Functional Domain Analysis of the Yeast ABC Transporter Ycf1p by Site-directed Mutagenesis

Juan M. Falcón-Pérez, Marı́a J. Mazón, Jesús Molano, Pilar Eraso
1999 Journal of Biological Chemistry  
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. Twentytwo single amino acid
more » ... 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.
doi:10.1074/jbc.274.33.23584 pmid:10438540 fatcat:4stausdbg5dpjjip4ysw6u4wfa