title: Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m) 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. SrtA7malso 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.
authors.affiliation: Whitehead Institute for Biomedical Research , 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.
authors.forename: Qin
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Whitehead Institute for Biomedical Research , 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.|Department of Biology, Massachusetts Institute of Technology (MIT) , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Hidde L
authors.identifier: 0000-0002-1090-6071
authors.initials: HL
authors.lastname: Ploegh
authors.affiliation: Whitehead Institute for Biomedical Research , 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.
authors.forename: Matthias C
authors.initials: MC
authors.lastname: Truttmann
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D011506:Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases