Photo-sensitive degron variants for tuning protein stability by light

Abstract

BackgroundRegulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus.ResultsWe used the model organism Saccharomyces cerevisiae to generate psd module variants with increased and decreased stabilities in darkness or when exposed to blue light using site-specific and random mutagenesis. The variants were characterized as fusions to fluorescent reporter proteins and showed half-lives between 6 and 75 minutes in cells exposed to blue light and 14 to 187 minutes in darkness. In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module. Measuring the dark/light ratio of selected constructs in yeast cells showed that two variants were obtained with ratios twice as high as in the wild type psd module. In silico modeling of photoreceptor variant characteristics suggested that for most cases alterations in behavior were induced by changes in the light-response of the LOV2 domain.ConclusionsIn total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light. Two variants showed characteristics that are profoundly improved compared to the original construct. The modular usage of the LOV2 domain in optogenetic tools allows the usage of the mutants in the context of other applications in synthetic and systems biology as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-014-0128-9) contains supplementary material, which is available to authorized users.

FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

“…The LOV2 domain from Avena sativa phototropin 1 (AsLOV2) was utilized for light-controlled caging of peptides and ß-galactosidase expression in yeast (38). Similarly, the LOV2 domain from A. thaliana phototropin 1 (AtLOV2) has been also used for blue-light control of the cell cycle progression and expression of conditional essential genes (39, 40).…”

Synthetic strategies for plant signalling studies: molecular toolbox and orthogonal platforms

“…In addition to the reconstruction strategy above described, there is an array of molecular tools available in yeast, but still scarcely applied to plant signalling studies, for conditional protein suppression controlled with chemicals or light (see ‘inducible systems’) (Renicke et al ., ; Usherenko et al ., ; Tanaka et al ., ). It is also possible to control the subcellular localization of proteins on command and therefore regulate their activity.…”

Noise-Reducing Negative-Feedback Optogenetic Circuits in Mammalian Cells

“…To achieve this, we introduce two novel peptide elements for controlling the Tet-repressor (TetR). We fuse TetR with a lightresponsive protein domain (LOV2) 27,30,31 , which is further fused to either a Tet-inhibiting peptide (TIP) 32 or a degradation tag consisting of four amino acids 33 . These components remain hidden until illuminated, at which point they ensure light-dependent inhibition or degradation of the TetR protein [32][33][34] .…”