A yeast recombinant aquaporin mutant that is not expressed or mistargeted in Xenopus oocyte can be functionally analyzed in reconstituted proteoliposomes.
We have recently identified AQPcic (for aquaporin cicadella), an insect aquaporin found in the digestive tract of homopteran insects and involved in the elimination of water ingested in excess with the dietary sap (Le Cahérec, F., Deschamps, S., Delamarche, C., Pellerin, I., Bonnec, G., Guillam, M. T., Gouranton, J., Thomas, D., and Hubert, J. F. (1996) Eur. J. Biochem. 241, 707-715). Like many other aquaporins, AQPcic is inhibited by mercury reagents. In this study, we have demonstrated that residue Cys82 is essential for mercury inhibition. Another mutant version of AQPcic (AQP-C134S), expression of which in Xenopus laevis failed to produce an active molecule, was successfully expressed in Saccharomyces cerevisiae. Using stopped-flow analysis of reconstituted proteoliposomes, we demonstrated that the biological activity and Hg sensitivity of yeast-expressed wild type and mutant type AQPcic was readily assessed. Therefore, we propose that the yeast system is a valid alternative to Xenopus oocytes for studying particular mutants of aquaporin.
Lagrée
V
V
UPRES-A CNRS, Biologie Cellulaire et Reproduction, Université de Rennes 1, 35042 Rennes cedex, France.
Pellerin
I
I
Hubert
J F
JF
Tacnet
F
F
Le Cahérec
F
F
Roudier
N
N
Thomas
D
D
Gouranton
J
J
Deschamps
S
S
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008628:Mercury / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011510:Proteolipids / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014981:Xenopus
D007473:Ion Channels; D011510:Proteolipids; C020485:proteoliposomes; D008628:Mercury; D003545:Cysteine