A FAMILY OF PUTATIVE CHLORIDE CHANNELS FROM ARABIDOPSIS AND FUNCTIONAL COMPLEMENTATION OF A YEAST-STRAIN WITH A CLC GENE DISRUPTION

We have cloned four novel members of the CLC family of chloride channe ls from Arabidopsis thaliana. The four plant genes are homologous to a recently isolated chloride channel gene from tobacco (CLC-Nt1; Lurin, C., Geelen, D., Barbier-Brygoo, H., Guern, J., and Maurel, C. (1996) Plant Cell 8, 701-711) and are about 30% identical in sequence to the most closely related CLC-6 and CLC-7: putative chloride channels from mammalia. AtCLC transcripts are broadly expressed in the plant. Simila rly, antibodies against the AtCLC-d protein detected the protein in al l tissues, but predominantly in the silique. AtCLC-a and AtCLC-b are h ighly homologous to each other (approximate to-87% identity), while be ing approximate to 50% identical to either AtCLC-c or AtCLC-d. None of the four cDNAs elicited chloride currents when expressed in Xenopus o ocytes, either singly or in combination. Among these genes, only AtCLC -d could functionally substitute for the single yeast CLC protein, res toring iron-limited growth of a strain disrupted for this gene. Introd uction of disease causing mutations, identified in human CLC genes, ab olished this capacity. Consistent with a similar function of both prot eins, the green fluorescent protein-tagged AtCLC-d protein showed the identical localization pattern as the yeast ScCLC protein. This sugges ts that in Arabidopsis AtCLC-d functions as an intracellular chloride channel.