Assembly surprise for membrane proteins

Membrane-spanning proteins have many crucial roles in the cell. New findings challenge our current understanding of the route by which such proteins are inserted into the membranes of animal cells

A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site specific crosslinking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild-type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild-type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.</p

Structures of the stator complex that drives rotation of the bacterial flagellum

The bacterial flagellum is the prototypical protein nanomachine and comprises a rotating helical propeller attached to a membrane-embedded motor complex. The motor consists of a central rotor surrounded by stator units that couple ion flow across the cytoplasmic membrane to generate torque. Here, we present the structures of the stator complexes from Clostridium sporogenes, Bacillus subtilis and Vibrio mimicus, allowing interpretation of the extensive body of data on stator mechanism. The structures reveal an unexpected asymmetric A5B2 subunit assembly where the five A subunits enclose the two B subunits. Comparison to structures of other ion-driven motors indicates that this A5B2 architecture is fundamental to bacterial systems that couple energy from ion flow to generate mechanical work at a distance and suggests that such events involve rotation in the motor structures

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Molecular characterisation of bacterial proteins that interact with sulfur or nitrogen compounds

Many bacteria use inorganic nitrogen and sulfur compounds for energy metabolism. These compounds are often toxic and so bacteria must adapt to survive their deleterious effects. Bacteria use specific proteins in order to metabolise, sense and detoxify these compounds. In this thesis protein interactions with inorganic nitrogen and sulfur compounds are examined at the mechanistic level. Intermediates in the Sox sulfur oxidation pathway are covalently attached to a cysteine on the swinging arm of the substrate carrier protein SoxYZ. An interaction between the Sox pathway enzyme SoxB and the carrier protein SoxYZ is demonstrated. A crystal structure of a trapped SoxB-SoxYZ complex at 3.3 &Aring; resolution identifies two sites of interaction, one between the SoxYZ carrier arm and the SoxB active site channel and the other at a patch distal to the active site. The presence of a distal interaction site suggests a mechanism for promiscuous specificity in the protein-protein interactions of the Sox pathway. Using biophysical methods it is shown that SoxB distinguishes between the substrate and product forms of the carrier protein through differences in interaction kinetics and that the carrier arm-bound substrate group is able to out-compete the adjacent C-terminal carboxylate for binding to the SoxB active site. The thiosulfate dehydrogenase TsdA has an unusual His/Cys coordinated heme. TsdA catalyses oxidative conjugation of two thiosulfate molecules to form tetrathionate. Mass spectrometry and UV/visible spectroscopy are used to identify an S-thiosulfonate reaction intermediate which is covalently attached to the cysteine heme ligand. A catalytic mechanism for TsdA is proposed using a crystal structure of TsdA at 1.3 &Aring; resolution alongside site-directed mutagenesis of active site residues. Nitric oxide is produced by the mammalian immune response to kill bacterial pathogens. Part of the killing mechanism occurs through the reaction of nitric oxide with protein-bound iron-sulfur clusters. However, the same type of reaction is also exploited by nitric oxide-sensing bacterial proteins. An infrared spectroscopy approach is developed to detect the products of iron-sulfur protein nitrosylation. Using this methodology it is shown that the presence of trace O2 strongly impacts which products are formed in these nitrosylation reactions. These observations are of physiological relevance because bacteria are often exposed to NO under aerobic conditions during an immune response.This file is not currently available in OR

Pathfinders and trailblazers:a prokaryotic targeting system for transport of folded proteins

The twin-arginine (Tat) protein translocase is a highly unusual protein transport machine that is dedicated to the movement of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In this minireview, we describe our current knowledge of the Tat system, paying particular attention to the function of the TatA protein and to the often overlooked step of signal peptide cleavage