Repurposing an endogenous degradation system for rapid and targeted depletion ofC. elegansproteins

Abstract

The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional lossof-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events.

A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans

“…An analysis of degradation kinetics for two in situ-tagged targets, GFP::MAD-1 and GFP:: PP1 , in the embryonic intestine revealed that GFP::MAD-1 was degraded with a half-life of ∼6 min, whereas GFP::PP1 GSP-2 , which is more abundant and cytoplasmic/nucleolar rather than nuclear, was degraded with an ∼40 min half-life. These degradation rates are comparable with rates published previously for ZF1 degron-tagged fusions targeted by ZIF-1 expression (15-30 min half-life; Armenti et al, 2014), auxin-inducible degradation (∼20 min half-life; Zhang et al, 2015) and sortase-based F-box ligation-mediated degradation (∼1 h half-life; Wu et al, 2017). Thus, degradation rates will differ between targets depending on abundance and accessibility, but are likely to be comparable for the different methods.…”

“…Of the described methods for conditional protein degradation in C. elegans (this report; Armenti et al, 2014;Wu et al, 2017;Zhang et al, 2015), each has advantages and limitations. The auxininducible degron enables simultaneous spatial (via tissue-specific expression of the TIR1 adaptor) and temporal (timing of auxin addition) control.…”

“…To this end, a method was developed that takes advantage of an endogenous C. elegans protein degradation system. With this approach, target proteins are tagged with a 36 amino acid degron sequence (ZF1); the SOCS-box adaptor protein ZIF-1, which targets ZF1-containing proteins for proteasomal degradation, is expressed in the target tissue (Armenti et al, 2014). However, a limitation of this system is that ZIF-1 plays an essential role during early embryogenesis (DeRenzo et al, 2003;Reese et al, 2000).…”

“…Here, we have developed a new system that combines potent ZIF-1-mediated protein degradation (Armenti et al, 2014) with the previously described deGradFP approach (Caussinus et al, 2012). In deGradFP, a GFP nanobody is fused to a F-box protein to degrade GFP-tagged proteins.…”

Noncentrosomal microtubules in C. elegans epithelia

“…viable alleles that partially suppress the effect of the MEI-1/MEI-2 katanin: tba-2(sb25) (E277K change), tba-2(sb27) (E194K change), tbb-2(sb26) (E439K change); the mutation tbb-2(sb26) has been used along with mei-1 or mei-2 mutations to probe the functions of MTs in the excretory canal (Shaye and Greenwald, 2015); iv. A targeted degradation strategy to overcome the essential function of GIP-2, which is part of the g-tubulin complex has been developed (Wang et al, 2015), by expressing in the epidermis a GFP nanobody::ZIF-1 fusion, ZIF-1 being an E3 ubiquitin ligase substrate-recognition subunit that will target any protein fused to GFP for degradation (Armenti et al, 2014b;Caussinus et al, 2012).…”