Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor

Abstract

The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia coli is responsible for the attachment of the holotoxin to the cell surface, by binding to the glycolipid, globotriaosyl ceramide. After receptor-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 live cells. Purified recombinant VT1B was labeled with either rhodamine or fluorescein for subcellular localization by confocal microscopy. After 1 h at 37°C, VT1B accumulated in a juxtanuclear structure that colocalized with several Golgi markers, including α-mannosidase II, β-COP, and NBD-ceramide. Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accumulation of VT1B in the Golgi complex. Imaging of the emission of fluorescein-labeled VT1B was used to measure intra-Golgi pH (pH(G)), which was calibrated in situ with ionophores. In intact Vero cells, pH(G) averaged 6.45 ± 0.03 (standard error). The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A1, a blocker of vacuolar- type ATPases. pH(G) remained constant despite acidification of the cytosol by reversal of the plasmalemmal Na+/H+ antiport. Similarly, pH(G) was unaffected by acute changes in cytosolic calcium. Furthermore, pH(G) recovered quickly toward the basal level after departures imposed with weak bases. These findings suggest that pH(G) is actively regulated, despite the presence of a sizable H+ 'leak' pathway. The ability of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic species.