The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations.

Cyanobacteria exhibit a complex form of membrane differentiation that sets them apart from most bacteria. Many processes take place in the plasma membrane, but photosynthetic light capture, electron transport and ATP synthesis take place in an abundant internal thylakoid membrane. This review considers how this system of subcellular compartmentalisation is maintained, and how proteins are directed towards the various subcompartments--specifically the plasma membrane, periplasm, thylakoid membrane and thylakoid lumen. The involvement of Sec-, Tat- and signal recognition particle- (SRP)-dependent protein targeting pathways is discussed, together with the possible involvement of a so-called 'spontaneous' pathway for the insertion of membrane proteins, previously characterised for chloroplast thylakoid membrane proteins. An intriguing aspect of cyanobacterial cell biology is that most contain only a single set of genes encoding Sec, Tat and SRP components, yet the proteomes of the plasma and thylakoid membranes are very different. The implications for protein sorting mechanisms are considered. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux.

The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic “SP motif” and a conserved “A motif” stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl− ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.

Sterols are an essential component of membranes in all eukaryotic cells and the precursor of multiple indispensable cellular metabolites. After endocytotic uptake, sterols are integrated into the lysosomal membrane by the Niemann-Pick type C (NPC) system before redistribution to other membranes. The process is driven by two proteins that, together, compose the NPC system: the lysosomal sterol shuttle protein NPC2 and the membrane protein NPC1 (named NCR1 in fungi), which integrates sterols into the lysosomal membrane. The Saccharomyces cerevisiae NPC system provides a compelling model to study the molecular mechanism of sterol integration into membranes and sterol homeostasis. This review summarizes recent advances in the field, and by interpreting available structural data, we propose a unifying conceptual model for sterol loading, transfer and transport by NPC proteins.

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Niemann Pick type C2 (NPC2) is a small sterol binding protein in the lumen of late endosomes and lysosomes. We showed recently that the yeast homologue of NPC2 together with its binding partner NCR1 mediates integration of ergosterol, the main sterol in yeast, into the vacuolar membrane. Here, we study the binding specificity and the molecular details of binding of a lipid to yeast NPC2. We find that NPC2 binds fluorescenceand spin-labeled analogues of phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), and sphingomyelin. Spectroscopic experiments show that NPC2 binds lipid monomers in solution but can also interact with lipid analogues in membranes. We further identify ergosterol, PC, and PI as endogenous NPC2 ligands. Using molecular dynamics simulations, we show that NPC2's binding pocket can adapt to the ligand shape and closes around bound ergosterol. Hydrophobic interactions stabilize the binding of ergosterol, but binding of phospholipids is additionally stabilized by electrostatic interactions at the mouth of the binding site. Our work identifies key residues that are important in stabilizing the binding of a phospholipid to yeast NPC2, thereby rationalizing future mutagenesis studies. Our results suggest that yeast NPC2 functions as a general "lipid solubilizer" and binds a variety of amphiphilic lipid ligands, possibly to prevent lipid micelle formation inside the vacuole. 56 oxysterols and, even more weakly, the hydrophobic amine 57 U18666A. 3,9−11 Yeast NPC2 has also been shown to bind 58 cholesterol, ergosterol, DHE, and U18666A, but also 59 edelfosine, a phosphatidylcholine-like lysophospholipid, sug-60 gesting that its binding spectrum is rather broad. 8 While the 61 overall structures of mammalian and yeast NPC2 are similar, 62 the binding site for yeast NPC2 is significantly larger and 63 more open. 8 This difference suggests that yeast NPC2 could 64 eventually bind ligands other than mammalian NPC2.

The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon α2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate substrates that contain surface domains with a level of hydrophobicity above a certain threshold.

The version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record.