Heitman J, Koller A, Kunz J, Henriquez R, Schmidt A, Movva N R, Hall M N
Department of Biochemistry, University of Basel, Switzerland.
Mol Cell Biol. 1993 Aug;13(8):5010-9. doi: 10.1128/mcb.13.8.5010-5019.1993.
The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.
免疫抑制剂环孢素A、FK506和雷帕霉素可抑制单细胞真核微生物的生长，也能阻断多细胞真核生物中T淋巴细胞的激活。在体外，这些化合物结合并抑制两种不同类型的肽基脯氨酰顺反异构酶。环孢素A结合亲环蛋白，而FK506和雷帕霉素结合FK506结合蛋白（FKBPs）。亲环蛋白和FKBPs普遍存在、含量丰富且定位于多个细胞区室，它们可能在体内折叠蛋白质。此前，一种12 kDa的细胞质FKBP被证明只是酿酒酵母中至少两个对FK506敏感的靶点之一。我们发现氨基酸导入需要第二个对FK506敏感的靶点。氨基酸营养缺陷型酵母菌株（trp1 his4 leu2）对FK506敏感，而原养型菌株（TRP1 his4 leu2、trp1 HIS4 leu2和trp1 his4 LEU2）对FK506有抗性。向生长培养基中外源添加氨基酸可减轻FK506的毒性。FK506诱导GCN4表达，而GCN4通常由氨基酸饥饿诱导。FK506抑制色氨酸、组氨酸和亮氨酸向酵母细胞内的转运。最后，几个编码参与氨基酸导入或生物合成的蛋白质的基因赋予了对FK506的抗性。这些发现表明，FK506抑制酵母细胞中的氨基酸导入，很可能是通过抑制氨基酸转运体来实现的。氨基酸转运体是整合膜蛋白，可导入细胞外氨基酸，它们构成一个具有30%至35%同源性的蛋白质家族，包括八个不变的脯氨酸。因此，酵母细胞中第二个对FK506敏感的靶点可能是一种脯氨酰异构酶，它在氨基酸转运体通过分泌途径转运过程中参与其折叠。
