[show abstract][hide abstract] ABSTRACT: The Golgi apparatus is thought to play a role in calcium homeostasis in plant cells. However, the calcium dynamics in this organelle is unknown in plants. To monitor the [Ca(2+)](Golgi)in vivo, we obtained and analyzed Arabidopsis thaliana plants that express aequorin in the Golgi. Our results show that free [Ca(2+)] levels in the Golgi are higher than in the cytosol (0.70μM vs. 0.05μM, respectively). Stimuli such as cold shock, mechanical stimulation and hyperosmotic stress, led to a transient increase in cytosolic calcium; however, no instant change in the [Ca(2+)](Golgi) concentration was detected. Nevertheless, a delayed increase in the [Ca(2+)](Golgi) up to 2-3μM was observed. Cyclopiazonic acid and thapsigargin inhibited the stimuli-induced [Ca(2+)](Golgi) increase, suggesting that [Ca(2+)](Golgi) levels are dependent upon the activity of Ca(2+)-ATPases. Treatment of these plants with the synthetic auxin analog, 2,4-dichlorophenoxy acetic acid (2,4-D), produced a slow decrease of free calcium in the organelle. Our results indicate that the plant Golgi apparatus is not involved in the generation of cytosolic calcium transients and exhibits its own dynamics modulated in part by the activity of Ca(2+) pumps and hormones.
[show abstract][hide abstract] ABSTRACT: The Arabidopsis thaliana AtHMA1 protein is a member of the P(IB)-ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase)-type pumps that is involved in inhibition by thapsigargin. To analyze the ion specificity of AtHMA1, we performed functional complementation assays using mutant yeast strains defective in Ca(2+) homeostasis or heavy metal transport. The heterologous expression of AtHMA1 complemented the phenotype of both types of mutants and, interestingly, increased heavy metal tolerance of wild-type yeast. Biochemical analyses were performed to describe the activity of AtHMA1 in microsomal fractions isolated from complemented yeast. Zinc, copper, cadmium, and cobalt activate the ATPase activity of AtHMA1, which corroborates the results of metal tolerance assays. The outcome establishes the role of AtHMA1 in Cd(2+) detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca(2+) transport mediated by AtHMA1. Ca(2+) transport displayed high affinity with an apparent K(m) of 370 nm and a V(max) of 1.53 nmol mg(-1) min(-1). This activity was strongly inhibited by thapsigargin (IC(50) = 16.74 nm), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca(2+)/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin.
Journal of Biological Chemistry 05/2008; 283(15):9633-41. · 4.65 Impact Factor