Varanasi U S, Klis M, Mikesell P B, Trumbly R J
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699, USA.
Mol Cell Biol. 1996 Dec;16(12):6707-14. doi: 10.1128/MCB.16.12.6707.
The Cyc8 (Ssn6)-Tup1 corepressor complex is required for repression in several important regulatory systems in yeast cells, including glucose repression and mating type. Cyc8-Tup1 is recruited to target genes by interaction with diverse repressor proteins that bind directly to DNA. Since the complex has a large apparent molecular mass of 1,200 kDa on nondenaturing gels (F. E. Williams, U. Varanasi, and R. J. Trumbly, Mol. Cell. Biol. 11:3307-3316, 1991), we used a variety of approaches to determine its actual subunit composition. Immunoprecipitation of epitope-tagged complex and reconstitution of the complex from in vitro-translated proteins demonstrated that only the Cyc8 and Tup1 proteins were present in the complex. Hydrodynamic properties showed that these proteins have unusually large Stokes radii, low sedimentation coefficients, and high frictional ratios, all characteristic of asymmetry which partly accounts for the apparent high molecular weight. Calculation of native molecular weights from these properties indicated that the Cyc8-Tup1 complex is composed of one Cyc8 subunit and four Tup1 subunits. This composition was confirmed by reconstitution of the complex from Cyc8 and Tup1 expressed in vitro and analysis by one- and two-dimensional gel electrophoresis.
Cyc8（Ssn6）-Tup1共抑制因子复合物在酵母细胞的几个重要调控系统中发挥抑制作用是必需的，包括葡萄糖抑制和交配型调控。Cyc8-Tup1通过与直接结合DNA的多种阻遏蛋白相互作用被招募到靶基因上。由于该复合物在非变性凝胶上具有1200 kDa的大表观分子量（F. E. Williams、U. Varanasi和R. J. Trumbly，《分子细胞生物学》11:3307-3316，1991年），我们采用了多种方法来确定其实际的亚基组成。对表位标记复合物进行免疫沉淀以及从体外翻译的蛋白质中重建该复合物表明，该复合物中仅存在Cyc8和Tup1蛋白。流体动力学性质表明，这些蛋白质具有异常大的斯托克斯半径、低沉降系数和高摩擦比，这些都是不对称性的特征，这部分解释了其表观高分子量的原因。根据这些性质计算天然分子量表明，Cyc8-Tup1复合物由一个Cyc8亚基和四个Tup1亚基组成。通过从体外表达的Cyc8和Tup1重建该复合物并进行一维和二维凝胶电泳分析，证实了这一组成。
