Dunn B, Stearns T, Botstein D
Department of Genetics, Stanford University School of Medicine, California 94305.
Nature. 1993 Apr 8;362(6420):563-5. doi: 10.1038/362563a0.
The essential Ras-related GTPases Ypt1 and Sec4 act at distinct stages of the secretion pathway in the yeast Saccharomyces cerevisiae: Ypt1 is required for vesicular transport from the endoplasmic reticulum to the Golgi apparatus, whereas Sec4 is required for fusion of secretory vesicles to the plasma membrane. Here we use chimaeras of the two proteins to identify a 9-residue segment of Ypt1 that, when substituted for the analogous segment of Sec4, allows the chimaera to perform the minimal functions of both proteins in vivo. This segment corresponds to loop L7 of the p21ras crystal structure. Substitution of a 24-residue Ypt1 segment, including the residues just mentioned, together with 12 residues of Ypt1 corresponding to the 'effector region' of p21ras (loop L2; refs 7,8), transforms Sec4 into a fully functional Ypt1 protein without residual Sec4 function.
在酿酒酵母中，重要的Ras相关GTP酶Ypt1和Sec4在分泌途径的不同阶段发挥作用：从内质网到高尔基体的囊泡运输需要Ypt1，而分泌囊泡与质膜的融合需要Sec4。在这里，我们使用这两种蛋白质的嵌合体来鉴定Ypt1的一个9个残基的片段，当它替代Sec4的类似片段时，能使嵌合体在体内执行这两种蛋白质的基本功能。该片段对应于p21ras晶体结构的L7环。用一个24个残基的Ypt1片段（包括上述残基）以及与p21ras的“效应区”（L2环；参考文献7,8）对应的12个Ypt1残基进行替换，可将Sec4转化为具有完整功能的Ypt1蛋白，且无Sec4的残留功能。
