Phenotypes on demand via switchable target protein degradation in multicellular organisms.

Frederik Faden,Nico Dissmeyer,Knud Nairz,Wolfgang Brandt,Marceli S Froehlich,R Jürgen Dohmen,Stefan Mielke,Arp Schnittger,Isabel Almudi,Wolfgang Hoehenwarter,Thomas Ramezani,Jörg Höckendorff

doi:10.1038/ncomms12202

Abstract

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation.

Highlights

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes
Our DHFR was initiated by phenylalanine (Phe), a strongly destabilizing residue in plants, which is presumably recognized by the bona fide N-end rule E3 Ub ligase PROTEOLYSIS1 (PRT1)[24,25,26]
As the first test system, we used the formation of Arabidopsis trichomes, which are a well-established model in cell and developmental biology[27,28,29], and an attractive target for plant metabolic engineering.[30]

Summary

Introduction

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. The classical way to generate temperature-sensitive alleles comprises random mutagenesis followed by large-scale screens at different temperatures. This procedure is usually limited to fast-growing single-celled organisms where mutant populations can be analysed simultaneously at restrictive and permissive temperatures. An alternative way to generate heat-sensitive mutants is to use a temperature-inducible N-degron (td), where a protein of interest (POI) is fused to a portable N-terminal degradation cassette[1]. To date this technique has been solely used in unicellular eukaryotes in a temperature range impractical for most multicellular organisms. We show how to efficiently circumvent these limitations by using a novel portable td fusion protein to render the levels of active POIs conditional

Methods

Results

Conclusion