Damon Huber

Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo

As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, β-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.

The DsbA Signal Sequence Directs Efficient, Cotranslational Export of Passenger Proteins to the Escherichia coli Periplasm via the Signal Recognition Particle Pathway

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.

Use of Thioredoxin as a Reporter To Identify a Subset of Escherichia coli Signal Sequences That Promote Signal Recognition Particle-Dependent Translocation

We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185:5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.

Integrating Protein Homeostasis Strategies in Prokaryotes

Bacterial cells are frequently exposed to dramatic fluctuations in their environment, which cause perturbation in protein homeostasis and lead to protein misfolding. Bacteria have therefore evolved powerful quality control networks consisting of chaperones and proteases that cooperate to monitor the folding states of proteins and to remove misfolded conformers through either refolding or degradation. The levels of the quality control components are adjusted to the folding state of the cellular proteome through the induction of compartment specific stress responses. In addition, the activities of several quality control components are directly controlled by these stresses, allowing for fast activation. Severe stress can, however, overcome the protective function of the proteostasis network leading to the formation of protein aggregates, which are sequestered at the cell poles. Protein aggregates are either solubilized by AAA+ chaperones or eliminated through cell division, allowing for the generation of damage-free daughter cells.

SecA cotranslationally interacts with nascent substrate proteins in vivo

ABSTRACT 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. IMPORTANCE 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.

Deletions in a ribosomal protein-coding gene are associated with tigecycline resistance in Enterococcus faecium

Enterococcus faecium is an emerging nosocomial pathogen associated with antibiotic therapy in the hospital environment. Whole-genome sequences were determined for three pairs of related, consecutively collected E. faecium clinical isolates to determine putative mechanisms of resistance to tigecycline. The first isolates (1S, 2S and 3S) in each of the three pairs were sensitive to tigecycline [minimum inhibitory concentration (MIC) of 0.125 mg/L]. Following tigecycline therapy, the second isolate in each pair demonstrated increased resistance to tigecycline. Two isolates (1R and 2R) were resistant (MIC of 8 mg/L) and one isolate (3I) demonstrated reduced susceptibility (MIC of 0.5 mg/L). Mutations distinguishing each pair of sensitive and resistant isolates were determined through alignment to a reference genome and variant detection. In addition, a de novo assembly of each isolate genome was constructed to confirm mutations. A total of 16 mutations in eleven coding sequences were determined. Mutations in the rpsJ gene, which encodes a structural protein forming part of the 30S ribosomal subunit, were detected in each of the pairs. Mutations were in regions proximal to the predicted tigecycline-binding site. Predicted amino acid substitutions were detected in 1R and 3I. The resistant strains were additionally associated with deletions of 15 nucleotides (2R) and 3 nucleotides (1R). This study confirms that amino acid substitutions in rpsJ contribute towards reduced susceptibility to tigecycline and suggests that deletions may be required for tigecycline resistance in E. faecium.

Amino acid residues important for folding of thioredoxin are revealed only by study of the physiologically relevant reduced form of the protein.

Thioredoxin-1 from Escherichia coli has frequently been used as a model substrate in protein folding studies. However, for reasons of convenience, these studies have focused largely on oxidized thioredoxin and not on reduced thioredoxin, the more physiologically relevant species. Here we describe the first extensive characterization of the refolding kinetics and conformational thermodynamics of reduced thioredoxin. We have previously described a genetic screen that yielded mutant thioredoxin proteins that fold more slowly in both the oxidized and reduced forms. In this study, we apply our more detailed analysis of reduced thioredoxin folding to a larger number of folding mutants that includes those obtained from continuation of the genetic screen. We have identified mutant proteins that display folding defects specifically in the reduced state but not the oxidized state. Some of these substitutions represent unusual folding mutants in that they result in semiconservative substitutions at solvent-exposed positions in the folded conformation and do not appear to affect the conformational stability of the protein. Further, the genetic selection yields mutants at only a limited number of sites, pointing to perhaps the most critical amino acids in the folding pathway and underscoring, in particular, the role of the carboxy-terminal amino acids in the folding of thioredoxin. Our results demonstrate the importance of studying the physiologically relevant folding species.

The way is the goal: how SecA transports proteins across the cytoplasmic membrane in bacteria

Abstract In bacteria, translocation of most soluble secreted proteins (and outer membrane proteins in Gram-negative bacteria) across the cytoplasmic membrane by the Sec machinery is mediated by the essential ATPase SecA. At its core, this machinery consists of SecA and the integral membrane proteins SecYEG, which form a protein conducting channel in the membrane. Proteins are recognised by the Sec machinery by virtue of an internally encoded targeting signal, which usually takes the form of an N-terminal signal sequence. In addition, substrate proteins must be maintained in an unfolded conformation in the cytoplasm, prior to translocation, in order to be competent for translocation through SecYEG. Recognition of substrate proteins occurs via SecA—either through direct recognition by SecA or through secondary recognition by a molecular chaperone that delivers proteins to SecA. Substrate proteins are then screened for the presence of a functional signal sequence by SecYEG. Proteins with functional signal sequences are translocated across the membrane in an ATP-dependent fashion. The current research investigating each of these steps is reviewed here.

Phage display extends its reach

Proteins that could not be displayed on the surface of phage are now amenable to this powerful selection technology.

Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting β-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.