Hepta-Mutant Staphylococcus aureus Sortase A (SrtA_7m) as a Tool for in Vivo Protein Labeling in Caenorhabditis elegans

Abstract

In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca2+ environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.

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.…”

“…Thus, proteins tagged with the ZF1 degron are degraded during embryogenesis as well as in the target tissue, which is problematic for analysis of proteins that function during embryogenesis as well as at later developmental stages. More recently, a bacterial sortase-based protein ligation approach was also used to achieve targeted protein degradation in C. elegans (Wu et al, 2017). This approach requires tagging the target protein with a small five amino acid tag and expressing a tagged F-box along with a modified sortase.…”

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

“…The ease of editing the C. elegans genome using CRISPR/Cas9-based approaches 564 (Calarco and Friedland 2015) and heterologous gene manipulation tools is ushering in a 565 new era of cellular and developmental biology. Several new tools available to C. elegans 566 researchers require the insertion of small amino acid tags into target loci, including ZF1 567 tagging (Armenti et al 2014), sortase A (Wu et al 2017), and the AID system (Zhang et 568 al. 2015).…”

“…Caenorhabditis elegans, including ZF1 tagging (Armenti et al 2014), a GFP nanobody 70 approach (Wang et al 2017), sortase A (Wu et al 2017), and auxin-mediated degradation 71 (Zhang et al 2015). 72…”