Dual Topology of the Escherichia coli TatA Protein

The Tat (twin arginine translocation) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The integral membrane proteins TatA, TatB, and TatC are essential components of the Tat pathway. TatA forms high order oligomers and is thought to constitute the protein-translocating unit of the Tat system. Cysteine scanning mutagenesis was used to systematically investigate the functional importance of residues in the essential N-terminal transmembrane and amphipathic helices of Escherichia coli TatA. Cysteine substitutions of most residues in the amphipathic helix, including all the residues on the hydrophobic face of the helix, severely compromise Tat function. Glutamine 8 was identified as the only residue in the transmembrane helix that is critical for TatA function. The cysteine variants in the transmembrane helix were used in disulfide mapping experiments to probe the oligomeric arrangement of TatA protomers within the larger TatA complex. Residues in the center of the transmembrane helix (including residues 10 -16) show a distinct pattern of cross-linking indicating that this region of the protein forms well defined interactions with other protomers. At least two interacting faces were detected. The results of our TatA studies are compared with analogous data for the homologous, but functionally distinct, TatB protein. This comparison reveals that it is only in TatA that the amphipathic helix is sensitive to amino acid substitutions. The TatA amphipathic helix may play a role in forming and controlling the path of substrate movement across the membrane.

Section: Discussionmentioning

confidence: 89%

mentioning

confidence: 99%

Cysteine Scanning Mutagenesis and Disulfide Mapping Studies of the TatA Component of the Bacterial Twin Arginine Translocase

Greene

Porcelli

Buchanan

et al. 2007

“…A population of TatA is certainly present at the cytoplasmic membrane and has been analyzed in several topology studies (35)(36)(37). Under normal growth conditions, the N and the C termini of TatA appear to be cytoplasmic (35,37) and possibly a small portion of TatA is exposed to the periplasm at its hinge region (37).…”

Section: Discussionmentioning

confidence: 99%

Recombinant Expression of tatABC and tatAC Results in the Formation of Interacting Cytoplasmic TatA Tubes in Escherichia coli

Berthelmann

Mehner

Richter

et al. 2008

The twin-arginine translocation (Tat) system of bacteria and plant plastids serves to translocate folded proteins across energized biological membranes. In Escherichia coli, the three components TatA, TatB, and TatC mediate this membrane passage. Here we demonstrate that TatA can assemble to form clusters of tube-like structures in vivo. While the presence of TatC is essential for their formation, TatB is not required. The TatA tubes have uniform outer and inner diameters of about 11.5 nm and 6.7 nm, respectively. They align to form a crystalline-like structure in which each tube is surrounded by six TatA tubes. The tube structures become easily detectable even at only a 15-fold overexpression of the tatABC genes. The TatA tubes could also be visualized by fluorescence when untagged TatA was mixed with low amounts of TatA-GFP. The structures were often found in contact with the cell poles. Because TatC is most likely polar in E. coli, as demonstrated by a RR-dependent targeting of translocation-incompatible Tat substrates to the cell poles, and because TatC is required for the formation of aligned TatA tubes, it is proposed that the TatA tubes are initiated at polarly localized TatC.Folded proteins can be transported across membranes by the twin-arginine translocation (Tat) 3 system (1). The mechanism of this transport is unknown but must be quite unusual, because the transported proteins can be even larger in diameter than the membrane they are transported across (2). The minimal Tat translocon is composed of two components, TatA and TatC (3, 4). Proteobacteria and thylakoids require a third component for efficient translocation, TatB, which associates with TatC to form a TatBC complex (1). Because Tat substrates bind tightly only to TatBC complexes, and because a covalent cross-link to TatC still allows translocation, it is believed that TatC is the motor for the translocation process (5). TatA, however, also plays an essential role, most likely by allowing the membrane passage of the substrate, which is moved by TatC across the membrane (6). TatA is also the most abundant Tat component in Escherichia coli (7). In the chloroplast system, the association of TatA with the TatBC complex occurs strictly after substrate binding (6), whereas TatA of Gram-positive bacteria has been detected inside the cytoplasm, where it has a high affinity for Tat substrates that might be targeted by TatA to the membranes (8 -10). TatA of the E. coli Tat system is known to form homooligomeric complexes (11). It has been purified from detergent-solubilized membrane fractions of strains overexpressing the tatABC genes (12). The TatA complexes identified in that study varied in diameter and formed a ladder in blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses. The variable sizes of these TatA complexes suggest the existence of some higher order homooligomeric TatA structure, which might have been disrupted by detergent treatment. We therefore analyzed strains overexpressing tatABC, tatAB, tatAC, or tatA by transmission...

“…Proteins with dual topology are known to have weak signals (net differences in charged amino acids next to membrane boundaries involved in determining membrane topology) adjacent to transmembrane domains (30). Interestingly, for at least one known bacterial protein, the relative proportions of the opposing topological orientations depend on growth conditions (10,33), raising the possibilities that (i) the ratio between the two orientations may not always be unity, and (ii) post/co-translational modification(s) may alter the net charges at critical membrane boundary regions of the protein sequence so as to control the relative proportions of the two alternative orientations depending on the prevailing activity of protein modifying enzymes. If these considerations can be extrapolated to DGAT1, they may explain our previous observations that the relative amounts of overt and latent DGAT activity in the ER of rat liver (which will be dependent on the relative distribution of DGAT1 activity as this accounts for the majority of the overt DGAT activity) are capable of modulation by physiological state and by hypolipidemic drug treatment (6).…”

Section: Discussionmentioning

confidence: 99%

Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

Wurie

Buckett

Zammit

2011

Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17-21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1 ؊/؊ mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology.Hypertriglyceridemia is a key biomarker for the metabolic/ insulin resistance syndrome and for associated morbidities, including type-2 diabetes and cardiovascular disease (1). Similarly, excessive accumulation of triglycerides in cytoplasmic lipid droplets results in hepatic steatosis, now recognized as being associated, possibly causatively, with whole body insulin resistance, and which may progress to nonalcoholic fatty liver or steatohepatitis (2, 3). Fasting hypertriglyceridemia is primarily due to the hypersecretion of triglyceride (TAG) 3 by the liver, within very low density lipoproteins (VLDL). Therefore, an understanding of the enzymology involved in triglyceride synthesis, remodeling, storage, and assembly into secreted VLDL is essential for the design of pharmacological strategies aimed at managing dyslipidaemia without the exacerbation of hepatic steatosis, and vice versa.Diacylglycerol acyltransferases (DGATs) catalyze the final reaction of TAG synthesis. Two distinct gene products, DGAT1 and DGAT2, that catalyze most of tissue TAG synthesis have been described (4, 5) but remain relatively poorly characterized, and their respective, nonredundant functions are still to be elucidated. In the present study, we have used two specific inhibitors (which belong to different chemical classes of compounds) of human DGAT1, in combination with the selective permeabilization of the plasma membrane and the ER membrane of whole hepatocytes, to study the sidednes...