Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins.
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 loss-of-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.
Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, NYU School of Medicine, New York, NY 10016, USA.
Stephen T
ST
Armenti
Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA.
Lauren L
LL
Lohmer
Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA.
David R
DR
Sherwood
Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, NYU School of Medicine, New York, NY 10016, USA Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA jeremy.nance@med.nyu.edu.
Jeremy
J
Nance
D000818:Animals; D015153:Blotting, Western; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005453:Fluorescence; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D005818:Genetic Engineering / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D059748:Proteolysis; D019076:Transgenes / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D017931:DNA Primers; C476529:ZIF-1 protein, C elegans; D044767:Ubiquitin-Protein Ligases
C. elegans; Genetic tool; Mosaic; Protein degradation