Mechanisms underlying the role of Drosophila timeless in the molecular clock and in the circadian response to light

Despite major advances in circadian biology, we still do not understand how the clock is sustained and how it generates a 24-hour oscillation. In Drosophila melanogaster, circadian rhythms are driven by a negative feedback loop that includes the key regulators, period ( per) and timeless (tim). A critical step in this molecular cycle is the timed nuclear localization of PER/TIM, but the mechanisms underlying this are not known. In addition, while entrainment of the molecular cycle to light is known to require light-induced degradation of TIM, how this event resets the clock is not understood. In this thesis, we identified specific components that mediate the nuclear translocation of clock proteins and also report that a specific residue in TIM regulates its light-induced degradation. We show that a specific Importin, importin &agr;1, targets TIM as a cargo, rather than the major timekeeper PER, and TIM in turn acts like a karyopherin to co-transport PER into the nucleus. Other components of the importin pathway (e.g. RAN-GTP and nuclear pore protein NUP153) are also shown to be required for nuclear entry of PER and TIM. Taken together, we report a unique twist to the known nuclear import pathway, in that a cargo (TIM) serves as an adaptor to transport another cargo (PER), and identify new components required for clock function. In our work on light-dependent regulation of TIM, we found that a specific tyrosine residue (Y48) on TIM is required for robust TIM degradation by light. Mutation of the Y48 residue on TIM has even more profound effects on light-induced modification of PER and on circadian behavioral activity of flies. These findings suggest that prior to its degradation TIM transmits photic signals to PER. These studies are expected to contribute to an understanding of how a circadian clock is generated and sustained, which is a long-debated but yet unsolved question.