DYNAMIC MEASUREMENT OF THE PH OF THE GOLGI-COMPLEX IN LIVING CELLS USING RETROGRADE TRANSPORT OF THE VEROTOXIN RECEPTOR

The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia c oli is responsible for the attachment of the holotoxin to the cell sur face, by binding to the glycolipid, globotriaosyl ceramide. After rece ptor-mediated endocytosis, the toxin is targeted to the Golgi complex by a process of retrograde transport. We took advantage of this unique property of VT1B to measure the pH of the Golgi complex in intact liv e cells. Purified recombinant VT1B was labeled with either rhodamine o r fluorescein for subcellular localization by confocal microscopy, Aft er 1 h at 37 degrees C, VT1B accumulated in a juxtanuclear structure t hat colocalized with several Golgi markers, including alpha-mannosidas e II, beta-COP, and NBD-ceramide. Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accum ulation of VT1B in the Golgi complex. Imaging of the emission of fluor escein-labeled VT1B was used to measure intra-Golgi pH (pH(G)), which was calibrated in situ with ionophores. In intact Vero cells, pH(G) av eraged 6.45 +/- 0.03 (standard error). The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A(1), a blocker of vac uolar-type ATPases. pH(G) remained constant despite acidification of t he cytosol by reversal of the plasmalemmal Na+/H+ antiport. Similarly, pH(G) was unaffected by acute changes in cytosolic calcium. Furthermo re, pH(G) recovered quickly toward the basal level after departures im posed with weak bases. These findings suggest that pH(G) is actively r egulated, despite the presence of a sizable H+ ''leak'' pathway. The a bility of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic spec ies.