Organization of the Escherichia coli aerobic enzyme complexes of oxidative phosphorylation in dynamic domains within the cytoplasmic membrane

MicrobiologyOpen

Heiko Erhardt²  Felix Dempwolff¹  Moritz Pfreundschuh²  Marc Riehle²  Caspar Schäfer²  Thomas Pohl²  Peter Graumann¹ 

[1] Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität, Freiburg, Germany;Institut für Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany

关键词: Escherichia coli;    FRAP;    in vivo fluorescence microsocopy;    membrane protein organization;    oxidative phosphorylation;    TIRF microscopy;   

DOI  :  10.1002/mbo3.163

来源: Wiley

【 摘 要 】

Abstract

The Escherichia coli cytoplasmic membrane contains the enzyme complexes of oxidative phosphorylation (OXPHOS). Not much is known about their supramolecular organization and their dynamics within the membrane in this model organism. In mitochondria and other bacteria, it was demonstrated by nondenaturing electrophoretic methods and electron microscopy that the OXPHOS complexes are organized in so-called supercomplexes, stable assemblies with a defined number of the individual enzyme complexes. To investigate the organization of the E. coli enzyme complexes of aerobic OXPHOS in vivo, we established fluorescent protein fusions of the NADH:ubiquinone oxidoreductase, the succinate:ubiquinone oxidoreductase, the cytochrome bd-I, and the cytochrome bo₃ terminal oxidases, and the F_oF₁ ATP-synthase. The fusions were integrated in the chromosome to prevent artifacts caused by protein overproduction. Biochemical analysis revealed that all modified complexes were fully assembled, active, and stable. The distribution of the OXPHOS complexes in living cells was determined using total internal reflection fluorescence microscopy. The dynamics within the membrane were detected by fluorescence recovery after photobleaching. All aerobic OXPHOS complexes showed an uneven distribution in large mobile patches within the E. coli cytoplasmic membrane. It is discussed whether the individual OXPHOS complexes are organized as clustered individual complexes, here called “segrazones.”

【 授权许可】

CC BY   
© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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