Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family

Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca2+ and Mg2+, which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H+ serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg2+. The protein transports Mg2+ and Mn2+ but not Ca2+ by a mechanism that is not coupled to H+. Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell.

A tonoplast-localized magnesium transporter is involved in stomatal opening in Arabidopsis

Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing are still not completely understood. Here, through large-scale screening, we identified an Arabidopsis mutant (cst2 for closed stomata2) defective in stomatal opening under light condition. A map-based cloning combined with complementation test revealed that the mutant phenotype was caused by a nucleotide substitution of a gene, which domains show similarity to human Mg efflux transporter ACDP/CNNM. Functional analysis showed that CST2 encodes a tonoplast-localized transporter for Mg. This protein is constitutively and highly expressed in the guard cells. Furthermore, CST2 is phosphorylated by calcineurin B-like protein (CBL)-interacting protein kinases 26 (CIPK26) in vitro, which is probably required for its activation. Knockout of this gene resulted in stomatal closing and growth retardation under high Mg concentration conditions, while over-expression of this gene increased tolerance to high Mg. Our results indicate that CST2 plays an important role in maintaining Mg homeostasis in plant cells through sequestering Mg into vacuoles especially in guard cells and that this homeostasis is required for stomatal opening, which provide a novel insight into mechanism of stomatal opening in plants.

Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family

Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca2+ and Mg2+, which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H+ serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg2+. The protein transports Mg2+ and Mn2+ but not Ca2+ by a mechanism that is not coupled to H+. Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell.

The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species

A significant challenge today within protein lipidology is to understand the relationship between cell structure, lipid membrane integrity, ion homeostasis and the embedded membrane proteins. The bacterial magnesium transporter A (MgtA) is a specialized P-type ATPase important for Mg2+ import into the cytoplasm; disrupted magnesium homeostasis is linked to intrinsic ribosome instability and nitro-oxidative stress in Salmonella strains. MgtAs function is highly dependent on anionic lipids, particularly cardiolipin, and further co-localization of cardiolipin with MgtA at the E. coli cell poles has been revealed. Here, we show that MgtA has functional specificity for cardiolipin 18:1, but it reaches maximum activity only in combination with cardiolipin 16:0, equivalent to the major components of native cardiolipin found in E. coli membranes. This is the first time it has been shown experimentally that two different lipid species from the same class, individually promoting low activity, can enhance activity in combination. Native mass spectrometry verifies the presence of two binding sites for cardiolipin and kinetic studies reveal that a cooperative relationship likely exists between the two different cardiolipin variants (cardiolipin 16:0 and cardiolipin 18:1). This is the first experimental evidence of cooperative effects between lipids of the same class, with only minor variations in their acyl chain composition, acting on a membrane protein. In summary, our results reveal that MgtA exhibits a highly complex interaction with one cardiolipin 18:1 and one cardiolipin 16:0, affecting protein activity and stability, and contributing to our understanding of the particular interactions between lipid environment and membrane proteins. Further, a better understanding of Mg2+ homeostasis in bacteria, due to its role as a virulence regulator, will provide further insights into the regulation and mechanism of bacterial infections.

Crystal structure of an archaeal CorB magnesium transporter

CNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg2+-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg2+ ion coordinated by a conserved π-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg2+-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg2+ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg2+ transport and associated diseases.

Crystal structure of a bacterial CNNM magnesium transporter

CBS-pair domain divalent metal cation transport mediators (CNNMs) are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. CNNM proteins are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determined the crystal structure of an archaeal CNNM protein with Mg2+-ATP bound. The structure reveals a novel transmembrane fold for the DUF21 domain, the largest family of domains of unknown function. The protein has a negatively charged cavity that penetrates halfway through the membrane suggesting it functions as a cation transporter. The cytosolic portion of the protein is comprised of highly charged four-helix bundle and a CBS-pair domain. HDX-MS experiments, molecular dynamics, and additional crystal structures show that the cytosolic domains undergo large conformational changes upon nucleotide binding suggesting a mechanism of regulation shared between human and bacterial orthologs. The molecular characterization of CNNM proteins has profound implications for understanding their biological functions in human diseases, including cancer, and in animals, bacteria and plants.

Slowing Development Facilitates Arabidopsis mgt Mutants to Accumulate Enough Magnesium for Pollen Formation and Fertility Restoration

Magnesium (Mg) is an abundant and important cation in cells. Plants rely on Mg transporters to take up Mg from the soil, and then Mg is transported to anthers and other organs. Here, we showed that MGT6+/− plants display reduced fertility, while mgt6 plants are fertile. MGT6 is expressed in the anther at the early stages. Pollen mitosis and intine formation are impaired in aborted pollen grains (PGs) of MGT6+/− plants, which is similar to the defective pollen observed in mgt5 and mgt9 mutants. These results suggest that Mg deficiency leads to pollen abortion in MGT6+/− plants. Our data showed that mgt6 organs including buds develop significantly slower and mgt6 stamens accumulate a higher level of Mg, compared with wild-type (WT) and MGT6+/− plants. These results indicate that slower bud development allows mgt6 to accumulate sufficient amounts of Mg in the pollen, explaining why mgt6 is fertile. Furthermore, we found that mgt6 can restore fertility of mgt5, which has been reported to be male sterile due to defects in Mg transport from the tapetum to microspores and that an additional Mg supply can restore its fertility. Interestingly, mgt5 fertility is recovered when grown under short photoperiod conditions, which is a well-known factor regulating plant fertility. Taken together, these results demonstrate that slow development is a general mechanism to restore mgts fertility, which allows other redundant magnesium transporter (MGT) members to transport sufficient Mg for pollen formation.

Small proteins regulateSalmonellasurvival inside macrophages by controlling degradation of a magnesium transporter

All cells require Mg2+to replicate and proliferate. The macrophage protein Slc11a1 is proposed to protect mice from invading microbes by causing Mg2+starvation in host tissues. However, the Mg2+transporter MgtB enables the facultative intracellular pathogenSalmonella entericaserovar Typhimurium to cause disease in mice harboring a functional Slc11a1 protein. Here, we report that, unexpectedly, theSalmonellasmall protein MgtR promotes MgtB degradation by the protease FtsH, which raises the question: How doesSalmonellapreserve MgtB to promote survival inside macrophages? We establish that theSalmonellasmall protein MgtU prevents MgtB proteolysis, even when MgtR is absent. Like MgtB, MgtU is necessary for survival inSlc11a1+/+macrophages, resistance to oxidative stress, and growth under Mg2+limitation conditions. TheSalmonellaMg2+transporter MgtA is not protected by MgtU despite sharing 50% amino acid identity with MgtB and being degraded in an MgtR- and FtsH-dependent manner. Surprisingly, themgtB,mgtR, andmgtUgenes are part of the same transcript, providing a singular example of transcript-specifying proteins that promote and hinder degradation of the same target. Our findings demonstrate that small proteins can confer pathogen survival inside macrophages by altering the abundance of related transporters, thereby furthering homeostasis.