Conditional control of fluorescent protein degradation by an auxin-dependent nanobody

Nat Commun. 2018 Aug 17;9(1):3297. doi: 10.1038/s41467-018-05855-5.

Abstract

The conditional and reversible depletion of proteins by auxin-mediated degradation is a powerful tool to investigate protein functions in cells and whole organisms. However, its wider applications require fusing the auxin-inducible degron (AID) to individual target proteins. Thus, establishing the auxin system for multiple proteins can be challenging. Another approach for directed protein degradation are anti-GFP nanobodies, which can be applied to GFP stock collections that are readily available in different experimental models. Here, we combine the advantages of auxin and nanobody-based degradation technologies creating an AID-nanobody to degrade GFP-tagged proteins at different cellular structures in a conditional and reversible manner in human cells. We demonstrate efficient and reversible inactivation of the anaphase promoting complex/cyclosome (APC/C) and thus provide new means to study the functions of this essential ubiquitin E3 ligase. Further, we establish auxin degradation in a vertebrate model organism by employing AID-nanobodies in zebrafish.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anaphase-Promoting Complex-Cyclosome / metabolism
  • Animals
  • Cell Compartmentation
  • Green Fluorescent Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Indoleacetic Acids / metabolism*
  • Kinetics
  • Lysine / metabolism
  • Proteolysis*
  • Recombinant Fusion Proteins / metabolism
  • Single-Domain Antibodies / metabolism*
  • Zebrafish / metabolism

Substances

  • Indoleacetic Acids
  • Recombinant Fusion Proteins
  • Single-Domain Antibodies
  • Green Fluorescent Proteins
  • Anaphase-Promoting Complex-Cyclosome
  • Lysine