Liku Muluye E, Nguyen Van Q, Rosales Audrey W, Irie Kaoru, Li Joachim J
Department of Biochemistry, University of California, San Francisco, CA 94143-2200, USA.
Mol Biol Cell. 2005 Oct;16(10):5026-39. doi: 10.1091/mbc.e05-05-0412. Epub 2005 Aug 10.
Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (pre-RCs) at replication origins to prevent inappropriate rereplication. In Saccharomyces cerevisiae, one of these mechanisms promotes the net nuclear export of a pre-RC component, the Mcm2-7 complex, during S, G2, and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raises the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.
细胞周期蛋白依赖性激酶（CDK）利用多种机制来阻止复制起始点处前复制复合体（pre-RC）的重新组装，以防止不适当的再复制。在酿酒酵母中，其中一种机制是在S期、G2期和M期促进前复制复合体组分Mcm2-7复合体的净核输出。在此，我们在Mcm2和Mcm3上鉴定出两个部分核定位信号（NLS），它们对于Mcm2-7复合体的核定位均是必需的，但并不充分。然而，当顺式排列在一起时，这两个部分信号构成一个有效的NLS，当与原本定位于细胞质的蛋白质融合时，足以实现稳固的核定位。我们还在Mcm3 NLS附近鉴定出一个依赖于Crm1的核输出信号（NES）。值得注意的是，Mcm2-Mcm3 NLS和Mcm3 NES足以形成一个转运模块，该模块概括了整个Mcm2-7复合体的细胞周期调控定位。此外，我们表明CDK调控通过磷酸化该模块的Mcm3部分来促进净输出，并且Mcm2-7复合体的核输出足以破坏复制起始。我们推测，部分转运信号在复合体不同亚基之间的分布可能会增强蛋白质定位的特异性，并增加其他多蛋白复合体使用先前未检测到的分布式转运信号的可能性。
