The
functioning of rat CLC-2 chloride channel in Saccharomyces cerevisiae cells requires an
increased level of the Kha1 protein.
Krzysztof Flis ,
Anna Kurlandzka
Department of Genetics, Institute of Biochemistry and Biophysics, Polish
Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland (kflis@bp.onet.pl)
Chloride channels are widely distributed in prokaryotic and eukaryotic cells and belong to several protein families. The CLC-2 channel, a member of the new family of voltage-gated chloride channels (CLC), is very interesting as a potential target of an alternative therapy for cystic fibrosis. The yeast S. cerevisiae has only one CLC chloride channel homologue encoded by the GEF1 gene. Yeast gef1 mutants are respiratory deficient and they exhibit a defect in iron metabolism. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12074596&dopt=Abstract Our data indicated recently that the Gef1 protein is associated with chloride channel activity. We expressed the rat CLC-2 gene in yeast. This procedure required codon and translation start optimization. The protein Clc2p is localized in yeast in the same compartment as the Gef1 protein (Golgi), but it does not substitute directly for Gef1p function. We assumed that proper functioning of CLC-2 could be dependent on an increased level of another yeast protein (a functional suppressor). To check that we transformed a strain devoid of GEF1 and bearing chromosome-integrated CLC-2 gene with a yeast genomic bank on a multicopy plasmid. We found that the presence of extra copies of the KHA1 gene restores the wild phenotype of the above strain (CLC-2 is required). KHA1, encoding a putative K+/H+ antiporter, has only be crudely characterized but we assume that it may change the pH of the Golgi thus enabling the functioning of CLC-2 in yeast cells. This work was supported by KBN grant No. 3 P05A 069 23.