Phenotypes on demand via switchable target protein degradation in multicellular organisms

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.

Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

“…Recombinant PRT1 had a preference for binding to the large aromatic acids tyrosine and phenylalanine, consistent with previously suggested specificity ( Fig. 5E; Potuschak et al 1998;Stary 2003;Faden et al 2016). To assess whether PRT1 had a role in DA1-mediated BB degradation, BB was expressed with an N-terminal ubiquitin fusion and a C-terminal luciferase fusion to reveal neo-N termini in Col-0 or prt1 mutant mesophyll protoplasts.…”

Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

“…Recombinant PRT1 had a preference for binding to the large aromatic acids tyrosine and phenylalanine, consistent with previously suggested specificity ( Fig. 5E) (Potuschak et al 1998;Stary 2003;Faden et al 2016). To assess whether PRT1 had a role in DA1-mediated BB degradation, BB was expressed with an N-terminal ubiquitin fusion and a Cterminal luciferase fusion to reveal neo-N termini in Col-0 or prt1 mutant mesophyll protoplasts.…”

Real‐time detection of N‐end rule‐mediated ubiquitination via fluorescently labeled substrate probes

“…The use of radioactive isotopes requires at least the running of an SDS‐PAGE and gel drying or western blotting followed by autoradiography for hours to days (Table S1). Besides the described in vitro methods, several protocols and tools were successfully applied in vivo , mainly based on translational fusions of fluorescent proteins to degrons of the Ub fusion degradation (UFD) pathway (Hamer et al ., ; Matilainen et al ., ), the N‐end rule pathway (Speese et al ., ; Faden et al ., ) or both (Dantuma et al ., ). Other methods make use of Ub‐binding systems to achieve various read‐outs (Marblestone et al ., ; Matilainen et al ., ; Table S1).…”

“…A novel in vivo protein stabilization tool for genetic studies in developmental biology and biotechnological applications, the low‐temperature (‘lt)‐degron’, works in plants and animals by directly switching the levels of functional proteins in vivo (Faden et al ., ). The method is based on conditional and specific PRT1‐mediated protein degradation, a process we studied in depth with the generated fluorescently labeled substrate reporter proteins.…”