Spang A, Schekman R
Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
J Cell Biol. 1998 Nov 2;143(3):589-99. doi: 10.1083/jcb.143.3.589.
Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone alpha-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369-377). Retrieval depends on the HDEL sequence; the alpha-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.
从高尔基体到内质网的逆行转运是一个必不可少的过程。必须回收那些逃离内质网的内质网驻留蛋白以及在高尔基体和内质网之间循环的蛋白。顺行和逆行囊泡运输的相互依存关系使得在体内剖析这两个过程都很困难。我们开发了一种体外系统，用于测量一种可溶性报告蛋白的回收情况，该报告蛋白是酵母信息素α因子的前体，在其COOH末端与一个回收信号（HDEL）融合（迪恩，N.，和H.R.B.佩勒姆。1990.《细胞生物学杂志》111:369 - 377）。回收依赖于HDEL序列；天然缺乏该序列的α因子前体不会被回收。顺行和逆行运输的完整循环需要一组简单的纯化胞质蛋白，包括Sec18p、Lma1p复合体、Uso1p、外被体蛋白和Arf1p。在膜结合的v - SNAP受体（v - SNARE）蛋白中，Bos1p仅对正向运输是必需的，Sec22p仅对逆行运输是必需的，而Bet1p与这两种运输途径都有关。从富含高尔基体的膜产生的假定逆行载体（COPI囊泡）既含有v - SNARE，也含有作为货物的Emp47p。
