Lone Baekgaard

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn2+ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn2+ and Cd2+ chelator with capacity to bind 10 Zn2+ ions per C terminus. When AtHMA4 is expressed in a Zn2+-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn2+ and Cd2+ tolerance and lowered Zn2+ and Cd2+ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn2+ and Cd2+ chelator (sensor) and as a regulator of the efficiency of Zn2+ and Cd2+ export. The identification of a post-translational handle on Zn2+ and Cd2+ transport efficiency opens new perspectives for regulation of Zn2+ nutrition and tolerance in eukaryotes.

The Plant Plasma Membrane Ca2+ Pump ACA8 Contains Overlapping as Well as Physically Separated Autoinhibitory and Calmodulin-binding Domains

In plant Ca2+ pumps belonging to the P2B subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg43 to Lys68) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.

Plasma membrane Ca2+ transporters mediate virus‐induced acquired resistance to oxidative stress

This paper reports the phenomenon of acquired cross-tolerance to oxidative stress in plants and investigates the activity of specific Ca²+ transport systems mediating this phenomenon. Nicotiana benthamiana plants were infected with Potato virus X (PVX) and exposed to oxidative [either ultraviolet (UV-C) or H₂O₂] stress. Plant adaptive responses were assessed by the combined application of a range of electrophysiological (non-invasive microelectrode ion flux measurements), biochemical (Ca²+- and H+-ATPase activity), imaging (fluorescence lifetime imaging measurements of changes in intracellular Ca²+ concentrations), pharmacological and cytological transmission electrone microscopy techniques. Virus-infected plants had a better ability to control UV-induced elevations in cytosolic-free Ca²+ and prevent structural and functional damage of chloroplasts. Taken together, our results suggest a high degree of crosstalk between UV and pathogen-induced oxidative stresses, and highlight the crucial role of Ca²+ efflux systems in acquired resistance to oxidative stress in plants.

Barley HvHMA1 Is a Heavy Metal Pump Involved in Mobilizing Organellar Zn and Cu and Plays a Role in Metal Loading into Grains

Heavy metal transporters belonging to the P1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. Heavy metal transporters belonging to the P1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. In this study we investigated the properties of HvHMA1, which is a barley orthologue of Arabidopsis thaliana AtHMA1 localized to the chloroplast envelope. HvHMA1 was localized to the periphery of chloroplast of leaves and in intracellular compartments of grain aleurone cells. HvHMA1 expression was significantly higher in grains compared to leaves. In leaves, HvHMA1 expression was moderately induced by Zn deficiency, but reduced by toxic levels of Zn, Cu and Cd. Isolated barley chloroplasts exported Zn and Cu when supplied with Mg-ATP and this transport was inhibited by the AtHMA1 inhibitor thapsigargin. Down-regulation of HvHMA1 by RNA interference did not have an effect on foliar Zn and Cu contents but resulted in a significant increase in grain Zn and Cu content. Heterologous expression of HvHMA1 in heavy metal-sensitive yeast strains increased their sensitivity to Zn, but also to Cu, Co, Cd, Ca, Mn, and Fe. Based on these results, we suggest that HvHMA1 is a broad-specificity exporter of metals from chloroplasts and serve as a scavenging mechanism for mobilizing plastid Zn and Cu when cells become deficient in these elements. In grains, HvHMA1 might be involved in mobilizing Zn and Cu from the aleurone cells during grain filling and germination.

Endomembrane Ca2+-ATPases play a significant role in virus-induced adaptation to oxidative stress.

Although the role of Ca2+ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the role of active Ca2+ efflux systems in this process. In our recent paper,17 we reported Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana and showed the critical role of plasma membrane Ca2+/H+ exchangers in this process. The current study continues this research. Using biochemical and electrophysiological approaches, we reveal that both endomembrane P2A and P2B Ca2+-ATPases play significant roles in adaptive responses to oxidative stress by removing excessive Ca2+ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca2+ efflux systems in acquired tolerance to oxidative stress and open up prospects for practical applications in agriculture, after in-depth comprehension of the fundamental mechanisms involved in common responses to environmental factors at the genomic, cellular and organismal levels.

Plasma Membrane ATPases

The plasma membrane separates the cellular contents from the surrounding environment. Nutrients must enter through the plasma membrane in order to reach the cell interior, and toxic metabolites and several ions leave the cell by traveling across the same barrier. Biological pumps in the plasma membrane include ABC transporters, vacuolar (V-type) H+ pumps, and P-type pumps. These pumps all utilize ATP as a fuel for energizing pumping. This review focuses on the physiological roles of plasma membrane P-type pumps, as they represent the major ATP hydrolytic activity in this membrane.

Transmembrane nine proteins in yeast and Arabidopsis affect cellular metal contents without changing vacuolar morphology

Transmembrane nine (TM9) proteins are localized in the secretory pathway of eukaryotic cells and are involved in cell adhesion and phagocytosis. The mechanism by which TM9 proteins operate is, however, not well understood. Here we have utilized elemental profiling by inductively coupled plasma mass spectrometry (ICP-MS) to further investigate the physiological function of TM9 proteins. Cellular copper contents in Saccharomyces cerevisiae varied depending on the presence of TM9 homologues from both yeast and Arabidopsis thaliana. A yeast tmn1-3 triple mutant lacking all three yeast endogenous TMNs showed altered metal homeostasis with a reduction in the cellular Cu contents to 25% of that in the wild-type. Conversely, when TMN1 was overexpressed in yeast, cellular Cu concentrations were more than doubled. Both Tmn1p-GFP and Tmn2p-GFP fusion proteins localized to the tonoplast. Yeast vacuolar biogenesis was not affected by the lack or presence of TM9 proteins neither in the tmn1-3 triple mutant nor in TM9 overexpressing strains. Heterologous expression in yeast of AtTMN7, a TM9 homologue from Arabidopsis, affected Cu homeostasis similar to the overexpression of TMN1. In Arabidopsis, the two TM9 homologues AtTMN1 and AtTMN7 were ubiquitously expressed. AtTMN7 promoter constructs driving the expression of GFP showed elevated expression of AtTMN7 in the root elongation zone. It is concluded that TM9 homologues from S. cerevisiae and A. thaliana have the ability to affect the intracellular Cu balance. Tmn1p and Tmn2p operate from the yeast vacuolar membrane without influencing vacuolar biogenesis. A new physiological function of the TM9 family coupled to vacuolar Cu homeostasis is proposed.

Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8.

Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca(2+) regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 A, beta = 113.2 degrees. A complete data set was collected to 3.0 A resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin.