Liu H Y, Chiang Y C, Pan J, Chen J, Salvadore C, Audino D C, Badarinarayana V, Palaniswamy V, Anderson B, Denis C L
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824.
J Biol Chem. 2001 Mar 9;276(10):7541-8. doi: 10.1074/jbc.M009112200. Epub 2000 Dec 11.
The CCR4-NOT transcriptional regulatory complex affects transcription both positively and negatively and consists of the following two complexes: a core 1 x 10(6) dalton (1 MDa) complex consisting of CCR4, CAF1, and the five NOT proteins and a larger, less defined 1.9-MDa complex. We report here the identification of two new factors that associate with the CCR4-NOT proteins as follows: CAF4, a WD40-containing protein, and CAF16, a putative ABC ATPase. Whereas neither CAF4 nor CAF16 was part of the core CCR4-NOT complex, both CAF16 and CAF4 appeared to be present in the 1.9-MDa complex. CAF4 also displayed physical interactions with multiple CCR4-NOT components and with DBF2, a likely component of the 1.9-MDa complex. In addition, both CAF4 and CAF16 were found to interact in a CCR4-dependent manner with SRB9, a component of the SRB complex that is part of the yeast RNA polymerase II holoenzyme. The three related SRB proteins, SRB9, SRB10, and SRB11, were found to interact with and to coimmunoprecipitate DBF2, CAF4, CCR4, NOT2, and NOT1. Defects in SRB9 and SRB10 also affected processes at the ADH2 locus known to be controlled by components of the CCR4-NOT complex; an srb9 mutation was shown to reduce ADH2 derepression and either an srb9 or srb10 allele suppressed spt10-enhanced expression of ADH2. In addition, srb9 and srb10 alleles increased ADR1(c)-dependent ADH2 expression; not4 and not5 deletions are the only other known defects that elicit this phenotype. These results suggest a close physical and functional association between components of the CCR4-NOT complexes and the SRB9, -10, and -11 components of the holoenzyme.
CCR4 - NOT转录调控复合体对转录具有正向和负向影响，它由以下两个复合体组成：一个核心的1×10⁶道尔顿（1 MDa）复合体，由CCR4、CAF1和五种NOT蛋白组成；另一个更大、结构不太明确的1.9 - MDa复合体。我们在此报告鉴定出两个与CCR4 - NOT蛋白相关的新因子：CAF4，一种含WD40的蛋白；以及CAF16，一种假定的ABC ATP酶。虽然CAF4和CAF16都不是核心CCR4 - NOT复合体的组成部分，但CAF16和CAF4似乎都存在于1.9 - MDa复合体中。CAF4还与多个CCR4 - NOT组分以及DBF2（1.9 - MDa复合体的一个可能组分）存在物理相互作用。此外，发现CAF4和CAF16都以CCR4依赖的方式与SRB9相互作用，SRB9是SRB复合体的一个组分，而SRB复合体是酵母RNA聚合酶II全酶的一部分。发现三种相关的SRB蛋白，SRB9、SRB10和SRB11，与DBF2、CAF4、CCR4、NOT2和NOT1相互作用并共免疫沉淀。SRB9和SRB10的缺陷也影响了ADH2基因座处已知受CCR4 - NOT复合体组分控制的过程；一个srb9突变被证明会降低ADH2的去抑制作用，并且一个srb9或srb10等位基因会抑制spt10增强的ADH2表达。此外，srb9和srb10等位基因会增加ADR1(c)依赖的ADH2表达；not4和not5缺失是引发这种表型的仅有的其他已知缺陷。这些结果表明CCR4 - NOT复合体的组分与全酶的SRB9、-10和-11组分之间存在紧密的物理和功能关联。
