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What is a CAX Transporter?
Plant cells have a large internal compartment (organelle) called a vacuole, and the contents of this organelle is separated from the cytosol, where life’s essential biochemical reactions take place, by the vacuolar membrane. The vacuole is the place where the plant cell stores various materials, including toxic molecules and ions.
The membrane that defines the vacuole is called the tonoplast, and a steep pH gradient exists across this membrane. Usually, the pH of the cytosolic side of tonoplast is maintained at the physiological level, and the pH inside the vacuole is significantly lower (pH=4-5). This acidic vacuolar environment is actively maintained by proton pumps. CAX transporters take advantage of this pH gradient to move cations into the vacuole in exchange for protons that are abundantly present in the vacuole.
Discovery of CAX in Plants
The first plant CAXs were discovered from Arabidopsis thaliana by Dr. Kendal D. Hirschi (Baylor College of Medicine) by using a yeast suppression assay (Hirschi et al., 19961), and so named because he was searching for the transporters that can function as CAlcium eXchangers. The two genes that he found were named CAX1 and CAX2.
There is an interesting story behind it, which is rarely mentioned in research publications. The CAX1 and CAX2 cDNAs that he used in the yeast assay were not full-length, but a short segment of their 5’-end was missing, which, during translation, will truncate the first 36 amino acids of the transporter proteins. It was later found that this 36 amino acid region acted as an autoinhibitory domain, and the proteins were active only when this region was removed. If he had used unbroken full-length cDNAs, he would never have discovered his CAXs.
CAX1 and CAX2 are calcium transporters, but it became clear later that the substrates of CAXs were not limited to calcium. The potential substrates of CAXs include cadmium, mercury, manganese, zinc, nickel, and even silver! Now CAX stands for CAtion eXchanger. (Fortunately, the abbreviation did not change.) This promiscuous nature of CAXs is both useful and problematic in practical applications, which I will discuss later.
Since the discovery of the first two CAXs in Arabidopsis, CAXs from other plants have been characterized. Significant discoveries have been made in Dr. Masayoshi Maeshima’s lab in Nagoya University, Japan. His group has identified new CAXs from mung bean and rice. Detailed mutagenesis studies by his group identified domains that form an ion filter in the pore structure of a CAX from rice.
1K. D. Hirschi, R. Zhen, K. W. Cunningham, P. A. Rea, and G. R. Fink. (1996) CAX1, an H+/Ca2+ antiporter from Arabidopsis. Proceedings of the National Academy of Sciences, USA 93:8782-8786.
Diversity of CAXs
The completion of the Arabidopsis genome project revealed that there are six CAXs in its genome. Many other plant genome projects have also shown that CAXs form a multigene family. As of 2010, there are over 200 entries of CAXs in the GenBank database. CAX open reading frames appear to be present in most taxa, with notable exceptions of mammals and other animals including insects and C. elegans. It is a mystery why CAXs are dispensable in these organisms.
The CAXs are grouped into three types according to their phylogenetic relationships. All the plant CAXs are classified as Type I CAXs. The secondary structure analysis shows that most Type I CAXs have 11 transmembrane domains, which is further divided into two weakly homologous units. The two units are thought to be the result of an ancient gene duplication, although the primordial "half proteins" are not found in plant genomes. However, such half proteins can be artificially made and both units can be co-expressed ectopically, and they were found to form functional proteins! This property affords us a means to study structure and function relationships in CAX proteins.
The Type II CAXs are the only CAXs found in animals, but are also present in fungi and protozoa. The size of Type II CAXs is approximately twice as that of Type I CAXs because of their long N-terminal hydrophilic domain, containing two additional transmembrane helices. The C-terminal half of the Type II CAXs is similar to that of Type I CAXs. Functional details of Type II CAXs are not known and the investigation is under way. The fact that these CAXs are present in animals will provide a unique opportunity to identify a fundamental physiological role that all CAXs might play.
The Type III CAXs are found exclusively in bacteria, although some bacteria have Type I CAXs as well in their genomes. Studies in E. coli suggest Type III CAXs may have roles in sodium homeostasis in bacteria. The size and the secondary structure of Type III CAXs are similar to those of Type I CAXs. Recent microbial genome sequencing projects identified numerous CAXs in bacteria, and it can be a rich source to explore the CAX diversity for protein engineering.
Interested readers may want to read the original paper on the classification of CAXs (Shigaki et al., 20062).
2 T. Shigaki, I. Rees, L. Nakhleh, and K. D. Hirschi. (2006) Identification of three distinct phylogenetic groups of CAX cation/proton antiporters. Journal of Molecular Evolution 63:815-825.
Practical applications of CAX transporters
The vacuole’s protected environment makes CAXs an ideal candidate for
two practical applications. First, the vacuole can store high levels of
calcium. For example, if we engineer a tomato plant to express a large number
of CAX proteins, the tomato will accumulate more calcium than an ordinary
tomato (biofortification). Such calcium-enriched vegetables have been already
developed and it is expected that more crops will be transformed with CAX genes
for the future commercialization. When CAXs are used for biofortification, we
have to keep in mind that CAXs are promiscuous metal transporters that can
transport toxic metals as well. (Calcium and cadmium have very similar ionic
radii, and therefore, it is difficult for transporters to distinguish them!) By
engineering CAX proteins for enhanced substrate specificity, such a potential
problem can be minimized.
Second, when plants express CAXs that are capable of transporting heavy metals, such as cadmium and mercury, the plants accumulate copious amounts of these metals without suffering an adverse effect in growth and development. If these plants are grown in the soil contaminated with heavy metals, they help to clean up the site (phytoremediation). It is a low-cost way of removing environmental pollutants from soil, and the optimization of the properties of CAX proteins and the selection of right plant species will lead to a much needed decontamination technology.