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J Biol Chem, Vol. 274, Issue 12, 7603-7606, March 19, 1999
From the Departments of Medicine and Physiology, Cardiovascular
Research Institute, University of California,
San Francisco, California 94143-0521
A strategy was developed to label
specified sites in living cells with a wide selection of fluorescent or
other probes and applied to study pH regulation in Golgi. cDNA
transfection was used to target a single-chain antibody to a specified
site such as an organelle lumen. The targeted antibody functioned as a
high affinity receptor to trap cell-permeable hapten-fluorophore
conjugates. Synthesized conjugates of a hapten
(4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and fluorescent
probes (Bodipy Fl, tetramethylrhodamine, fluorescein) were bound with
high affinity (~5 nM) and specific localization to
the single-chain antibody expressed in the endoplasmic reticulum,
Golgi, and plasma membrane of living Chinese hamster ovary cells. Using
the pH-sensitive phOx-fluorescein conjugate and ratio imaging
microscopy, pH was measured in the lumen of Golgi (pH 6.25 ± 0.06). Measurements of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi
provided direct evidence that resting Golgi pH is determined by
balanced leak-pump kinetics rather than the inability of the
H+/ATPase to pump against an electrochemical gradient. Like
expression of the green fluorescent protein, the receptor-mediated
fluorophore targeting approach permits specific intracellular
fluorescence labeling. A significant advantage of the new approach is
the ability to target chemical probes with custom-designed spectral and
indicator properties.
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