MUTATIONAL ANALYSIS OF THE P-GLYCOPROTEIN FIRST INTRACELLULAR LOOP AND FLANKING TRANSMEMBRANE DOMAINS

The role of individual intracellular (IC) loops linking transmembrane (TM) domains in P-glycoprotein (P-gp) function remains largely unknown . The high degree of sequence conservation of these regions in the P-g p family and other ABC transporters suggests an important role in a co mmon mechanism of action of these proteins. To gain insight into this problem, we have randomly mutagenized a portion of TM2, the entire IC1 loop, TM3, the entire extracellular loop (EC2), and part of TM4, and analyzed the effect of such mutations on P-gp function. Random mutagen esis was carried out using Tag DNA polymerase and dITP under condition s of low polymerase fidelity, and the mutagenized segments were reintr oduced in the full length mdr3 cDNA by homologous recombination in the yeast Saccharomyces cerevisiae strain JPY201. The biological activity of mutant P-gp variants was analyzed in yeast by their ability to con fer cellular resistance to the antifungal drug FK506 and the peptide i onophore valinomycin, and by their ability-to complement the yeast Ste 6 gene and restore mating in a yeast strain bearing a null mutation [R aymond, M., et al. (1992) Science 256, 232-4] at this locus. The analy sis of 782 independent yeast transformants allowed the identification of 49 independent mutants bearing single amino acid substitutions in t he mutagenized segment resulting in an altered P-gp function. The muta nts could be phenotypically classified into two major groups, those th at resulted in partial or complete overall loss of function and those that seemed to affect substrate specificity. Several of the mutants af fecting overall activity mapped in IC1; in particular we identified a segment of four consecutive mutation sensitive residues (TRLT, positio ns 169-172) with such a phenotype. On the other hand, we identified a cluster of mutants affecting substrate specificity within the short EC 2 segment and in the adjacent portion of the neighboring TM4 domain. E xpression and partial purification of a representative subset of these mutants showed that in all but two cases, loss of function was associ ated with loss of drug-induced ATPase activity of P-gp. Therefore, it appears that TM domains, IC and EC loops, are structurally and functio nally tightly coupled in the process of drug stimulatable ATPase chara cteristic of P-gp.