Ross E. Dalbey

YidC mediates membrane protein insertion in bacteria.

The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulum using the signal recognition particle (SRP) and the SRP receptor, as well as the integral membrane Sec61 trimeric complex (composed of alpha, beta and gamma subunits). In bacteria, most proteins are inserted by a related pathway that includes the SRP homologue Ffh, the SRP receptor FtsY, and the SecYEG trimeric complex, where Y and E are related to the Sec61 alpha and gamma subunits, respectively. Proteins in bacteria that exhibit no dependence on the Sec translocase were previously thought to insert into the membrane directly without the aid of a protein machinery. Here we show that membrane insertion of two Sec-independent proteins requires YidC. YidC is essential for E. coli viability and homologues are present in mitochondria and chloroplasts. Depletion of YidC also interferes with insertion of Sec-dependent membrane proteins, but it has only a minor effect on the export of secretory proteins. These results provide evidence for an additional component of the translocation machinery that is specialized for the integration of membrane proteins.

Crystal structure of a bacterial signal peptidase in complex with a beta-lactam inhibitor

The signal peptidase (SPase) from Escherichia coli is a membrane-bound endopeptidase with two amino-terminal transmembrane segments and a carboxy-terminal catalytic region which resides in the periplasmic space. SPase functions to release proteins that have been translocated into the inner membrane from the cell interior, by cleaving off their signal peptides. We report here the X-ray crystal structure of a catalytically active soluble fragment of E. coli SPase (SPase Δ2–75),. We have determined this structure at 1.9 Å resolution in a complex with an inhibitor, a β-lactam (5S,6S penem),, which is covalently bound as an acyl-enzyme intermediate to the γ-oxygen of a serine residue at position 90, demonstrating that this residue acts as the nucleophile in the hydrolytic mechanism of signal-peptide cleavage. The structure is consistent with the use by SPase of Lys 145 as a general base in the activation of the nucleophilic Ser 90, explains the specificity requirement at the signal-peptide cleavage site, and reveals a large exposed hydrophobic surface which could be a site for an intimate association with the membrane. As enzymes that are essential for cell viability, bacterial SPases present a feasible antibacterial target: our determination of the SPase structure therefore provides a template for the rational design of antibiotic compounds.

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

Serine proteases comprise nearly one‐third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so‐called “classical” catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of “nonclassical” serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.

Signal peptidases in prokaryotes and eukaryotes - a new protease family

Signal peptidases remove targeting peptides from pre-proteins and play central roles in the secretory pathway, as well as in the delivery of proteins to the mitochondrial intermembrane space and to the lumen of thylakoids. The catalytic mechanism of pre-protein cleavage has long been an enigma, but recent data from site-directed mutagenesis and sequence alignment studies suggest that signal peptidases may constitute a new type of serine protease, mechanistically related to the beta-lactamases.

Escherichia coli YidC is a membrane insertase for Sec-independent proteins

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.

YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids

Like its mitochondrial homolog Oxa1p, the inner membrane protein YidC of Escherichia coli is involved in the integration of membrane proteins. We have analyzed individual insertion steps of the polytopic E. coli membrane protein MtlA targeted as ribosome‐nascent chain complexes to inner membrane vesicles. YidC can accommodate at least the first two transmembrane segments of MtlA at the protein lipid interface and retain them even though the length of the nascent chain would amply allow insertion into membrane lipids. An even longer insertion intermediate of MtlA is described that still has the first transmembrane helix bound to YidC while the third contacts SecE and YidC during integration. Our findings suggest that YidC forms a contiguous integration unit with the SecYE translocon and functions as an assembly site for polytopic membrane proteins mediating the formation of helix bundles prior to their release into the membrane lipids.

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 Bacterial Inner Membrane Proteins

Numerous membrane proteins form multisubunit protein complexes, which contain both integral and peripheral subunits, in addition to prosthetic groups. Bacterial membrane proteins are inserted into the inner membrane by the Sec translocase and YidC insertase. Their folding can be facilitated by YidC and the phospholipid phosphatidylethanolamine (PE). Glycine zippers and other motifs promote transmembrane-transmembrane (TM-TM) helix interactions that may lead to the formation of α-helical bundles of membrane proteins. During or after membrane insertion, the subunits of oligomeric membrane proteins must find each other to build the homo-oligomeric and the hetero-oligomeric membrane complexes. Although chaperones may function as assembly factors in the formation of the oligomer, many protein oligomers appear to fold and oligomerize spontaneously. Current studies show that most subunits of hetero-oligomers follow a sequential and ordered pathway to form the membrane protein complex. If the inserted protein is misfolded or the membrane protein is misassembled, quality control mechanisms exist that can degrade the proteins.

Protein translocation into and across the bacterial plasma membrane and the plant thylakoid membrane

Over the past decade, some familiar themes have emerged on how proteins are inserted into or translocated across the plant chloroplast thylakoid membrane and bacterial inner membranes. In the SecA and signal recognition particle (SRP) pathways, nucleotides and soluble factors are used to translocate proteins across the membrane bilayer in the unfolded state. However, the delta pH-dependent pathway in thylakoids uses a radically different mechanism: transport of proteins across the membrane is driven by the transmembrane pH gradient, and neither stromal factors nor nucleotide triphosphates are needed. In addition, this pathway, which requires the membrane-bound protein Hcf106, appears to translocate proteins in a tightly folded form. Recently, a similar pathway has been shown to operate in eubacteria, and several of its components have been identified.

The structure and mechanism of bacterial type I signal peptidases. A novel antibiotic target.

Type I signal peptidases are essential membrane-bound serine proteases that function to cleave the amino-terminal signal peptide extension from proteins that are translocated across biological membranes. The bacterial signal peptidases are unique serine proteases that utilize a Ser/Lys catalytic dyad mechanism in place of the classical Ser/His/Asp catalytic triad mechanism. They represent a potential novel antibiotic target at the bacterial membrane surface. This review will discuss the bacterial signal peptidases that have been characterized to date, as well as putative signal peptidase sequences that have been recognized via bacterial genome sequencing. We review the investigations into the mechanism of Escherichia coli and Bacillus subtilis signal peptidase, and discuss the results in light of the recent crystal structure of the E. coli signal peptidase in complex with a beta-lactam-type inhibitor. The proposed conserved structural features of Type I signal peptidases give additional insight into the mechanism of this unique enzyme.