Redding K, Holcomb C, Fuller R S
Department of Biochemistry, Stanford University School of Medicine, California 94305.
J Cell Biol. 1991 May;113(3):527-38. doi: 10.1083/jcb.113.3.527.
The Kex2 protein of the yeast Saccharomyces cerevisiae is a membrane-bound, Ca2(+)-dependent serine protease that cleaves the precursors of the mating pheromone alpha-factor and the M1 killer toxin at pairs of basic residues during their transport through the secretory pathway. To begin to characterize the intracellular locus of Kex2-dependent proteolytic processing, we have examined the subcellular distribution of Kex2 protein in yeast by indirect immunofluorescence. Kex2 protein is located at multiple, discrete sites within wild-type yeast cells (average, 3.0 +/- 1.7/mother cell). Qualitatively similar fluorescence patterns are observed at elevated levels of expression, but no signal is found in cells lacking the KEX2 gene. Structures containing Kex2 protein are not concentrated at a perinuclear location, but are distributed throughout the cytoplasm at all phases of the cell cycle. Kex2-containing structures appear in the bud at an early, premitotic stage. Analysis of conditional secretory (sec) mutants demonstrates that Kex2 protein ordinarily progresses from the ER to the Golgi but is not incorporated into secretory vesicles, consistent with the proposed localization of Kex2 protein to the yeast Golgi complex.
酿酒酵母的Kex2蛋白是一种膜结合的、Ca2(+)依赖的丝氨酸蛋白酶，它在交配信息素α因子和M1杀伤毒素的前体通过分泌途径运输过程中，于成对的碱性残基处将它们切割。为了开始表征Kex2依赖性蛋白水解加工的细胞内位点，我们通过间接免疫荧光检查了酵母中Kex2蛋白的亚细胞分布。Kex2蛋白位于野生型酵母细胞内的多个离散位点（平均每个母细胞3.0±1.7个）。在表达水平升高时观察到定性相似的荧光模式，但在缺乏KEX2基因的细胞中未发现信号。含有Kex2蛋白的结构并不集中在核周位置，而是在细胞周期的所有阶段分布于整个细胞质中。含有Kex2的结构在芽的有丝分裂前期早期出现。对条件性分泌（sec）突变体的分析表明，Kex2蛋白通常从内质网转运到高尔基体，但不被整合到分泌小泡中，这与Kex2蛋白定位于酵母高尔基体复合体的提议一致。
