Metal binding to the dynamic cytoplasmic domain of the cation diffusion facilitator (CDF) protein MamM induces a 'locked-in' configuration

Cation diffusion facilitator (CDF) proteins are a conserved family of transmembrane transporters that ensure cellular homeostasis of divalent transition metal cations. Metal cations bind to CDF protein's cytoplasmic C-terminal domain (CTD), leading to closure from its apo open V-shaped dimer to a tighter packed structure, followed by a conformational change of the transmembrane domain thus enabling transport of the metal cation. By implementing a comprehensive range of biochemical and biophysical methods, we studied the molecular mechanism of metal binding to the magnetotactic bacterial CDF protein MamM CTD. Our results reveal that the CTD is rather dynamic in its apo form, and that two dependent metal binding sites, a single central binding site and two symmetrical, peripheral sites, are available for metal binding. However, only cation binding to the peripheral sites leads to conformational changes that lock the protein in a compact state. Thus, this work reveals how metal binding is regulating the sequential uptakes of metal cations by MamM, and extends our understanding of the complex regulation mechanism of CDF proteins. This article is protected by copyright. All rights reserved

Aluminium-induced ion transport in Arabidopsis: the relationship between Al tolerance and root ion flux

Aluminium (Al) rhizotoxicity coincides with low pH; however, it is unclear whether plant tolerance to these two factors is controlled by the same mechanism. To address this question, the Al-resistant alr104 mutant, two Al-sensitive mutants (als3 and als5), and wild-type Arabidopsis thaliana were compared in long-term exposure (solution culture) and in short-term exposure experiments (H+ and K+ fluxes, rhizosphere pH, and plasma membrane potential, Em). Based on biomass accumulation, als5 and alr104 showed tolerance to low pH, whereas alr104 was tolerant to the combined low-pH/Al treatment. The sensitivity of the als5 and als3 mutants to the Al stress was similar. The Al-induced decrease in H+ influx at the distal elongation zone (DEZ) and Al-induced H+ efflux at the mature zone (MZ) were higher in the Al-sensitive mutants (als3 and als5) than in the wild type and the alr104 mutant. Under combined low-pH/Al treatment, alr104 and the wild type had depolarized plasma membranes for the entire 30 min measurement period, whereas in the Al-sensitive mutants (als3 and als5), initial depolarization to around –60 mV became hyperpolarization at –110 mV after 20 min. At the DEZ, the Em changes corresponded to the changes in K+ flux: K+ efflux was higher in alr104 and the wild type than in the als3 and als5 mutants. In conclusion, Al tolerance in the alr104 mutant correlated with Em depolarization, higher K+ efflux, and higher H+ influx, which led to a more alkaline rhizosphere under the combined low-pH/Al stress. Low-pH tolerance (als5) was linked to higher H+ uptake under low-pH stress, which was abolished by Al exposure

Data from: Synergy between RecBCD subunits is essential for efficient DNA unwinding

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD's individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25-40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast, but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state