Comparative Photochemistry of Animal Type 1 and Type 4 Cryptochromes

Abstract

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities.

Reaction mechanism of Drosophila cryptochrome

“…It was reported that proteolytic degradation of CRY stopped when the light was turned off (23), suggesting that CRY had to be in a photochemically excited state for enzymatic modification that ultimately leads to its proteolysis. However, a subsequent study using camera flash photolysis revealed that proteolysis of CRY continues for 60 min after the light pulse (30), leading to the conclusion that light induces a long-lived signaling state conformation (Lit state) that continues to interact with downstream partners including ubiquitin ligase so that CRY could be marked for proteolysis. However, in that study the kinetics of conversion of the Lit state to the Dark state could not be determined because following the light flash, cells were incubated at 25°C, and during incubation in the dark CRY was continuously being degraded.…”

“…Although when plant and insect CRYs, expressed in heterologous systems, are purified they contain flavin in the two-electron oxidized state (36, 37) they are readily reduced by light to FADH • (36) and FAD •− (5-7) and there are some experimental data indicating that the FAD ox -form is an artifact generated by exposure to air during purification (8). (ii) Some CRYs (Arabidopsis CRY1 and human CRY1 and CRY2) but not others (Arabidopsis CRY2 and insect Type 1 CRYs) have autokinase activity (30,(38)(39)(40). Furthermore, the crystal structure of Arabidopsis CRY1 crystallized in the presence of ATP reveals that the ATP is located in the cavity leading to flavin, which corresponds to the photodimer binding site in DNA photolyases (41,42).…”

Mechanism of Photosignaling by Drosophila Cryptochrome

“…We constructed pAc5.1-V5-dCRY, which expresses V5 tag at the N terminus, to avoid any unseen/unexpected contribution of a tag located at the C terminus because one of the mutations (W536F) we used in our study is located at the very C terminus and the C-terminal extension folds onto the PHR domain (20). pFast-FH-dCRY was described previously (30). Site-directed mutations in the cloned genes for W397F, W420R, W536F, and W397F/W536F of dCRY were introduced by standard methods using the QuikChange method (Stratagene).…”

Ramshackle (Brwd3) promotes light-induced ubiquitylation of Drosophila Cryptochrome by DDB1-CUL4-ROC1 E3 ligase complex

“…The procedures for baculovirus preparation and protein purification were described previously (29,30). RNAi experiments in Drosophila S2 cells were performed following established protocols (31,32).…”