Mechanism of Protein Export in E. coli
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Mechanism of Protein Export in E. coli

The translocation of proteins from one compartment to another is an essential feature of cellular life. The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment.  Gram-negative bacteria secrete a wide range of proteins whose functions include biogenesis of organelles, such as Pili and flagella; nutrient acquisition; virulence; and efflux of drugs and other toxins. Export of these proteins to the bacterial surface involves transport across the IM (Inner Membrane), Periplasm, and OM (Outer Membrane) of the cell envelope. All proteins in Escherichia coli are initially synthesized in the cytoplasm then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. Two distinct pathways exist for the export of proteins across the cytoplasmic membrane of Escherichia coli. These are Sec dependent and Sec independent pathways. The bulk of protein translocation across the cytoplasmic membrane occurs by the general Sec (Secretory) pathway. However, a fundamentally different mechanism for periplasmic protein localization was recently discovered, first in the thylakoid membranes of photosynthetic organisms and subsequently in Bacteria and Archaea. In the latter, this mechanism has been named the Tat (Twin-Arginine Translocation) pathway because of the signature Arg-Arg motif found near the N-terminus of the leader peptide of proteins that are engaged in this mode of export from the cytoplasm. Besides the Sec and Tat pathways, protein targeting to the Escherichia coli inner membrane can also occur via another pathway named SRP (Signal Recognition Particle) Pathway (Ref.1 & 2).

The Sec-dependent protein export pathway of Escherichia coli is responsible for translocation of Secretory proteins, in a (partially) unfolded conformation through a channel formed by the membrane-embedded SecYEG protein complex, across the inner membrane to final destinations in the Periplasm or Outer membrane. Secretory proteins, also called Preproteins, are synthesized with a cleavable amino terminal signal sequence that functions both to slow folding of the preprotein and to aid in recognition of the secretory protein by export factors. Export of secretory proteins is dependent on interaction with SecB, a cytoplasmic Chaperone that maintains the Preprotein in a loosely folded conformation competent for translocation. Both SecB and the Preprotein provide binding sites for SecA, a peripheral membrane ATPase. SecA targets the preprotein to the membranous translocase complex composed of SecY, SecE, SecG, SecD, SecF, YidC and YajC. Formation of the complete translocase complex promotes an ATP binding and hydrolysis cycle by SecA that results in segmental translocation of the secretory protein across the membrane. Translocation of the remaining protein through the translocase channel is dependent on the proton motive force. As soon as the C-terminus of the signal peptide reaches the periplasmic surface of the membrane, it is cut off by the Signal Peptidases I and II (LepB and LSPA) (Ref. 3 & 4).

Besides Sec Pathway, cotranslational protein export and integration of membrane proteins into the cytoplasmic membrane is also initiated by the bacterial SRP pathway. Bacterial SRP is smaller than the eukaryotic SRP, which mediates protein translocation into the endoplasmic reticulum. The SRP, composed of Ffh and 4.5S RNA, arrests the synthesis of the nascent preprotein at the ribosome by binding to the signal peptide. The complex then binds to the SRP-receptor, which in E.coli consists of only one peripheral membrane protein, FtsY. After GTP-hydrolysis, the ribosomes-preprotein complex is transferred to the translocase SecYEG, independent of SecA. In bacteria, the Sec and SRP pathways converge at the SecYEG translocon, which is intimately involved in the insertion of plasma membrane proteins (Ref.5 & 6).

Apart from the Sec system, an alternative system for the transfer of proteins over the inner membrane has been described, i.e. the Tat System. In stark contrast to the Sec-dependent threading of unstructured substrates, the Tat pathway has the unique ability to transport  proteins that have attained a substantial degree of tertiary or even quaternary structure in the cytoplasm prior to membrane translocation. The bacterial Tat system is structurally and mechanistically related to the DpH-dependent thylakoid import pathway of chloroplasts. Remarkably, the Tat machinery uses the energy of the proton motive force to translocate folded proteins across the ionically sealed cytoplasmic membrane. In Escherichia coli the integral membrane proteins TatA, TatB, TatC and TatE have been shown to be components of the Tat pathway. To date, no precise function for any of these Tat components is known. TatA and TatE are around 60% identical and can at least in part substitute for each other. TatB, sharing only 25% of sequence homology with TatA/TatE, is functionally distinct. TatA, TatB, and TatE are sequence-related proteins that are each predicted to comprise a transmembrane N-terminal a helix followed by an amphipathic a helix at the cytoplasmic side of the membrane. TatA and TatE have overlapping functions on the  Tat pathway while TatB is an essential Tat component with a distinct role in protein export. TatE may be a cryptic gene duplication of TatA. TatC is also an essential component of the Tat system and is predicted to be a polytopic membrane protein with six transmembrane helices. In E. coli, the genes encoding TatA, TatB, and TatC are arranged as an operon with a fourth gene, TatD, that has no discernible role in protein export. The Tat translocon must possess an amazing structural flexibility, as Tat substrates can vary dramatically in size, surface properties, and three-dimensional structure and most bacterial genomes typically encode numerous Tat substrates. Following preprotein folding in the cytoplasm, Tat substrates (S) are recognized by the translocon in a process that involves TatB, TatC, and the leader peptide. Preprotein binding to the TatB/TatC complex triggers assembly of multiple TatA monomers that likely form a translocation pore through which a folded substrate is able to pass. Following successful transport, the Tat-ABC complex disassembles. In many instances, substrates traverse the Tat pathway because they are inherently incompatible with the Sec machinery. This can occur if the substrate simply folds too rapidly to remain Sec export competent or if the substrate is unable to reach its native conformation in the compartment to which it is targeted. For instance, some transported proteins need to incorporate cofactors or assemble subunits in the cytoplasm prior to export. Others benefit from prefolding in the cytoplasmic compartment, which can provide a more favorable folding environment relative to certain extracytoplasmic locations (Ref.7 & 8).

A combination of genomic analysis and physiological characterization of Tat mutants has started to reveal the wide diversity of substrates transported by the Tat system in different prokaryotes. The mechanism of Tattransport is especially intriguing since Tat is the only general protein translocation system known to transport folded proteins across an ionically tight membrane. How the Tat system is able to achieve protein transport without rendering the membrane freely permeable to protons and other ions and how the energy of the transmembrane proton electrochemical gradient is transduced to effect unidirectional transport are challenges for the future. Addressing these challenges will require detailed structural data and new approaches to probe protein conformational changes and protein– protein dynamics in the translocation cycle (Ref.1 & 9).