A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein.

M.S Szczypka,J.A Wemmie,W.S Moye-Rowley,D.J Thiele

doi:10.1016/s0021-9258(17)31723-4

Abstract

Members of the ATP binding cassette (ABC) protein superfamily transport a variety of substances across biological membranes, including drugs, ions, and peptides. The yeast cadmium factor (YCF1) gene from Saccharomyces cerevisiae is required for cadmium resistance and encodes a 1,515 amino acid protein with extensive homology to both the human multidrug resistance-associated protein (MRP1) and the cystic fibrosis transmembrane conductance regulator (hCFTR). S. cerevisiae cells harboring a deletion of the YCF1 gene are hypersensitive to cadmium compared with wild type cells. Mutagenesis experiments demonstrate that conserved amino acid residues, functionally critical in hCFTR, play a vital role in YCF1-mediated cadmium resistance. Mutagenesis of phenylalanine 713 in the YCF1 nucleotide binding fold 1, which correlates with the delta F508 mutation found in the most common form of cystic fibrosis, completely abolished YCF1 function in cadmium detoxification. Furthermore, substitution of a serine to alanine residue in a potential protein kinase A phosphorylation site in a central region of YCF1, which displays sequence similarity to the central regulatory domain of hCFTR, also rendered YCF1 nonfunctional. These results suggest that YCF1 is composed of modular domains found in human proteins which function in drug and ion transport.

Highlights

Members of the ATP binding cassette (ABC) protein potentially carcinogenic metal into the environment
Mutagenesis experiments demonstrate that conservedaminoacidresidues,functionallycriticalin hCFTR, playa vital role in YCF1-mediated cadmium toxic environmental metals involves the chelation of intracellular metal ions through low molecular weight cysteine-rich proteins known as metallothioneins
a deletion of codon 508 (AF508) mutation found in the most common form of cypse-ptidesare enzymatically synthesizedinresponseto toxic tic fibrosis, completely abolished YCFl function in cad- metals and form metal chelate complexes that are thought to mium detoxification

Summary

MATERIALS AND METHODS

Yeast Strains and Growth Media-The isogenic yeast strains DTY7 lem in metal toxicity due to the deposition of this toxic and (MATa,ura3-52,leu2-3,-112,his and DTY168(MATa,ycflA::hisG, ura, leu2-3,-112, his6) were used in this study. Cancer Society,Grant GM41840 fromthe National Institutes of Health, compassing the entire YCFl gene was subclonedinto plasmid pIBI30 to. Grant Mol-RR00042 to the General Clinical Research Center, Univer- generate pIBrYCFl[14].pIBNCFl was linearizedat a unique Sal1site sity of Michigan MedicalCenter andthe Elsa U-Pardee Foundation for in the 3’ endof the YCFl open reading framef,illed in withthe Klenow. Institutes of Health to the University of Iowa Diabetesand Endocrinol- linkers (5’-GGGATCCC-3’). The nucleotide sequencels) reported in this paper has been submitted mium factor; CFTR, cystic fibrosis transmembrane conductance reguto the GenBankmIEMBL Data Bank with accession nurnber(s) L.35237.lator; MRP1, multidrug resistance-associated proteinb;p, base pair(s);. Kinase; NBF, nucleotide binding fold R, regulatory region

A Yeast Cadmium Resistance ABC Dansporter

RESULTS

DISCUSSION