Ca 2؉ signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca 2؉ signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome encodes 14 Ca 2؉ pumps, 10 of which belong to a family of autoinhibited Ca 2؉ ATPases (ACA) that are predicted to be activated by Ca 2؉ ͞calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential and a high frequency of aborted fertilization, resulting in a >80% reduction in seed set. These findings identify a plasma membrane Ca 2؉ transporter as a key regulator of pollen development and fertilization in flowering plants.

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn 2؉ 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 Zn 2؉ P1B-type ATPases form a subfamily of P-type ATPases and pump metal ions across biological membranes (1-4). These pumps maintain metal homeostasis in all domains of life (5-8).Humans have only two P1B-ATPases, namely ATP7A and ATP7B, both of which transport Cu ϩ , and cause Menke and Wilson diseases, respectively, when mutated (8). In contrast, in the model plant Arabidopsis thaliana eight P1B-ATPase genes (heavy metal ATPases 1-8 (HMA1-HMA8)3 ) are present (9, 10). Monovalent metal ions, such as Cu ϩ and Ag ϩ , are transported by HMA5-HMA8, which belong to the subclass P1B1, whereas divalent metal ions, such as Zn 2ϩ and Cd 2ϩ , are transported by HMA2-HMA4 belonging to the P1B2 subgroup that is related to prokaryotic divalent heavy metal pumps (4, 11).In A. thaliana, HMA2 and HMA4 are closely related in primary sequence and might have evolved as a result of gene duplication (10). In the physiology of A. thaliana, these two pumps are redundant in several functions (12, 13). Both genes are expressed in roots in the pericycle cells surrounding the xylem, a vascular tissue specialized in transport of inorganic nutrients and water to the shoot. Single knock-out mutants of AtHMA2 and AtHMA4 have weak phenotypes, whereas a double hma2 hma4 mutant accumulates zinc in root pericycle cells, which causes shoots to suffer from zinc deficiency. This strongly suggests that AtHMA2 and AtHMA4 are responsible for catalyzing zinc efflux from pericycle cells, thereby loading the xylem with zinc (12-13). In the zinc hyperaccumulator Arabidopsis halleri the gene encoding HMA4 has been copied three times (14). This, together with increased promoter strength, results in an increased capacity of this metallophyte to accumulate zinc in shoots (14).P1B-ATPases have six to eight transmembrane segments responsible for metal ion coordination during transport, a large cytosolic portion divided into three catalytic domains (A, P, and N), and two terminal domains that contain metal-coordinating residues (the N terminus and the C terminus). The N-terminal domains of P1B-ATPases are not essential for the transport mechanism but play important roles in their post-translational regulation. In bacterial P1B-ATPases the N-terminal domains are characterized by Cys-X-X-Cys sequences (4,7,8,15). The Cys residues are responsible for metal coordination and can be involved in binding both monovalent (Ag ϩ and Cu ϩ ) and divalent metal cations (Cu 2ϩ , Cd 2ϩ , and Zn 2ϩ ) (16 -21). In plant Zn 2ϩ -ATPases the Cys-X-X-Cys conserved sequence is replaced by a Cys-Cys-X-X-Glu motif (9,12,22,23). Truncation of P1B-ATP...

In plant Ca2؉ pumps belonging to the P 2B 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 (Arg 43 to Lys 68 ) 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. Ca2ϩ acts as a secondary messenger in eukaryotic cells. One of the key proteins that mediate Ca 2ϩ signals is calmodulin (CaM) 2 a small, ubiquitous, and highly conserved protein found in all eukaryotes (1, 2). Upon Ca 2ϩ binding, CaM changes conformation, which enables CaM to bind to and regulate a wide array of target enzymes. The interaction between CaM and the CaM-binding domain (CaMBD) of the target enzyme is mainly hydrophobic in which two bulky hydrophobic residues in the CaMBD, the so-called anchor points, are especially important to anchor the protein to CaM. In addition, the complex is stabilized by electrostatic interactions between negatively charged glutamates in CaM and basic residues in the CaMBD (3-5). No consensus CaMBD exists for proteins that are targets of CaM, but CaMBDs typically have a hydrophobic and basic nature consisting of 15-30 amino acid residues that have a tendency to form an ␣-helix. Based on the position of the two hydrophobic anchor points, the majority of Ca 2ϩ -dependent CaMBDs is divided into three classes, namely 1-10, 1-14, and 1-16 (6, 7). Many CaM-regulated enzymes are autoinhibited with autoinhibition released by CaM. The autoinhibitory domain is often located either adjacent to or overlapping with the CaMBD (5, 8).The P 2B Ca 2ϩ -ATPases including PMCAs (plasma membrane Ca 2ϩ -ATPases) from animals and ACAs (autoinhibited Ca 2ϩ -ATPases) from plants are autoinhibitory proteins regulated by CaM. The regulatory region consisting of a CaMBD and an autoinhibitory domain is located in the C terminus in animals and N terminus in plants (9, 10). These two domains are likely to be at least partly overlapping in both plant and mammalian Ca 2ϩ -ATPases, as has been shown for the human PMCA4b (isoform 4, splice variant b) (11), Arabidopsis ACA2 (i...

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.