a consequence, signal sequences can have important roles in modulating protein biogenesis. Based on a synthesis of studies in numerous experimental systems, we propose that substrate-specific sequence elements embedded in a conserved domain structure impart unique and physiologically important functionality to signal sequences.

Animals↗

PubMed2006

Translation arrest requires two-way communication between a nascent polypeptide and the ribosome.

When the export of E. coli SecM is blocked, a 17 amino acid motif near the C terminus of the protein induces a translation arrest from within the ribosome tunnel. Here we used a recently described application of fluorescence resonance energy transfer (FRET) to gain insight into the mechanism of translation arrest. We found that the SecM C terminus adopted a compact conformation upon synthesis of the arrest motif. This conformational change did not occur spontaneously, but rather was induced by the ribosome. Translation arrest required both compaction of the SecM C terminus and the presence of key residues in the arrest motif. Further analysis showed that the arrested peptidyl-tRNA was resistant to puromycin treatment and revealed additional changes in the ribosome-nascent SecM complex. Based on these observations, we propose that translation arrest results from a series of reciprocal interactions between the ribosome and the C terminus of the nascent SecM polypeptide.

Amino Acid Motifs↗

PubMed2006

An unusual signal peptide extension inhibits the binding of bacterial presecretory proteins to the signal recognition particle, trigger factor, and the SecYEG complex.

Considerable evidence indicates that the Escherichia coli signal recognition particle (SRP) selectively targets proteins that contain highly hydrophobic signal peptides to the SecYEG complex cotranslationally. Presecretory proteins that contain only moderately hydrophobic signal peptides typically interact with trigger factor (TF) and are targeted post-translationally. Here we describe a striking exception to this rule that has emerged from the analysis of an unusual 55-amino acid signal peptide associated with the E. coli autotransporter EspP. The EspP signal peptide consists of a C-terminal domain that resembles a classical signal peptide plus an N-terminal extension that is conserved in other autotransporter signal peptides. Although a previous study showed that proteins containing the C-terminal domain of the EspP signal peptide are targeted cotranslationally by SRP, we found that proteins containing the full-length signal peptide were targeted post-translationally via a novel TF-independent mechanism. Mutation of an invariant asparagine residue in the N-terminal extension, however, restored cotranslational targeting. Remarkably, proteins containing extremely hydrophobic derivatives of the EspP signal peptide were also targeted post-translationally. These and other results suggest that the N-terminal extension alters the accessibility of the signal peptide to SRP and TF and promotes post-translational export by reducing the efficiency of the interaction between the signal peptide and the SecYEG complex. Based on data, we propose that the N-terminal extension mediates an interaction with an unidentified cytoplasmic factor or induces the formation of an unusual signal peptide conformation prior to the onset of protein translocation.

Amino Acid Sequence↗

PubMed2005

Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter.

Bacterial autotransporters are proteins that contain a small C-terminal 'beta domain' that facilitates translocation of a large N-terminal 'passenger domain' across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPDelta1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPDelta1-851 behaves as a compact monomer and strongly suggest that the channel formed by the beta domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspPDelta1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP beta domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.

Blotting, Western↗

PubMed2004

An unusual signal peptide facilitates late steps in the biogenesis of a bacterial autotransporter.

Bacterial autotransporters are proteins that use a C-terminal porin-like domain to facilitate the transport of an upstream "passenger domain" across the outer membrane. Although autotransporters are translocated across the inner membrane (IM) via the Sec pathway, some of them contain exceptionally long signal peptides distinguished by a unique N-terminal sequence motif. In this study, we used the Escherichia coli O157:H7 autotransporter EspP as a model protein to investigate the function of the unusual signal peptides. We found that removal of the N-terminal motif or replacement of the EspP signal peptide did not affect translocation of the protein across the IM. Remarkably, modification of the signal peptide caused EspP to misfold in the periplasm and blocked transport of the passenger domain across the outer membrane. Further analysis suggested that the EspP signal peptide transits slowly through the Sec machinery. Based on these results, we propose that the unusual signal peptides not only function as targeting signals, but also prevent misfolding of the passenger domain in the periplasm by transiently tethering it to the IM.

Escherichia coli↗

PubMed2004

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain.

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop-loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop-loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.

Alu Elements↗

PubMed2003

Basic amino acids in a distinct subset of signal peptides promote interaction with the signal recognition particle.

Previous studies have demonstrated that signal peptides bind to the signal recognition particle (SRP) primarily via hydrophobic interactions with the 54-kDa protein subunit. The crystal structure of the conserved SRP ribonucleoprotein core, however, raised the surprising possibility that electrostatic interactions between basic amino acids in signal peptides and the phosphate backbone of SRP RNA may also play a role in signal sequence recognition. To test this possibility we examined the degree to which basic amino acids in a signal peptide influence the targeting of two Escherichia coli proteins, maltose binding protein and OmpA. Whereas both proteins are normally targeted to the inner membrane by SecB, we found that replacement of their native signal peptides with another moderately hydrophobic but unusually basic signal peptide (DeltaEspP) rerouted them into the SRP pathway. Reduction in either the net positive charge or the hydrophobicity of the DeltaEspP signal peptide decreased the effectiveness of SRP recognition. A high degree of hydrophobicity, however, compensated for the loss of basic residues and restored SRP binding. Taken together, the data suggest that the formation of salt bridges between SRP RNA and basic amino acids facilitates the binding of a distinct subset of signal peptides whose hydrophobicity falls slightly below a threshold level.

Amino Acid Sequence↗

PubMed2002

Signal recognition particle (SRP)-mediated targeting and Sec-dependent translocation of an extracellular Escherichia coli protein.

Hemoglobin protease (Hbp) is a hemoglobin-degrading protein that is secreted by a human pathogenic Escherichia coli strain via the autotransporter mechanism. Little is known about the earliest steps in autotransporter secretion, i.e. the targeting to and translocation across the inner membrane. Here, we present evidence that Hbp interacts with the signal recognition particle (SRP) and the Sec-translocon early during biogenesis. Furthermore, Hbp requires a functional SRP targeting pathway and Sec-translocon for optimal translocation across the inner membrane. SecB is not required for targeting of Hbp but can compensate to some extent for the lack of SRP. Hbp is synthesized with an unusually long signal peptide that is remarkably conserved among a subset of autotransporters. We propose that these autotransporters preferentially use the co-translational SRP/Sec route to avoid adverse effects of the exposure of their mature domains in the cytoplasm.

Adenosine Triphosphatases↗

PubMed2002

DnaK promotes the selective export of outer membrane protein precursors in SecA-deficient Escherichia coli.

Consistent with many other results indicating that SecA plays an essential role in the translocation of presecretory proteins across the Escherichia coli inner membrane, we previously found that a approximately 95% depletion of SecA completely blocks the export of periplasmic proteins in vivo. Surprisingly, we found that about 25% of the outer membrane protein (OMP) OmpA synthesized after SecA depletion was gradually translocated across the inner membrane. In this study we analyzed the export of several other OMPs after SecA depletion. We found that 25-50% of each OMP as well as an OmpA-alkaline phosphatase fusion protein was exported from SecA-deficient cells. This partial export was completely abolished by the SecA inhibitor sodium azide and therefore still required the participation of SecA. Examination of a variety of OmpA derivatives, however, ruled out the possibility that OMPs are selectively translocated in SecA-deficient cells because SecA binds to their N termini with unusually high affinity. Export after SecA depletion was observed in cells that lack SecB, the primary targeting factor for OMPs, but was abolished by partial inactivation of DnaK. Furthermore, OmpA could be isolated in a stable complex with DnaK. The data strongly suggest that OMPs require only a relatively low level of translocase activity to cross the inner membrane because they can be preserved in a prolonged export-competent state by DnaK.

Adenosine Triphosphatases↗

PubMed2002

Trigger factor retards protein export in Escherichia coli.

Trigger factor (TF) is a ribosome-associated protein that interacts with a wide variety of nascent polypeptides in Escherichia coli. Previous studies have indicated that TF cooperates with DnaK to facilitate protein folding, but the basis of this cooperation is unclear. In this study we monitored protein export in E. coli that lack or overproduce TF to obtain further insights into its function. Whereas inactivation of genes encoding most molecular chaperones (including dnaK) impairs protein export, inactivation of the TF gene accelerated protein export and suppressed the need for targeting factors to maintain the translocation competence of presecretory proteins. Furthermore, overproduction of TF (but not DnaK) markedly retarded protein export. Manipulation of TF levels produced similar effects on the export of a cytosolic enzyme fused to a signal peptide. The data strongly suggest that TF has a unique ability to sequester nascent polypeptides for a relatively prolonged period. Based on our results, we propose that TF and DnaK promote protein folding by distinct (but complementary) mechanisms.

Base Sequence↗