To avoid the unpredictability of de novo sensor design, we
adopted the aminoethyltriphenylphosphonium (TPP) ion as
a small chemical tag to direct a mobile zinc sensor to the mitochondrion,
a well-established strategy (21–27) based on known
intracellular physiochemical properties. We created a derivative
of the widely applied zinc sensor ZP1 (16) with the TPP ion attached
via an amide linkage to the 6-position on the benzoic acid
ring of the fluorophore (Fig. 1). Unexpectedly, the resulting construct,
ZP1-TPP, sequestered within endosomes/lysosomes and
lost its ability to respond to changes in mobile zinc concentrations
(vide infra).
To prevent such endosomal localization and afford TPP-mediated
mitochondrial targeting in live cells, we altered the physical properties
of ZP1-TPP through conversion to the fluorescein diacetate