Energy shifts induce membrane sequestration of DraG in Rhodospirillum rubrum independent of the ammonium transporters and diazotrophic conditions.

Helen Wang,Agneta Norén,Dominik Waluk,Ray Dixon,Stefan Nordlund

doi:10.1093/femsle/fny176

Helen Wang, Agneta Norén + Show 3 more

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https://doi.org/10.1093/femsle/fny176

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Abstract

ABSTRACTMetabolic regulation of Rhodospirillum rubrum nitrogenase is mediated at the post-translational level by the enzymes DraT and DraG when subjected to changes in nitrogen or energy status. DraT is activated during switch-off, while DraG is inactivated by reversible membrane association. We confirm here that the ammonium transporter, AmtB1, rather than its paralog AmtB2, is required for ammonium induced switch-off. Amongst several substitutions at the N100 position in DraG, only N100K failed to locate to the membrane following ammonium shock, suggesting loss of interaction through charge repulsion. When switch-off was induced by lowering energy levels, either by darkness during photosynthetic growth or oxygen depletion under respiratory conditions, reversible membrane sequestration of DraG was independent of AmtB proteins and occurred even under non-diazotrophic conditions. We propose that under these conditions, changes in redox status or possibly membrane potential induce interactions between DraG and another membrane protein in response to the energy status.

Highlights

Rhodospirillum rubrum is a diazotrophic, photosynthetic αproteobacterium that forms a highly invaginated membrane structure induced by anaerobic growth in the light or by microaerobic growth under reducing conditions in the dark (Grammel and Ghosh 2008; Carius, Henkel and Grammel 2011; Carius, Hadicke and Grammel 2013; Grammel, Gilles and Ghosh2003)
When switch-off was induced by lowering energy levels, either by darkness during photosynthetic growth or oxygen depletion under respiratory conditions, reversible membrane sequestration of DraG was independent of AmtB proteins and occurred even under non-diazotrophic conditions
Reversible DraG association to the chromatophore membrane depends on the energy status According to our model, the activity of DraG is influenced by its reversible association with the chromatophore membrane during nitrogenase switch-off/on cycles

Summary

Introduction

Rhodospirillum rubrum is a diazotrophic, photosynthetic αproteobacterium that forms a highly invaginated membrane structure induced by anaerobic growth in the light or by microaerobic growth under reducing conditions in the dark (Grammel and Ghosh 2008; Carius, Henkel and Grammel 2011; Carius, Hadicke and Grammel 2013; Grammel, Gilles and Ghosh2003). Latter involves the action of two enzymes, DraG (dinitrogenase reductase activating glycohydrolase) and DraT (dinitrogenase reductase ADP-ribose transferase) that act in a reciprocal manner (Liang et al 1991; Lowery, Lehman and Ludden 1986; Masepohl, Krey and Klipp 1993). When subjected to an external ‘switch-off’ signal, active DraT catalyses ADP-ribosylation of dinitrogenase reductase (encoded by nifH), thereby interfering with electron transfer to dinitrogenase by preventing the interaction with dinitrogenase reductase. Our understanding of the ammonia-induced regulation and the players/proteins involved in this regulatory system has increased considerably in recent years. In both R. rubrum and Azospirillum brasilense, DraG regulation has been shown to be mediated by a reversible membrane association, inactivating DraG (Huergo et al 2006b; Wang et al 2005). When the ammonia has been metabolised DraG is released into the cytosol, regaining its activity

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