The cyclic expression of the period (PER) and timeless (TIM) proteins is cr
itical for the molecular circadian feedback Loop in Drosophila. The entrain
ment by Light of the circadian clock is mediated by a reduction in TIM Leve
ls. To elucidate the mechanism of this process, the sensitivity of TIM regu
lation by Light was tested in an in vitro assay with inhibitors of candidat
e proteolytic pathways. The data suggested that TIM is degraded through a u
biquitin-proteasome mechanism. In addition, in cultures from third-instar L
arvae, TIM degradation was blocked specifically by inhibitors of proteasome
activity. Degradation appeared to be preceded by tyrosine phosphorylation.
Finally, TIM was ubiquitinated in response to Light in cultured cells.