Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo. Author summary Much of what we know about biological processes has come from the analysis of mutants whose loss-of-function phenotypes provide insight into their normal functions. However, for genes that are required for viability and which have multiple functions in the life of a cell or organism one can only observe mutant phenotypes produced up to the time of death. Normal functions performed in wild-type individuals later than the time of death of mutants cannot be observed. In one approach to overcoming this limitation, a class of peptide degradation signals (degrons) have been developed, which when fused to proteins-of-interest, can target those proteins for degradation in response to various stimuli (temperature, chemical agents, co-expressed proteins, or light). Here we describe a new inducible degron (the photo-N-degron or PND), which when fused to the N-terminus of a protein, can induce N-end rule-mediated degradation in response to blue-light illumination and have validated its use in both yeast and Drosophila embryos. Moreover, using the Drosophila embryonic patterning protein Cactus, we show that like the PND, the previously-described B-LID domain, but not the previously-described photosensitive degron (psd), can produce detectable light-inducible phenotypes in Drosophila embryos that are consistent with the role of Cactus in dorsal-ventral patterning.