Baker D, Hicke L, Rexach M, Schleyer M, Schekman R
Department of Biochemistry, University of California, Berkeley 94720.
Cell. 1988 Jul 29;54(3):335-44. doi: 10.1016/0092-8674(88)90196-1.
Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.
α-因子前体从内质网到高尔基体的转运已在轻度裂解的酵母原生质球中得以重建。转运通过将外链碳水化合物偶联添加到转运至破碎原生质球内质网中的[35S]甲硫氨酸标记的α-因子前体来进行测定。该反应绝对依赖于ATP，受胞质溶胶刺激6倍，且发生在物理上可分离的封闭区室之间。转运受到鸟嘌呤核苷酸类似物GTPγS的抑制。sec23突变细胞在体内内质网到高尔基体的转运中存在温度敏感缺陷。这种缺陷已在体外使用sec23膜和胞质溶胶得以重现。在30℃下使用sec23膜进行转运需要添加含有SEC23（野生型）基因产物的胞质溶胶。这表明体外细胞器间转运反应依赖于体内转运所需的一个因子。体外sec突变的互补作用为纯化单个区室间转运因子提供了一种功能测定方法。
