Abstract
Cryptochromes are a highly conserved class of UV-A/blue light photoreceptors. In Drosophila, cryptochrome is required for the normal entrainment of circadian rhythms to light dark cycles. The photocycle and molecular mechanism of animal cryptochrome photoreception are presently unknown. Drosophila cryptochrome undergoes light-dependent degradation when heterologously expressed in Schneider-2 cells. We have generated Drosophila luciferase-cryptochrome fusion proteins to more precisely monitor light-dependent cryptochrome degradation. We found that the luciferase-cryptochrome fusion protein undergoes light-dependent degradation with luciferase activity declining approximately 50% within 5 min of light exposure and approximately 85% within 1 h of light exposure. Degradation is inhibited by MG-132, consistent with a proteasomal degradation mechanism. Irradiance-response curves yield an action spectrum similar to absorption spectra for prokaryotic and eukaryotic cryptochromes with highest sensitivity in the UV-A. A luciferase-cryptochrome fusion protein lacking the terminal 15 amino acids is stably expressed in the dark but demonstrates increased sensitivity to light-induced degradation. The conferral of light-dependent degradation on a heterologous protein by fusion to cryptochrome may be a useful tool for probing protein function in cell expression systems.
Highlights
Cryptochromes are blue-light photoreceptors found throughout the animal and plant kingdoms [1]
We have demonstrated that a fusion protein of luciferase and Drosophila cryptochrome undergoes light-dependent loss of luciferase activity
Given the previous demonstration by Busza et al [9] that light induces degradation of cryptochrome and given the reduction of light-dependent loss of luciferase activity following proteasome inhibition, we believe that light induces proteasome-dependent degradation of the luciferase-cryptochrome fusion protein
Summary
Drosophila cryptochrome undergoes light-dependent degradation when heterologously expressed in Schneider-2 cells. Cryptochromes are blue-light photoreceptors found throughout the animal and plant kingdoms [1] These proteins were first identified in Arabidopsis [2, 3], where they have been shown to be required for specific light-dependent developmental and circadian entrainment effects. APRIL 6, 2007 VOLUME 282 NUMBER 14 expression constructs, light induces degradation of both proteins in a dCRY-dependent manner [9]. By fusing firefly luciferase to dCRY, we are able to measure the kinetics of light-dependent degradation of cryptochrome as a function of luciferase activity. This has allowed the generation of an action spectrum for Drosophila cryptochrome
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