Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein

The ATP binding cassette (ABC) transporter protein Yor1p was identified on the basis of its ability to elevate oligomycin resistance when it was overp roduced from a high-copy-number plasmid. Analysis of the predicted amino ac id sequence of Yor1p indicated that this protein was a new member of a subf amily of BBC transporter proteins defined by the multidrug resistance prote in (MRP). In this work, Yor1p is demonstrated to localize to the Saccharomy ces cerevisiae plasma membrane by both indirect immunofluorescence and bioc hemical fractionation studies. Several mutations were generated in the amin o-terminal nucleotide binding domain (NBD1) of Yor1p to test if the high de gree of sequence conservation in this region of the protein was important f or function, Deletion of a phenylalanine residue at Yor1p position 670 led to a mutant protein that appeared to be retained in the endoplasmic reticul um (ER) and that was unstable. As shown by others, deletion of the analogou s residue from a second mammalian MRP family member, the cystic fibrosis tr ansmembrane conductance regulator (CFTR), also led to retention of this nor mally plasma membrane-localized protein in the ER. Changes in the spacing b etween or the sequences flanking functional motifs of Yor1p NBD1 led to def ective trafficking or decreased activity of the mutant proteins. Analyses o f the degradation of wild-type and Delta F670 Yor1p indicated that the half -life of Delta F670 Yor1p was dramatically shortened. While the vacuole was the primary site for turnover of wild-type Yor1p, degradation of Delta F67 0 Yor1p was found to be more complex with both proteasomal and vacuolar con tributions.