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Plant and Cell Physiology, 2003, Vol. 44, No. 6 597-606
© 2003 Oxford University Press

Insertional Mutagenesis in a Homologue of a Pi Transporter Gene Confers Arsenate Resistance on Chlamydomonas

Isao Kobayashi1, Shoko Fujiwara1, Kosuke Shimogawara2, Toshikazu Kaise1, Hideaki Usuda2 and Mikio Tsuzuki1,3

1 School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo, 192-0392 Japan
2 Laboratory of Chemistry, Teikyo University School of Medicine, Ohtsuka, Hachioji, Tokyo, 192-0395 Japan

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1,666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na+/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.

3 Corresponding author: E-mail,mtsu{at}ls.toyaku.ac.jp; Fax, +81-426-76-6721.


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