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.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Drosophila melanogaster/radiation effects , Optogenetics/methods , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/radiation effects , Animals , Arginine/metabolism , Avena , Cell Nucleus/metabolism , Cell Nucleus/radiation effects , Darkness , Drosophila melanogaster/embryology , Embryo, Nonmammalian/metabolism , Embryo, Nonmammalian/radiation effects , Female , Fluorescence , Lasers , Light , Loss of Function Mutation , Male , Neoplasm Proteins/metabolism , Phenotype , Proteasome Endopeptidase Complex/metabolism , Protein Domains/radiation effects , Protein Serine-Threonine Kinases/chemistry , Proteolysis/radiation effects , Ubiquitin-Protein Ligases/metabolismABSTRACT
Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.
Subject(s)
Body Patterning/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Transcription Factors/metabolism , Twist-Related Protein 1/metabolism , Animals , Body Patterning/radiation effects , Drosophila Proteins/genetics , Embryo, Nonmammalian , Light , Nuclear Proteins/genetics , Phosphoproteins/genetics , Proteolysis/radiation effects , Snail Family Transcription Factors/metabolism , Transcription Factors/genetics , Twist-Related Protein 1/geneticsABSTRACT
A complex program of translational repression, mRNA localization, and translational activation ensures that Oskar (Osk) protein accumulates only at the posterior pole of the Drosophila oocyte. Inappropriate expression of Osk disrupts embryonic axial patterning, and is lethal. A key factor in translational repression is Bruno (Bru), which binds to regulatory elements in the osk mRNA 3' UTR. After posterior localization of osk mRNA, repression by Bru must be alleviated. Here we describe an in vivo assay system to monitor the spatial pattern of Bru-dependent repression, separate from the full complexity of osk regulation. This assay reveals a form of translational activation-region-specific activation-which acts regionally in the oocyte, is not mechanistically coupled to mRNA localization, and functions by inhibiting repression by Bru. We also show that Bru dimerizes and identify mutations that disrupt this interaction to test its role in vivo. Loss of dimerization does not disrupt repression, as might have been expected from an existing model for the mechanism of repression. However, loss of dimerization does impair regional activation of translation, suggesting that dimerization may constrain, not promote, repression. Our work provides new insight into the question of how localized mRNAs become translationally active, showing that repression of osk mRNA is locally inactivated by a mechanism acting independent of mRNA localization.
