Functional domain analysis of the yeast ABC transporter Ycf1p by site-directed mutagenesis.
The yeast cadmium factor (Ycf1p) is a vacuolar protein involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors in yeast. It belongs to the superfamily of ATP binding cassette transporters, which includes the human cystic fibrosis transmembrane conductance regulator and the multidrug resistance-associated protein. To examine the functional significance of conserved amino acid residues in Ycf1p, we performed an extensive mutational analysis. Twenty-two single amino acid substitutions or deletions were generated by site-directed mutagenesis in the nucleotide binding domains, the proposed regulatory domain, and the fourth cytoplasmic loop. Mutants were analyzed phenotypically by measuring their ability to grow in the presence of Cd(2+). Expression and subcellular localization of the mutant proteins were examined by immunodetection in vacuolar membranes. For functional characterization of the Ycf1p variants, the kinetic parameters of glutathione S-conjugated leukotriene C(4) transport were measured. Our analysis shows that residues Ile(711), Leu(712), Phe(713), Glu(927), and Gly(1413) are essential for Ycf1p expression. Five other amino acids, Gly(663), Gly(756), Asp(777), Gly(1306), and Gly(1311), are critical for Ycf1p function, and two residues, Glu(709) and Asp(821), are unnecessary for Ycf1p biogenesis and function. We also identify several regulatory domain mutants in which Cd(2+) tolerance of the mutant strain and transport activity of the protein are dissociated.
Falcón-Pérez
J M
JM
Instituto de Investigaciones Biomédicas "Alberto Sols, " Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier 4, 28029-Madrid, Spain.
Mazón
M J
MJ
Molano
J
J
Eraso
P
P
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001692:Biological Transport; D002104:Cadmium / Q000378:metabolism; D003470:Culture Media; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D017997:Leukotriene C4 / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D003470:Culture Media; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C089179:YCF1 protein, S cerevisiae; D002104:Cadmium; D017997:Leukotriene C4