Pál Margit, Nagy Olga, Ménesi Dalma, Udvardy Andor, Deák Péter
Institute of Biochemistry, Biological Research Center, H-6726 Szeged, Hungary.
J Cell Sci. 2007 Sep 15;120(Pt 18):3238-48. doi: 10.1242/jcs.004762.
The anaphase-promoting complex/cyclosome or APC/C is a key regulator of chromosome segregation and mitotic exit in eukaryotes. It contains at least 11 subunits, most of which are evolutionarily conserved. The most abundant constituents of the vertebrate APC/C are the four structurally related tetratrico-peptide repeat (TPR) subunits, the functions of which are not yet precisely understood. Orthologues of three of the TPR subunits have been identified in Drosophila. We have shown previously that one of the TPR subunits of the Drosophila APC/C, Apc3 (also known as Cdc27 or Mákos), is essential for development, and perturbation of its function results in mitotic cyclin accumulation and metaphase-like arrest. In this study we demonstrate that the Drosophila APC/C associates with a new TPR protein, a genuine orthologue of the vertebrate Apc7 subunit that is not found in yeasts. In addition to this, transgenic flies knocked down for three of the TPR genes Apc6 (Cdc16), Apc7 and Apc8 (Cdc23), by RNA interference were established to investigate their function. Whole-body expression of subunit-specific dsRNA efficiently silences these genes resulting in only residual mRNA concentrations. Apc6/Cdc16 and Apc8/Cdc23 silencing induces developmental delay and causes different pupal lethality. Cytological examination showed that these animals had an elevated level of apoptosis, high mitotic index and delayed or blocked mitosis in a prometaphase-metaphase-like state with overcondensed chromosomes. The arrested neuroblasts contained elevated levels of cyclin B but, surprisingly, cyclin A appeared to be degraded normally. Contrary to the situation for the Apc6/Cdc16 and Apc8/Cdc23 genes, the apparent loss of Apc7 function does not lead to the above abnormalities. Instead, the Apc7 knocked down animals and null mutants are viable and fertile, although they display mild chromosome segregation defects and anaphase delay. Nevertheless, the Apc7 subunit shows synergistic genetic interaction with Apc8/Cdc23 that, together with the phenotypic data, assumes a limited functional role for Apc7. Taken together, these data suggest that the structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex.
后期促进复合物/环体（APC/C）是真核生物中染色体分离和有丝分裂退出的关键调节因子。它至少包含11个亚基，其中大部分在进化上是保守的。脊椎动物APC/C中最丰富的成分是四个结构相关的四三肽重复（TPR）亚基，其功能尚未完全明确。在果蝇中已鉴定出其中三个TPR亚基的直系同源物。我们之前已经表明，果蝇APC/C的一个TPR亚基Apc3（也称为Cdc27或Mákos）对发育至关重要，其功能的扰动会导致有丝分裂周期蛋白积累和中期样停滞。在本研究中，我们证明果蝇APC/C与一种新的TPR蛋白相关联，该蛋白是脊椎动物Apc7亚基的真正直系同源物，在酵母中未发现。除此之外，通过RNA干扰建立了敲低三个TPR基因Apc6（Cdc16）、Apc7和Apc8（Cdc23）的转基因果蝇，以研究它们的功能。亚基特异性dsRNA的全身表达有效地使这些基因沉默，导致仅残留mRNA浓度。Apc6/Cdc16和Apc8/Cdc23的沉默诱导发育延迟并导致不同程度的蛹期致死率。细胞学检查表明，这些动物的凋亡水平升高，有丝分裂指数高，并且在有丝分裂前期-中期样状态下有丝分裂延迟或受阻，染色体过度浓缩。停滞的神经母细胞中细胞周期蛋白B水平升高，但令人惊讶的是，细胞周期蛋白A似乎正常降解。与Apc6/Cdc16和Apc8/Cdc23基因的情况相反，Apc7功能的明显丧失不会导致上述异常。相反，敲低Apc7的动物和无效突变体是可存活和可育的，尽管它们表现出轻微的染色体分离缺陷和后期延迟。然而，Apc7亚基与Apc8/Cdc23表现出协同遗传相互作用，结合表型数据，表明Apc7具有有限的功能作用。综上所述，这些数据表明结构相关的TPR亚基对后期促进复合物的功能贡献不同。
