Chen S, Brockenbrough J S, Dove J E, Aris J P
Department of Anatomy and Cell Biology, Health Science Center, University of Florida, Gainesville, Florida 32610, USA.
J Biol Chem. 1997 Apr 18;272(16):10839-46. doi: 10.1074/jbc.272.16.10839.
We have generated monoclonal antibodies against nuclear proteins from the yeast Saccharomyces cerevisiae. The monoclonal antibodies react with proteins of 47 and 49 kDa on immunoblots and with partially overlapping sets of proteins on two-dimensional nonequilibrium pH gradient electrophoresis-SDS blots. Immunofluorescence localization shows a nuclear staining pattern. Immunoscreening a yeast expression library yielded five independent full-length clones of two open reading frames from chromosome IV, corresponding to YDL182w (LYS20) and YDL131w in the Saccharomyces genome data base. These two open reading frames are predicted to encode homocitrate synthase isozymes of 47 and 49 kDa, respectively. A clone carrying YDL182w was sequenced in its entirety and directs the expression of a 47-kDa protein in Escherichia coli. A clone carrying YDL131w expresses a 49-kDa protein in E. coli. Yeast grown in minimal medium plus lysine show significant reductions in nuclear immunofluorescence staining. Cell fractionation studies localize the 47- and 49-kDa proteins to the nucleus. Nuclear fractionation studies reveal that a portion of the 47- and 49-kDa proteins can only be extracted with DNase digestion and high salt. The localization of homocitrate synthase to the nucleus is unexpected given previous reports that homocitrate synthase is present in mitochondria and the cytoplasm in S. cerevisiae.
我们已制备出针对酿酒酵母核蛋白的单克隆抗体。这些单克隆抗体在免疫印迹中与47 kDa和49 kDa的蛋白质发生反应，在二维非平衡pH梯度电泳-SDS印迹中与部分重叠的蛋白质组发生反应。免疫荧光定位显示出核染色模式。对酵母表达文库进行免疫筛选，从第四条染色体中获得了两个开放阅读框的五个独立全长克隆，分别对应于酿酒酵母基因组数据库中的YDL182w（LYS20）和YDL131w。预计这两个开放阅读框分别编码47 kDa和49 kDa的同柠檬酸合酶同工酶。对携带YDL182w的克隆进行了全序列测定，并在大肠杆菌中指导表达一种47 kDa的蛋白质。携带YDL131w的克隆在大肠杆菌中表达一种49 kDa的蛋白质。在添加赖氨酸的基本培养基中生长的酵母，其核免疫荧光染色显著减少。细胞分级分离研究将47 kDa和49 kDa的蛋白质定位于细胞核。细胞核分级分离研究表明，一部分47 kDa和49 kDa的蛋白质只能通过DNA酶消化和高盐提取。鉴于之前有报道称同柠檬酸合酶存在于酿酒酵母的线粒体和细胞质中，同柠檬酸合酶定位于细胞核这一情况出乎意料。
