Organelle-specific zinc detection using zinpyr-labeled fusion proteins in live cells.
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.
Tomat
Elisa
E
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Nolan
Elizabeth M
EM
Jaworski
Jacek
J
Lippard
Stephen J
SJ
D002470:Cell Survival; D005452:Fluoresceins / Q000032:analysis* / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D015394:Molecular Structure; D015388:Organelles / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry; D015032:Zinc / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
D005452:Fluoresceins; D011993:Recombinant Fusion Proteins; D015032:Zinc; C501987:zinpyr-1