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

Elisa Tomat, Elizabeth M. Nolan, Jacek Jaworski, Stephen J. Lippard

Research output: Contribution to journalArticlepeer-review

164 Scopus citations

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 fluroescence detection system is zinc-responsive in living cells.

Original languageEnglish (US)
Pages (from-to)15776-15777
Number of pages2
JournalJournal of the American Chemical Society
Volume130
Issue number47
DOIs
StatePublished - Nov 26 2008

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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