Organelle-specific zinc detection using zinpyr-labeled fusion proteins in live cells

J Am Chem Soc. 2008 Nov 26;130(47):15776-7. doi: 10.1021/ja806634e.

Abstract

A protein labeling approach is employed for the localization of a zinc-responsive fluorescent probe in the mitochondria and in the Golgi apparatus of living cells. ZP1, a zinc sensor of the Zinpyr family, was functionalized with a benzylguanine moiety and thus converted into a substrate (ZP1BG) for the human DNA repair enzyme alkylguaninetransferase (AGT or SNAP-Tag). The labeling reaction of purified glutathione S-transferase tagged AGT with ZP1BG and the zinc response of the resulting protein-bound sensor were confirmed in vitro. The new detection system, which combines a protein labeling methodology with a zinc fluorescent sensor, was tested in live HeLa cells expressing AGT in specific locations. The enzyme was genetically fused to site-directing proteins that anchor the probe onto targeted organelles. Localization of the zinc sensors in the Golgi apparatus and in the mitochondria was demonstrated by fluorescence microscopy. The protein-bound fluorescence detection system is zinc-responsive in living cells.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Survival
  • Fluoresceins / analysis*
  • Fluoresceins / chemistry
  • HeLa Cells
  • Humans
  • Molecular Structure
  • Organelles / metabolism*
  • Recombinant Fusion Proteins / analysis*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism*
  • Spectrophotometry
  • Zinc / analysis*
  • Zinc / chemistry
  • Zinc / metabolism*

Substances

  • Fluoresceins
  • Recombinant Fusion Proteins
  • Zinc
  • zinpyr-1