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

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.

Opticool: Cutting-edge transgenic optical tools

“…B-LID contains a small peptide degron [141] rendered cryptic by fusion to a LOV2 domain; in this way, degradation of fusion proteins is triggered by exposure of the degron upon exposure to blue light (see Fig 3A). Photo-N-degron was recently developed to take advantage of light-dependent N-end rule-mediated protein degradation [142]. Light-induced uncoiling of the Jα helix in LOV2 exposes an N-terminal arginine amino acid and triggers N-end rule degradation [143,144] (see Fig 3A).…”

Precise modulation of embryonic development through optogenetics

“…Other than intermolecular association and dissociation, the intramolecular conformational change of PAP can also be used to control target proteins' activity. The allosteric transition of the Ja helix in AsLOV2 (Figure 1f) can be used to achieve control of the cell signaling pathway (Čapek et al, 2019; Murakoshi et al, 2017), programmed cell death (Smart et al, 2017), cytoskeleton reorganization (Harris et al, 2020; Rich et al, 2020), transcription factor activity (Irizarry et al, 2020), protein degradation (Stevens et al, 2021).…”

BMP Signaling: Lighting up the Way for Embryonic Dorsoventral Patterning

“…The maternal gene dorsal is ubiquitously expressed in the oocyte, as is the Dorsal protein ( Zeitlinger et al, 2007 ; Irizarry and Stathopoulos, 2021 ). Cactus, the fly orthologue of the mammalian transcription factor NFκB, inhibits Dorsal ( Stevens et al, 2021 ). Cactus forms a complex with Dorsal to prevent the transportation of the Dorsal protein into the nucleus of the oocyte ( Stein and Stevens, 2014 ; Liu et al, 2016 ).…”