Jan-Willem de Gier

YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase

In Escherichia coli, both secretory and inner membrane proteins initially are targeted to the core SecYEG inner membrane translocase. Previous work has also identified the peripherally associated SecA protein as well as the SecD, SecF and YajC inner membrane proteins as components of the translocase. Here, we use a cross‐linking approach to show that hydrophilic portions of a co‐translationally targeted inner membrane protein (FtsQ) are close to SecA and SecY, suggesting that insertion takes place at the SecA/Y interface. The hydrophobic FtsQ signal anchor sequence contacts both lipids and a novel 60 kDa translocase‐associated component that we identify as YidC. YidC is homologous to Saccharomyces cerevisiae Oxa1p, which has been shown to function in a novel export pathway at the mitochondrial inner membrane. We propose that YidC is involved in the insertion of hydrophobic sequences into the lipid bilayer after initial recognition by the SecAYEG translocase.

Tuning Escherichia coli for membrane protein overexpression

A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

Competition between Sec- and TAT-dependent protein translocation in Escherichia coli.

Recently, a new protein translocation pathway, the twin‐arginine translocation (TAT) pathway, has been identified in both bacteria and chloroplasts. To study the possible competition between the TAT‐ and the well‐characterized Sec translocon‐dependent pathways in Escherichia coli, we have fused the TorA TAT‐targeting signal peptide to the Sec‐dependent inner membrane protein leader peptidase (Lep). We find that the soluble, periplasmic P2 domain from Lep is re‐routed by the TorA signal peptide into the TAT pathway. In contrast, the full‐length TorA–Lep fusion protein is not re‐routed into the TAT pathway, suggesting that Sec‐targeting signals in Lep can override TAT‐targeting information in the TorA signal peptide. We also show that the TorA signal peptide can be converted into a Sec‐targeting signal peptide by increasing the hydrophobicity of its h‐region. Thus, beyond the twin‐arginine motif, the overall hydrophobicity of the signal peptide plays an important role in TAT versus Sec targeting. This is consistent with statistical data showing that TAT‐targeting signal peptides in general have less hydrophobic h‐regions than Sec‐targeting signal peptides.

Optimization of membrane protein overexpression and purification using GFP fusions

Optimizing conditions for the overexpression and purification of membrane proteins for functional and structural studies is usually a laborious and time-consuming process. This process can be accelerated using membrane protein–GFP fusions 1–3 , which allows direct monitoring and visualization of membrane proteins of interest at any stage during overexpression, solubilization and purification (Fig. 1). The exceptionally stable GFP moiety of the fusion protein can be used to detect membrane proteins by observing fluorescence in whole cells during overexpression, with a detection limit as low as 10 µg of GFP per liter of culture, and in solution during solubilization and purification. Notably, the fluorescence of the GFP moiety can also be detected in standard SDS polyacrylamide gels with a detection limit of less than 5 ng of GFP per protein band (Fig. 2). In-gel fluorescence allows assessment of the integrity of membrane protein–GFP fusions and provides a rapid and generic alternative for the notoriously difficult immunoblotting of membrane proteins. With whole-cell and in-gel fluorescence the overexpression potential of many membrane protein–GFP fusions can be rapidly assessed and yields of promising targets can be improved. In this protocol the Escherichia coli BL21(DE3)-pET system—the most widely used (membrane) protein overexpression system—is used as a platform to illustrate the GFP-based method. The methodology described in this protocol can be transferred easily to other systems.

Consequences of Membrane Protein Overexpression in Escherichia coli

Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a down-regulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.

Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis.

We present an approach that allows rapid determination of the topology of Escherichia coli inner-membrane proteins by a combination of topology prediction and limited fusion-protein analysis. We derive new topology models for 12 inner-membrane proteins: MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA. We estimate that our approach should make it possible to arrive at highly reliable topology models for roughly 10% of the ≈800 inner-membrane proteins thought to exist in E. coli.

Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor

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.

Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli

Escherichia coli is one of the most widely used vehicles to overexpress membrane proteins (MPs). Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favoured to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies. Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion versus inclusion body formation of overexpressed MPs in E. coli. Furthermore, we show that an overexpressed MP can be recovered from a MP–GFP fusion using a site specific protease. This makes GFP an excellent tool for large‐scale MP target selection in structural genomics projects.

Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC.

Recent studies identified YidC as a novel membrane factor that may play a key role in membrane insertion of inner membrane proteins (IMPs), both in conjunction with the Sec‐translocase and as a separate entity. Here, we show that the type II IMP FtsQ requires both the translocase and, to a lesser extent, YidC in vivo. Using photo‐crosslinking we demonstrate that the transmembrane (TM) domain of the nascent IMP FtsQ inserts into the membrane close to SecY and lipids, and moves to a combined YidC/lipid environment upon elongation. These data are consistent with a crucial role for YidC in the lateral transfer of TM domains from the Sec translocase into the lipid bilayer.

Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle

Targeting of the cytoplasmic membrane protein leader peptidase (Lep) and a Lep mutant (Lep‐inv) that inserts with an inverted topology compared to the wild‐type protein was studied in Escherichia coli strains that are conditional for the expression of either Ffh or 4.5S RNA, the two components of the E. coli SRP. Depletion of either component strongly affected the insertion of both Lep and Lep‐inv into the cytoplasmic membrane. This indicates that SRP is required for the assembly of cytoplasmic membrane proteins in E. coli.