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.

The chaperone SecB keeps precursor proteins in a translocation-competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA is thought to recognize SecB via its carboxy-terminus. To determine the minimal requirement for a SecB-binding site, fusion proteins were created between glutathione-S-transferase and different parts of the carboxy-terminus of SecA and analysed for SecB binding. A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB-precursor protein complex. SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB. Heterodimers of wild-type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy-termini of the SecA dimer are needed to form a genuine SecBbinding site. SecB is released from the translocase at a very early stage in protein translocation when the membrane-bound SecA binds ATP to initiate translocation. It is concluded that the SecB-binding site on SecA is confined to the extreme carboxy-terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.

of a translocating polypeptide chain across the membrane Arnold J.M.Driessen 2 (Schiebel et al., 1991) by a two-stroke reaction (van der

The ABC transporter Mdl1p, a structural and functional homologue of the transporter associated with antigen processing (TAP) plays an important role in intracellular peptide transport from the mitochondrial matrix of Saccharomyces cerevisiae. To characterize the ATP hydrolysis cycle of Mdl1p, the nucleotide-binding domain (NBD) was overexpressed in Escherichia coli and purified to homogeneity. The isolated NBD was active in ATP binding and hydrolysis with a turnover of 25 ATP per minute and a K m of 0.6 mM and did not show cooperativity in ATPase activity. However, the ATPase activity was non-linearly dependent on protein concentration (Hill coefficient of 1. ATP-binding cassette (ABC)1 transporters comprise a large family of membrane proteins that catalyze the active transfer of a variety of solutes across biological membranes (1). The function of ABC transporters is central to various human pathologies such as cystic fibrosis, adrenoleukodystrophy, retinal dystrophies, and multidrug resistance. The transporter associated with antigen processing (TAP) is an ABC transporter in vertebrates, which translocates peptides from the cytosol into the ER and performs a key function in the antigen presentation and adaptive immune response (2). Recently, a close homologue, Mdl1p (multidrug resistance like), localized in the inner mitochondrial membrane of Saccharomyces cerevisiae, has been identified as an intracellular peptide transporter (3). This transporter exports peptides derived from the degradation of non-assembled membrane proteins. These peptides are generated by ATP-dependent m-AAA (matrix-oriented ATPases associated with a variety of cellular activities) proteases, which mediate the degradation and turnover of inner mitochondrial membrane proteins and short-lived regulatory proteins in an ubiquitin/proteasome-independent manner (4). Protein fragments with a length of 6 -21 amino acids are released by Mdl1p into the intermembrane space (3).Half-size ABC transporters, like the heterodimeric TAP and homodimeric Mdl1p, have a common molecular architecture consisting of two polytopic transmembrane domains (TMD) and two nucleotide-binding domains (NBD). The transmembrane domains interact with the substrates and form the substrate translocation pore across the membrane. The TMDs generally share little homology (5), probably caused by the broad substrate spectrum of the ABC transporter family. Binding and hydrolysis of nucleotides drive the transport process by transducing conformational changes from the NBDs to the TMDs. The similarity of different NBDs is significantly higher compared with the TMDs, suggesting that even in transporters of unrelated function the structure and function of the NBDs be highly conserved. Each NBD contains a highly conserved Walker A and Walker B motif (6) characteristic of ATP-binding P-loop proteins, as well as the C-loop motif (LSGGQ) unique to ABC proteins, which is also known as the ABC signature motif. The crystal structures of bacterial ABC transporters (e.g. MsbA, BtuCD) and of is...

Preprotein translocation in Escherichia coli is mediated by translocase, a multimeric membrane protein complex with SecA as the peripheral ATPase and SecYEG as the translocation pore. Unique cysteines were introduced into transmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible sites of interaction between the integral membrane subunits. The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expression vector and functionally overexpressed. Oxidation of the single-Cys pairs revealed periodic contacts between SecY and SecE that are confined to a specific alpha-helical face of TMS 2 and 3, respectively. A Cys at the opposite alpha-helical face of TMS 3 of SecE was found to interact with a neighboring SecE molecule. Formation of this SecE dimer did not affect the high-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked preprotein translocation and thus uncouples the SecA ATPase activity from translocation. Conditions that prevent membrane deinsertion of SecA markedly stimulated the interhelical contact between the SecE molecules. The latter demonstrates a SecA-mediated modulation of the protein translocation channel that is sensed by SecE.