Figure 2.

The tim^PL mutation and a mutation in a neighboring threonine prevent nuclear localization and light-induced degradation of TIM. A, S2R+ cells were transiently transfected with wild-type tim (wt), tim^PL (PL), or tim carrying a mutation in a neighboring threonine tim (TA). All tim constructs were tagged with YFP. Cells were treated with 10 nm LMB for 2 h, and then fixed and examined by fluorescence confocal microscopy. Scale bar, 25 μm. The distribution of cells showing mostly nuclear, mostly cytoplasmic, or both nuclear and cytoplasmic TIM is indicated. Two additional independent experiments showed similar results. B, Light-induced degradation of TIM is reduced in tim^PL mutants. Flies were collected at ZT20 with or without a 1 h light exposure, and fly head extracts were examined by Western blot analysis. C, Light-induced TIM degradation is inhibited by the PL (left) and TA (right) mutations. S2 cells were transiently transfected with pAc-tim along with MYC epitope-tagged pIZ-cry and FLAG epitope-tagged pIZ-jet, with or without 10 nm LMB. MAPK was probed to control for loading. Two other identical independent experiments showed similar results.