Drosophila TIM Binds Importin α1, and Acts as an Adapter to Transport PER to the Nucleus

Regulated nuclear entry of clock proteins is a conserved feature of eukaryotic circadian clocks and serves to separate the phase of mRNA activation from mRNA repression in the molecular feedback loop. In Drosophila, nuclear entry of the clock proteins, PERIOD (PER) and TIMELESS (TIM), is tightly controlled, and impairments of this process produce profound behavioral phenotypes. We report here that nuclear entry of PER-TIM in clock cells, and consequently behavioral rhythms, require a specific member of a classic nuclear import pathway, Importin α1 (IMPα1). In addition to IMPα1, rhythmic behavior and nuclear expression of PER-TIM require a specific nuclear pore protein, Nup153, and Ran-GTPase. IMPα1 can also drive rapid and efficient nuclear expression of TIM and PER in cultured cells, although the effect on PER is mediated by TIM. Mapping of interaction domains between IMPα1 and TIM/PER suggests that TIM is the primary cargo for the importin machinery. This is supported by attenuated interaction of IMPα1 with TIM carrying a mutation previously shown to prevent nuclear entry of TIM and PER. TIM is detected at the nuclear envelope, and computational modeling suggests that it contains HEAT-ARM repeats typically found in karyopherins, consistent with its role as a co-transporter for PER. These findings suggest that although PER is the major timekeeper of the clock, TIM is the primary target of nuclear import mechanisms. Thus, the circadian clock uses specific components of the importin pathway with a novel twist in that TIM serves a karyopherin-like role for PER.

生物钟蛋白的调控性核输入是真核生物昼夜节律钟的保守特征，其功能是在分子反馈环路中区分mRNA激活与mRNA抑制的时相。在果蝇（Drosophila）中，生物钟蛋白PERIOD（PER）与TIMELESS（TIM）的核输入过程受到严格调控，该过程的功能缺陷会引发显著的行为表型。本研究证实，生物钟细胞中PER-TIM复合物的核输入以及由此产生的行为节律，依赖于经典核输入通路的特定成员——Importin α1（IMPα1）。除IMPα1之外，PER-TIM的核表达与节律性行为还需要特定的核孔蛋白Nup153以及Ran-GTP酶（Ran-GTPase）。IMPα1还可在培养细胞中快速且高效地介导TIM与PER的核表达，不过其对PER的作用需由TIM介导。对IMPα1与TIM/PER相互作用结构域的定位分析显示，TIM是该输入蛋白复合物的主要转运底物。此前研究已证实，携带特定突变的TIM会阻断TIM与PER的核输入过程，而IMPα1与该突变型TIM的相互作用显著减弱，这一结果进一步支持了上述结论。研究在核被膜处检测到了TIM，且计算机建模显示TIM含有通常在核转运蛋白（karyopherins）中存在的HEAT-ARM重复序列，这与其作为PER共转运蛋白的功能相符。上述研究结果表明，尽管PER是昼夜节律钟的核心计时分子，但TIM是核输入机制的主要作用靶点。因此，昼夜节律钟利用了输入蛋白通路的特定组分，并展现出全新的调控机制：TIM可发挥类似核转运蛋白的功能，辅助PER完成核输入。