Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on Chlamydomonas

• Published in: Plant and cell physiology 2003-06, Vol.44 (6), p.597-606
• Main Authors: Kobayashi, I. (Tokyo Univ. of Pharmacy and Life Science, Hachioji (Japan)), Fujiwara, S, Shimogawara, K, Kaise, T, Usuda, H, Tsuzuki, M
• Format: Article
• Language: English
• Subjects: Algal Proteins - genetics; Algal Proteins - isolation & purification; Animals; ARG7 gene; arsenate; Arsenates - pharmacology; ARSENIC COMPOUNDS; Carrier Proteins - genetics; Cells, Cultured; CHLAMYDOMONAS; Chlamydomonas - drug effects; Chlamydomonas - genetics; Chlamydomonas - metabolism; Chlamydomonas reinhardtii; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; Drug Resistance - genetics; GENE EXPRESSION; hexose transporter; HXT; insertional mutagenesis; Keywords: Arsenate resistance — Chlamydomonas reinhardtii — Pi transporter — Pi uptake; Membrane Proteins - genetics; Molecular Sequence Data; Mutagenesis, Insertional - genetics; MUTATION; Mutation - genetics; Phospholipid Transfer Proteins; PHOSPHOLIPIDS; phytochelatin; PTB1 gene; PTB1 protein; RESISTANCE TO CHEMICALS; reverse transcriptional polymerase chain reaction; RT-PCR; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; TAP medium; Tris-acetate-phosphate medium
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/pcp/pcg081

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.