![]() ![]() CRY signaling in plants requires redox activity and is mediated, at least in part, by the fl avin moiety bound to CRY (23, 45). For CRY b, the mutation is thought to prevent associ- ation with fl avin, which may be required for a redox-mediated conformation change (see below). The large dele- tions in CRY-N and CRY-C may prevent such a conformation change. Since there is no common sequence that is deleted in all these constructs, this indicates either that CRY degradation requires more than one part of the molecule or that the overall conformation of the molecule is important for its recognition by the degradation system. However, none of these proteins showed a response to light. CRY-C was ex- pressed at high levels, and levels of CRY b were equivalent to those of the wild type. For CRY-N the levels were consistently low with and without light treatment, indi- cating a general instability of the protein. Treatment with light did not reduce the levels of any of these mutant CRY proteins (Fig. It corresponds to the orig- inal cry mutation isolated through a genetic screen of Drosoph- ila (39). The cry b mutation is a missense mutation within the sequence that encodes the highly conserved fl avin- binding region of Drosophila CRY. 2B, CRY-N (ami- no acids 1 to 423) has the C-terminal 119 amino acids deleted and CRY-C (amino acids 244 to 542) has the N-terminal 243 amino acids deleted. ![]() To determine whether a speci fi c region of CRY mediates its degradation, we transfected different CRY mutants and as- sayed their response to light. The presence of endogenous photic signaling mechanisms in S2 cells provided us with a system in which we could assay the degradation of transfected CRY regardless of its ability to transduce a photic signal. Consistent with the presence of endogenous CRY in our S2 cells, we found that light-dependent inhibition of PER- TIM feedback activity, which is known to be CRY dependent (4), occurred to a signi fi cant extent in the absence of trans- fected CRY (S. 2A, we con fi rmed the presence of cry RNA in S2 cells. The photoreceptor that transduced pho- tic signals to TIM was not known, but, based on data implying a role for CRY in circadian photoreception (11, 39) together with the reported light-activated direct interaction of TIM and CRY (4), we suspected that S2 cells expressed endogenous cry. We previously reported light-induced ubiquitination of TIM in S2 cells (fur- ther discussed below). This effect of light on both TIM (27) and CRY suggests that the action of a ubiquitin/proteasome deg- radation pathway may be one of the fi rst events in photic resetting in Drosophila. Both these agents were effective in blocking CRY degradation (Fig. To identify the mechanisms that degrade CRY, we treated CRY -transfected cells with light in the presence of proteasome inhibitors MG115 and lactacystin. We transfected cells with a pIZ- cry construct, in which CRY is tagged with a V5 epitope, and noticed that levels of the protein were reduced by light treatment. Light-induced instability of CRY was supported by experiments in which CRY was expressed in S2 cells (4). The pro fi le of CRY protein expression in light-dark (LD) cycles suggested that the protein is unstable in the presence of light (9).
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