The CPH1 Gene of Chlamydomonas reinhardtii Encodes Two Forms of Cryptochrome Whose Levels Are Controlled by Light-Induced Proteolysis  

Abstract

Cryptochromes are proteins related to DNA photolyases and have been shown to function as blue-light photoreceptors and to play important roles in circadian rhythms in both plants and animals. The CPH1 gene from Chlamydomonas reinhardtii was originally predicted to encode a putative cryptochrome protein of 867 amino acids with a predicted molecular mass of 91 kD (Small et al., 1995). However, western blotting with antibodies specific to the CPH1 protein revealed the presence of two proteins that migrate at apparent molecular mass of approximately 126 and 143 kD. A reexamination of the assigned intronexon boundaries has shown that the previously assigned intron 7 is in fact part of exon 7 which leads to a predicted protein of 1,007 amino acids corresponding to a size of 104.6 kD. The two forms of CPH1 that migrate slower on SDS-PAGE presumably result from unknown posttranslational modifications. In C. reinhardtii cells synchronized by light to dark cycles, the two slow migrating forms of CPH1 protein accumulate in the dark and disappear rapidly in the light. Both red and blue light are effective at inducing the degradation of the CPH1 proteins. Proteasomes are implicated because degradation is inhibited by MG132, a proteasome inhibitor. Studies with deletion mutants indicate that the C-terminal region is important for both the posttranslational modification and the protein's stability under both light and dark conditions.The discovery of the first blue light photoreceptor in Arabidopsis (Ahmad and Cashmore, 1993) has led to the subsequent discovery of similar proteins in several other species. Also known as cryptochromes, or CRYs, these include two each in mice (Kobayashi et al., 1998) and humans (Todo et al., 1996;van der Spek et al., 1996), one in Chlamydomonas reinhardtii (Small et al., 1995) and Drosophila melanogaster Stanewsky et al., 1998), and five in fern (Adiantum capillus-veneris; Kanegae and Wada, 1998;Imaizumi et al., 2000). Cryptochromes show significant amino acid similarity to DNA photolyases in their N termini although cryptochromes lack photolyase activity. In addition, cryptochromes contain a C-terminal extension that appears to be unique for each cryptochrome in size and amino acid sequence (Lin and Shalitin, 2003). Recently, a second class of cryptochromes, DASH, has been identified, whose members share similarities to DNA photolyases in DNA binding and recognition (Brudler et al., 2003). These proteins have been hypothesized to have redox activity and act as transcriptional regulators.Cryptochromes in Arabidopsis (AtCRY1 and At-CRY2) belong to the first class of cryptochromes, act as blue light photoreceptors, and have roles in the regulation of flowering time and plant development (Cashmore et al., 1999). The Arabidopsis CRY1 is known to regulate hypocotyl elongation and cotyledon growth in seedlings under high light intensities (Ahmad et al., 1995) while its close relative, AtCRY2, is involved in hypocotyl and cotyledon growth under conditions of low light . The AtCRY1 protein has been show...

A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii

“…The accumulation of aCRY exhibits an amplitude change of about twofold within a light/dark cycle (Figures 2A and 2B). In contrast to CPH1 (Reisdorph and Small, 2004), aCRY is not rapidly degraded in the light. The highest levels of this protein are observed at the beginning of and extending to the middle of the day (LD2 and LD6), with its lowest values at the beginning of the night (LD14) (Figures 2A and 2B).…”

“…The responses of this photoreceptor to blue light have been thoroughly investigated in vitro (Immeln et al, 2007(Immeln et al, , 2010Langenbacher et al, 2009). However, in vivo studies have been limited, although it was shown that the expression pattern of C. reinhardtii CPH1 tracks the diurnal light/dark cycle, with lightdependent CPH1 degradation mediated by the proteasome pathway; this degradation involves blue light, but surprisingly, red light also participates in the degradation process (Reisdorph and Small, 2004).…”

Phase-resetting mechanism of the circadian clock in Chlamydomonas reinhardtii

“…Although it is an attractive candidate of photoreceptor for light-induced ROC15 degradation, we could not exclude the possibility of the existence of other unknown red-light photoreceptors in C. reinhardtii. Another cryptochrome homolog, Chlamydomonas photolyase homolog 1 (CPH1), shows a rapid decrease in protein accumulation level, similar to ROC15, after exposure to blue or red light because of proteasomedependent protein degradation (39). The light-induced degradation of ROC15 and CPH1 may occur through the same pathway.…”

UV-B Perception and Acclimation in Chlamydomonas reinhardtii

“…Specifically, Chlamydomonas possesses a single phototropin (Huang and Beck, 2003), rhodopsins, including channelrhodopsins (Nagel et al, 2002(Nagel et al, , 2003Kateriya et al, 2004), a plant-like cryptochrome (Reisdorph and Small, 2004), and an animal-like cryptochrome that responds to both blue and red light (Beel et al, 2012). Furthermore, a histidine kinase rhodopsin was recently characterized that exists in both UV-A-and blue-light-absorbing isoforms (Luck et al, 2012).…”