Abstract
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.
Highlights
More than a century of genetic analysis underlies much of our understanding of biology
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
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
Summary
More than a century of genetic analysis underlies much of our understanding of biology. While the use of site-specific recombination systems to generate clones of cells lacking expression of a protein in the background of an otherwise viable individual [1,2,3,4] can, in some cases, overcome this barrier, proteins already present may perdure for some time and even multiple cell generations after mutant clone induction, which can complicate the analysis of the loss-of-function phenotypes. This is especially problematic in situations in which it is desirable to achieve rapid protein inactivation, such as investigations of protein function at specific stages of the cell cycle, during cell migration and morphogenesis, or during neuronal signaling. RNA interference via the expression of dsRNA or siRNAs, which has been used to interrogate the function of vital genes in a cellular or tissue-specific manner [5,6] often achieves only partial elimination of the protein-of-interest and is susceptible to the problem of protein perdurance noted above
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