Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas.
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 1666 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.
Kobayashi
Isao
I
School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo, 192-0392 Japan.
Fujiwara
Shoko
S
Shimogawara
Kosuke
K
Kaise
Toshikazu
T
Usuda
Hideaki
H
Tsuzuki
Mikio
M
D020418:Algal Proteins / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D001149:Arsenates / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D002696:Chlamydomonas / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004351:Drug Resistance / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D049294:Phospholipid Transfer Proteins; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D020418:Algal Proteins; D001149:Arsenates; D002352:Carrier Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; C025657:arsenic acid