Subunit a of cytochrome o oxidase requires both YidC and SecYEG for membrane insertion

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that facilitate the insertion of membrane proteins. Depletion of YidC in E. coli leads to a specific defect in the functional assembly of major energy transducing complexes such as the F1F0 ATPase and cytochrome bo(3) oxidase. Here we report on the in vitro reconstitution of the membrane insertion of the CyoA subunit of cytochrome bo(3) oxidase. Efficient insertion of in vitro synthesized pre-CyoA into proteoliposomes requires YidC, SecYEG, and SecA and occurs independently of the proton motive force. These data demonstrate that pre-CyoA is a substrate of a novel pathway that involves both SecYEG and YidC.</p

Stepwise evolution of the Sec machinery in Proteobacteria

The Sec machinery facilitates the translocation of proteins across and into biological membranes. In several of the Proteobacteria, this machinery contains accessory features that are not present in any other bacterial division. The genomic distribution of these features in the context of bacterial phylogeny suggests that the Sec machinery has evolved in discrete steps. The canonical Sec machinery was initially supplemented with SecB; subsequently, SecE was extended with two transmembrane segments and, finally, SecM was introduced. The Sec machinery of Escherichia coli and other Enterobacteriales represents the end product of this stepwise evolution.</p

Direct demonstration of ATP-dependent release of SecA from a translocating preprotein by surface plasmon resonance

Translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds with high affinity to the membrane-embedded protein-conducting SecYEG complex and serves as both a receptor for secretory proteins and as an ATP-driven molecular motor. Cycles of ATP binding and hydrolysis by SecA drive the progressive movement of the preprotein across the membrane. Surface plasmon resonance allows an online monitoring of protein interactions. Here we report on the kinetic analysis of the interaction between SecA and the membrane-embedded SecYEG complex. Immobilization of membrane vesicles containing overproduced SecYEG on the Biacore Pioneer L1 chip allows the detection of high affinity SecA binding to the SecYEG complex and online monitoring of the translocation of the secretory protein proOmpA. SecA binds tightly to the SecYEG . proOmpA complex and is released only upon ATP hydrolysis. The results provide direct evidence for a model in which SecA cycles at the SecYEG complex during translocation

Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 100 mu M verapamil. Quantification of intracellular cadmium concentration by atomic absorption spectrometry showed a reduced cadmium accumulation in cells expressing the MDR genes. Inside-out membrane vesicles of L. lactis overexpressing lmrA displayed an ATP-dependent Cd-109(2+) uptake that was stimulated by glutathione. An evolutionary model is discussed in which MDR proteins have evolved independently from an ancestor protein displaying both organic xenobiotic- and divalent metal-extrusion abilities

ENERGY-TRANSDUCING PROPERTIES OF PRIMARY PROTON PUMPS RECONSTITUTED INTO ARCHAEAL BIPOLAR LIPID VESICLES

Archaeal lipids differ considerably from eubacterial and eukaryotic lipids in their structure and physical properties. From the membranes of the extreme thermophilic archaea Sulfolobus acidocaldarius a tetraether lipid fraction was isolated, which can form closed and stable monolayer liposomes in aqueous media. The function of three different primary proton pumps originating from archaeal, bacterial and eukaryotic lipid sources have been studied after reconstitution in these liposomes: bacteriorhodopsin from the archaea Halobacterium halobium; cytochrome-c oxidase from the thermophilic bacterium Bacillus stearothermophilus and cytochrome-c oxidase from beef heart mitochondria. Liposomes composed of tetraether lipids form a competent matrix for all three exogenous proton pumps. Bacteriorhodopsin was inserted inside-out in these liposomes, as normally observed in bilayer-forming lipid. The activities of the two oxidases were inhibited at high tetraether-lipid concentration, probably due to the low fluidity of these membranes. Only bacteriorhodopsin, which originates from diether archaeal lipids is fully functional in the tetraether membranes