YidC is a recently discovered bacterial membrane protein that is related to the mitochondrial Oxa1p and the Alb3 protein of chloroplasts. These proteins are required in the membrane integration process of newly synthesized proteins that do not require the classical Sec machinery. Here we demonstrate that YidC is sufficient for the membrane integration of a Sec-independent protein. Microgram amounts of the purified single-spanning Pf3 coat protein were efficiently inserted into proteoliposomes containing the purified YidC. A mutant Pf3 coat protein with an extended hydrophobic region was inserted independently of YidC into the membrane both in vivo and in vitro, but its insertion was accelerated by YidC. These results show that YidC can function separately from the Sec translocase to integrate membrane proteins into the lipid bilayer.

Bacterial integral inner membrane proteins are either translocated across the lipid bilayer using an energydriven enzyme, such as the Sec translocase, or they might interact directly with the membrane due to hydrophobic forces. We report that the single-spanning Pf3 coat protein is spontaneously inserted into the membrane of Escherichia coli and requires the electrical component of the membrane potential (∆Ψ) to translocate its N-terminal region. This results in a final N out C in orientation of the protein in the cytoplasmic membrane, due the potential-driven translocation of the aspartyl residue at position 18 in the hydrophilic N-terminal tail. Uncharged protein tails are only translocated when the hydrophobic transmembrane region of the protein has been extended. An extended transmembrane anchor allows membrane insertion in the absence of an electrochemical membrane potential, but also causes the loss of a strict determination of the topology.

The coat protein of Pseudomonas aeruginosa phage Pf3 is transiently inserted into the bacterial inner membrane with a single transmembrane anchor sequence in the NoutCin orientation. The N‐terminal sequence immediately flanking the membrane anchor contains one negatively charged residue, whereas the C‐terminal hydrophilic segment has two positively charged residues. To investigate how the orientation of this protein is achieved, the three flanking charged amino acid residues were altered. Membrane insertion was analyzed in vivo using the accessibility to externally added protease and in vitro by testing the insertion into inverted Escherichia coli membrane vesicles. In both systems, the orientation of the protein was completely reversed for the oppositely charged mutant coat protein (RD mutant). In addition, we show in vivo that the electrochemical membrane potential is necessary for the translocation of both the wild‐type and the mutant Pf3 coat proteins, suggesting that membrane insertion is driven by electrophoretic forces.

The YidC protein of Escherichia coli is required for inserting Sec-independent membrane proteins and has a supportive role for the insertion of Sec-dependent proteins into the membrane bilayer. Because a portion of YidC copurifies with the Sec translocase, this interaction might be necessary to assist in the membrane insertion of Sec-dependent proteins. This study describes a deletion analysis that investigates which parts of YidC are required for its interaction with the SecDF complex of the Sec translocase and for the function of YidC as an insertase for the Sec-dependent membrane proteins. The results suggest that the first periplasmic region, which includes residues 24-346, is required for the interaction of YidC with the Sec translocase, in particular with the SecF protein. Further studies showed that residues 215-265 of YidC are sufficient for SecF binding. Surprisingly, the interaction of YidC with SecF is not critical for cell viability as YidC, lacking residues 24-264, was fully functional to support the growth of E. coli. It was also observed that this YidC mutant was fully functional to insert the Sec-dependent subunit A of the F(1)F(o) ATP synthase and an M13 procoat derivative, as well as the Sec-independent M13 procoat protein and subunit C of the ATP synthase. Only when additional residues of the periplasmic region were deleted (265-346) was the membrane insertase function of YidC inhibited.

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SummaryThe marine Gram-negative bacteria Rhodopirellula baltica and Oceanicaulis alexandrii have, in contrast to Escherichia coli, membrane insertases with extended positively charged C-terminal regions similar to the YidC homologues in mitochondria and Gram-positive bacteria. We have found that chimeric forms of E. coli YidC fused to the C-terminal YidC regions from the marine bacteria mediate binding of YidC to ribosomes and therefore may have a functional role for targeting a nascent protein to the membrane. Here, we show in E. coli that an extended C-terminal region of YidC can compensate for a loss of SRP-receptor function in vivo. Furthermore, the enhanced affinity of the ribosome to the chimeric YidC allows the isolation of a ribosome nascent chain complex together with the C-terminally elongated YidC chimera. This complex was visualized at 8.6 Å by cryo-electron microscopy and shows a close contact of the ribosome and a YidC monomer.

SummaryThe insertion of proteins into the prokaryotic plasma membrane is catalyzed by translocases and insertases. On one hand, the Sec translocase operates as a transmembrane channel that can open laterally to first bind and then release the hydrophobic segments of a substrate protein into the lipid bilayer. On the other hand, YidC insertases interact with their substrates in a groove-like structure at an amphiphilic protein-lipid interface thus allowing the transmembrane segments of the substrate to slide into the lipid bilayer. The recently published highresolution structures of YidC provide new mechanistic insights of how transmembrane proteins achieve the transition from an aqueous environment in the cytoplasm to the hydrophobic lipid bilayer environment of the membrane.

The KdpD protein is a K ؉ sensor kinase located in the cytoplasmic membrane of Escherichia coli. It contains four transmembrane stretches and two short periplasmic loops of 4 and 10 amino acid residues, respectively. To determine which part of KdpD functions as a K ؉ sensor, genetic variants were constructed with truncations or altered arrangements of the transmembrane segments. All KdpD constructs were tested by complementation of an E. coli kdpD deletion strain for their ability to grow at a K ؉ concentration of 0.1 mM in the medium. A soluble protein composed of the C-terminal cytoplasmic domain was able to complement the kdpD deletion strain. In addition, analysis of the ␤-galactosidase activity of an E. coli strain which carries a transcriptional fusion of the upstream region of the kdpFABC operon and a promoterless lacZ gene revealed that this soluble KdpD mutant responds to changes in the K ؉ concentration in the extracellular medium. The results suggest that the sensing and response functions are both located in the C-terminal domain and might be modulated by the N-terminal domain as well as by membrane anchoring.