Mechanism of Acidification of the trans-Golgi Network (TGN): IN SITU MEASUREMENTS OF pH USING RETRIEVAL OF TGN38 AND FURIN FROM THE CELL SURFACE

Abstract

Sorting of secretory cargo and retrieval of components of the biosynthetic pathway occur at the trans-Golgi network (TGN). The pH within the TGN is thought to be an important determinant of these functions. However, studies of the magnitude and regulation of the pH of the TGN have been hampered by the lack of appropriate detection methods. This report describes a noninvasive strategy to measure the luminal pH of the TGN in intact cells. We took advantage of endogenous cellular mechanisms for the specific retrieval of TGN resident proteins, such as TGN38 and furin, that transit briefly to the plasma membrane. Cells were transfected with chimeric constructs that contained the internalization and retrieval signals of TGN resident proteins, and a luminal (extracellular) epitope (CD25). Like TGN38 and furin, the chimeras were shown by fluorescence microscopy to accumulate within the TGN. During their transient exposure at the cell surface, the chimeras were labeled with extracellular anti-CD25 antibodies conjugated with a pH-sensitive fluorophore. Subsequent endocytosis and retrograde transport resulted in preferential labeling of the TGN with the pH-sensitive probe. Continuous, quantitative measurements of the pH of the TGN were obtained by ratio fluorescence imaging. The resting pH, calibrated using either ionophores or the "null point" technique, averaged 5.95 in Chinese hamster ovary cells and 5.91 in HeLa cells. The acidification was dissipated upon addition of concanamycin, a selective blocker of vacuolar-type ATPases. The counterion conductance was found to be much greater than the rate of H+ pumping at the steady state, suggesting that the acidification is not limited by an electrogenic potential. Both Cl- and K+ were found to contribute to the overall counterion permeability of the TGN. No evidence was found for the presence of active Na+/H+ or Ca2+/H+ exchangers on the TGN membrane. In conclusion, selective retrieval of recombinant proteins can be exploited to target ion-sensitive fluorescent probes to specific organelles. The technique provides real-time, noninvasive, and quantitative determinations of the pH, allowing the study of pH regulation within the TGN in intact cells. The acidic pH of the TGN reflects active H+ pumping into an organelle with a low intrinsic H+ permeability and a high conductance to monovalent ions.