Kwan T, Gros P
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Biochemistry. 1998 Mar 10;37(10):3337-50. doi: 10.1021/bi972680x.
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-gp family and other ABC transporters suggests an important role in a common 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 mutagenesis was carried out using Taq DNA polymerase and dITP under conditions of low polymerase fidelity, and the mutagenized segments were reintroduced 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 confer cellular resistance to the antifungal drug FK506 and the peptide ionophore valinomycin, and by their ability to complement the yeast Ste6 gene and restore mating in a yeast strain bearing a null mutation [Raymond, M., et al. (1992) Science 256, 232-4] at this locus. The analysis of 782 independent yeast transformants allowed the identification of 49 independent mutants bearing single amino acid substitutions in the mutagenized segment resulting in an altered P-gp function. The mutants could be phenotypically classified into two major groups, those that resulted in partial or complete overall loss of function and those that seemed to affect substrate specificity. Several of the mutants affecting overall activity mapped in IC1; in particular we identified a segment of four consecutive mutation sensitive residues (TRLT, positions 169-172) with such a phenotype. On the other hand, we identified a cluster of mutants affecting substrate specificity within the short EC2 segment and in the adjacent portion of the neighboring TM4 domain. Expression and partial purification of a representative subset of these mutants showed that in all but two cases, loss of function was associated with loss of drug-induced ATPase activity of P-gp. Therefore, it appears that TM domains, IC and EC loops, are structurally and functionally tightly coupled in the process of drug stimulatable ATPase characteristic of P-gp.
连接跨膜（TM）结构域的单个细胞内（IC）环在P-糖蛋白（P-gp）功能中的作用仍 largely 未知。这些区域在P-gp家族和其他ABC转运蛋白中的高度序列保守性表明它们在这些蛋白质的共同作用机制中起重要作用。为了深入了解这个问题，我们随机诱变了TM2的一部分、整个IC1环、TM3、整个细胞外环（EC2）和TM4的一部分，并分析了这些突变对P-gp功能的影响。使用Taq DNA聚合酶和dITP在低聚合酶保真度条件下进行随机诱变，并通过在酿酒酵母菌株JPY201中的同源重组将诱变片段重新引入全长mdr3 cDNA中。通过突变型P-gp变体赋予细胞对抗真菌药物FK506和肽离子载体缬氨霉素的抗性的能力，以及通过它们补充酵母Ste6基因并在该位点具有无效突变的酵母菌株中恢复交配的能力，在酵母中分析突变型P-gp变体的生物学活性。对782个独立的酵母转化体的分析使得能够鉴定出49个在诱变片段中具有单个氨基酸取代的独立突变体，这些突变导致P-gp功能改变。这些突变体在表型上可分为两大类，一类导致部分或完全整体功能丧失，另一类似乎影响底物特异性。几个影响整体活性的突变体定位于IC1中；特别是我们鉴定出一段具有这种表型的四个连续突变敏感残基（TRLT，位置169 - 172）。另一方面，我们在短的EC2片段和相邻的TM4结构域的相邻部分中鉴定出一组影响底物特异性的突变体。这些突变体的一个代表性子集的表达和部分纯化表明，除了两个案例外，在所有情况下，功能丧失与P-gp的药物诱导的ATP酶活性丧失相关。因此，似乎TM结构域、IC和EC环在P-gp特征性的药物刺激ATP酶过程中在结构和功能上紧密耦合。
