Studying organelle physiology with fusion protein-targeted avidin and fluorescent biotin conjugates.

We have developed and used a novel method for studying the lumenal pH of specific cellular organelles: a membrane-permeable, pH-sensitive fluorescein-biotin derivative is targeted to specific organelles expressing avidin chimera proteins. Until recently, the major hurdle to studying organelle pH in live cells had been the lack of appropriate methods for targeting pH probes to specific organelles. Several groups have now targeted pH dyes specifically to the Golgi, truns-Golgi network (TGN), and endoplasmic reticulum (ER) of live, intact cells by using the following methods: microinjection of cells with pH dye-filled liposomes, which appeared to fuse preferentially with the Golgi,’ targeting a fluorescently labeled bacterial toxin to the Golgi or ER via retrograde transport,2-4 and expressing pH-sensitive green fluorescent protein (GFP) mutants in specific organelles.5-7 All these methods have provided important contributions to the understanding of the regulation of organelle pH, however, each approach has certain drawbacks. Microinjection of dye-containing liposomes is technically difficult, and fusion of the liposomes to Golgi membranes may result in perturbation of