Action spectrum of Drosophila cryptochrome.
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.
VanVickle-Chavez
Sarah J
SJ
Departments of Ophthalmology and Visual Sciences, Washington University Medical School, St. Louis, Missouri 63110, USA.
Van Gelder
Russell N
RN
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002940:Circadian Rhythm / Q000502:physiology*; D056931:Cryptochromes; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007976:Leupeptins / Q000494:pharmacology; D017440:Photoperiod; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011499:Protein Processing, Post-Translational / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014466:Ultraviolet Rays
D056931:Cryptochromes; D015853:Cysteine Proteinase Inhibitors; D029721:Drosophila Proteins; D005420:Flavoproteins; D007976:Leupeptins; D061988:Proteasome Inhibitors; D011993:Recombinant Fusion Proteins; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde