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.

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.

SummaryThe 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.

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. ß

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 Fill 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. coil K, y words: Escherichia coli; Signal recognition particle; Membrane protein; Leader peptidase; Protein targeting ln~oducfionTargeting of secretory proteins to the cytoplasmic memb~ ane of E. coli can follow different pathways that probably c~nverge at the membrane embedded translocation machinery, the translocon [1][2][3]. The so-called SRP pathway involves the signal recognition particle (SRP), a complex of a 4.5S R NA and the Ffh protein which are homologous to the 7S R NA and 54 kDa constituents of the mammalian SRP, respectively [2,3]. FtsY, an E. coli homologue of the mammalian SRP receptor, has been identified on the basis of sequence similarity [4] and has been found to have affinity for the E. c~li SRP in vitro [5]. The SRP subunits 4.5S RNA and Ffh as ~' 11 as their receptor FtsY are essential for viability and their depletion results in defective protein secretion [6][7][8][9].In E. coli there seems to be a correlation between the affi~Lity of a signal sequence for the SRP-targeting pathway and tl~e hydrophobicity of the signal sequence core region [10]. Tfis seems to hold also for the SRP-targeting pathways in 5tceharomyces cerevisiae [11] and chloroplasts (S. High, pers~,nal communication). Whether the SRP is also involved in the targeting and assembly of cytoplasmic membrane proteins fi E. coli is not known, however. Based on the relatively strong hydrophobicity of signal anchor domains and their ability to be cross-linked to SRP in vitro, it has been proposed tl at, like in mammalian cells, membrane proteins are targeted v a the SRP pathway [10]. A role for the SRP in the targeting *~ 7orresponding author. Fax: (46)(8) 153679. E mail: gunnar@biokemi.su.seAbbreviations." Ffh, Fifty-four homologue; SRP, signal recognition particle; Lep, leader peptidase; LacY, lactose permease; IPTG, isopropyl-l-thio-~-D-galactopyranoside; PMSF, phenylmethylsulfonyl flaoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel el zetrophoresis of the cytoplasmic membrane protein lactose permease (LacY) has been suggested using an indirect approach [12]. In addition, overproduction of another cytoplasmic membrane protein, leader peptidase (Lep), results in reduced levels of free SRP and a SRP depletion secretion phenotype (our unpublished data).In this study, the membrane assembly of wild-type Lep and a Lep mutant (Lepqnv) [13] that inserts with an inverted topology compared to the wild-type protein has been studied in E. coli strains that are conditional for the expression of either Ff...

Assembly of several inner membrane proteins-leader peptidase (Lep), a Lep derivative (Lep-inv) that inserts with an inverted topology compared with the wild-type protein, the phage M13 procoat protein, and a procoat derivative (H1-procoat) with the hydrophobic core of the signal peptide replaced by a stretch from the first transmembrane segment in Lep-has been studied in vitro and in Escherichia coli strains that are conditional for the expression of either the 54 homologue (Ffh) or 4.5S RNA, which are the two components of the E. coli signal recognition particle (SRP), or SecE, an essential core component of the E. coli preprotein translocase. Membrane insertion has also been tested in a SecB null strain. Lep, Lep-inv, and H1-procoat require SRP for correct assembly into the inner membrane; in contrast, we find that wild-type procoat does not. Lep and, surprisingly, Lep-inv and H1-procoat fail to insert properly when SecE is depleted, whereas insertion of wild-type procoat is unaffected under these conditions. None of the proteins depend on SecB for assembly. These observations indicate that inner membrane proteins can assemble either by a mechanism in which SRP delivers the protein at the preprotein translocase or by what appears to be a direct integration into the lipid bilayer. The observed change in assembly mechanism when the hydrophobicity of the procoat signal peptide is increased demonstrates that the assembly of an inner membrane protein can be rerouted between different pathways.

Summary In Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e coupling efficiencies of the cbb3‐type

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (delta ctaDI, delta ctaDII), indicating that they are isoforms of subunit I of the aa3-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. This protohaem-containing oxidase, called cytochrome bb3, is the only quinol oxidase expressed under the conditions used. In a triple oxidase mutant (delta ctaDI, delta ctaDII, cyoB::KmR) an alternative cytochrome c oxidase has been characterized; this cbb3-type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.