Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase*

Brandt, Karsten; Maiwald, Sarah; Herkenhoff-Hesselmann, Brigitte; Gnirß, Kerstin; Greie, Jörg-Christian; Dunn, Stanley D.; Deckers-Hebestreit, Gabriele

doi:10.1074/jbc.m113.465633

Cited by 22 publications

(16 citation statements)

References 69 publications

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“…Inverted membrane vesicles were prepared as previously described (Brandt et al, 2013) using 850 ml of cell cultures grown under conditions stated above. After harvest, cell pellets were resuspended in 25 ml 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10% (v/v) glycerol and disrupted in the presence of 10 µg/ml DNaseI (Sigma) with a constant cell disruptor system (Daventry) at 4°C and 1.35 kbar.…”

Section: Membrane Vesicles and Dccd-sensitive Atpase Activitymentioning

confidence: 99%

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

Gohrbandt

Lipski

Grimshaw

et al. 2019

Preprint

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Important physicochemical properties of cell membranes such as fluidity sensitively depend on fluctuating environmental factors including temperature, pH or diet. To counteract these disturbances, living cells universally adapt their lipid composition in return. In contrast to eukaryotic cells, bacteria tolerate surprisingly drastic changes in their lipid composition while retaining viability, thus making them a more tractable model to study this process. Using the model organisms Escherichia coli and Bacillus subtilis, which regulate their membrane fluidity with different fatty acid types, we show here that inadequate membrane fluidity interferes with essential cellular processes such as morphogenesis and maintenance of membrane potential, and triggers large-scale lipid phase separation that drives protein segregation into the fluid phase. These findings illustrate why lipid homeostasis is such a critical cellular process. Finally, our results provide direct in vivo support for current in vitro and in silico models regarding lipid phase separation and associated protein segregation.Keywords: Lipid phase separation, lipid domains, protein partitioning, membrane fluidity, homeoviscous adaptation, Escherichia coli, Bacillus subtilis, WALP, FOF1 ATP synthase liquid-disordered phase characterized by low packing density and high diffusion rates that forms the regular state of biological membranes, (ii) the more ordered, cholesterol/hopanoid-dependent liquidordered phase found in biological membranes in form of lipid rafts, and (iii) the gel phase characterized by dense lipid packing with little lateral or rotational diffusion, which is generally assumed to be absent in biologically active membranes (Schmid, 2017;Veatch, 2007). In fact, the temperature associated with gel phase formation has been postulated to define the lower end of the temperature range able to support vital cell functions (Burns et al., 2017;Drobnis et al., 1993;Ghetler et al., 2005). At last, the lipid phases can co-exist, resulting in separated membrane areas exhibiting distinctly different composition and characteristics (Baumgart et al., 2007;Elson et al., 2010;Heberle and Feigenson, 2011). This principal mechanism of lipid domain formation is best studied in the context of lipid rafts (Lingwood and Simons, 2010).While in vitro and in silico approaches with simplified lipid models have provided detailed insights into the complex physicochemical behavior of lipid bilayers, testing the formed hypotheses and models in the context of protein-rich biological membranes is now crucial. Bacteria tolerate surprisingly drastic changes in their lipid composition and only possess one or two membrane layers as part of their cell envelope. Consequently, bacteria are both a suitable and a more tractable model to study the fundamental biological process linked to membrane fluidity and phase separation in vivo.We analyzed the biological importance of membrane homeoviscous adaptation in Escherichia coli (phylum Proteobacteria) and Bacillus subtilis (phylum Firmic...

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Section: Membrane Vesicles and Dccd-sensitive Atpase Activitymentioning

confidence: 99%

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

Gohrbandt

Lipski

Grimshaw

et al. 2019

Preprint

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“…In the case of b-␣ cross-linking, as a side reaction, an ␣-␣ cross-linking was observed, which was analyzed by Brandt et al (48) to be an interenzyme cross-link reaction. Substitution of a cysteine residue in ␣ resulted in incorporation of three thiol groups into F O F 1 .…”

Section: Presence Of F O F 1 Subunits In Membranes Ofmentioning

confidence: 99%

Subunit δ Is the Key Player for Assembly of the H+-translocating Unit of Escherichia coli FOF1 ATP Synthase

Hilbers¹,

Eggers²,

Pradela

et al. 2013

Journal of Biological Chemistry

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“…After induction of AOX expression for 1 h with 0.3 mM isopropyl-b-D-1-thiogalactopyranoside, cells were harvested, frozen in liquid nitrogen, and stored at 280°C. Inverted membrane vesicles were prepared at 4°C according to Brandt et al (2013). Isolated membranes were resuspended in 300 mL of TMG buffer (50 mM TrisHCl [pH 7.5], 10 mM MgCl 2 , and 10% [v/v] glycerol), and the concentration of the membrane protein was determined using a small-scale approach of the bicinchoninic acid assay according to the protocol of the supplier (Pierce).…”

Section: Activity Measurements Of Recombinantly Expressed Alternativementioning

confidence: 99%

Analysis of Posttranslational Activation of Alternative Oxidase Isoforms

et al. 2017

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ORCID IDs: 0000-0002-1247-7282 (J.S.); 0000-0001-5754-025X (J.W.); 0000-0002-6140-6181 (R.S.).Mitochondrial alternative oxidase (AOX) in plants is a non-proton-motive ubiquinol oxidase that is activated by redox mechanisms and 2-oxo acids. A comparative analysis of the AOX isoenzymes AOX1A, AOX1C, and AOX1D from Arabidopsis (Arabidopsis thaliana) revealed that cysteine residues, CysI and CysII, are both involved in 2-oxo acid activation, with AOX1A activity being more increased by 2-oxo acids than that of AOX1C and AOX1D. Substitution of cysteine in AOX1A by glutamate mimicked its activation by pyruvate or glyoxylate, but not in AOX1C and AOX1D. CysIII, only present in AOX1A, is not involved in activation by reduction or metabolites, but substitutions at this position affected activity. AOX1A carrying a serine residue at position CysI was activated by succinate, while correspondingly substituted variants of AOX1C and AOX1D were insensitive. Activation by glutamate at CysI and CysII is consistent with the formation of the thiohemiacetal, while succinate activation after changing CysI to serine suggests hemiacetal formation. Surprisingly, in AOX1A, replacement of CysI by alanine, which cannot form a (thio)hemiacetal, led to even higher activities, pointing to an alternative mechanism of activation. Taken together, our results demonstrate that AOX isoforms are differentially activated and that activation at CysI and CysII is additive.

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Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase*

Cited by 22 publications

References 69 publications

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

Subunit δ Is the Key Player for Assembly of the H+-translocating Unit of Escherichia coli FOF1 ATP Synthase

Analysis of Posttranslational Activation of Alternative Oxidase Isoforms

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