MECHANISM OF ACIDIFICATION OF THE TRANS-GOLGI NETWORK (TGN) - IN-SITUMEASUREMENTS OF PH USING RETRIEVAL OF TGN38 AND FURIN FROM THE CELL-SURFACE

Sorting of secretory cargo and retrieval of components of the biosynth etic pathway occur at the trans-Golgi network (TGN), The pH within the TGN is thought to be an important determinant of these functions, How ever, studies of the magnitude and regulation of the pH of the TGN hav e been hampered by the lack of appropriate detection methods, This rep ort describes a noninvasive strategy to measure the luminal pH of the TGN in intact cells, We took advantage of endogenous cellular mechanis ms for the specific retrieval of TGN resident proteins, such as TGN38 and furin, that transit briefly to the plasma membrane, Cells were tra nsfected with chimeric constructs that contained the internalization a nd retrieval signals of TGN resident proteins, and a luminal (extracel lular) epitope (CD25), Like TGN38 and furin, the chimeras were shown b y fluorescence microscopy to accumulate within the TGN, During their t ransient exposure at the cell surface, the chimeras were labeled with extracellular anti-CD25 antibodies conjugated with a pH-sensitive fluo rophore. Subsequent endocytosis and retrograde transport resulted in p referential labeling of the TGN with the pH-sensitive probe, Continuou s, quantitative measurements of the pH of the TGN were obtained by rat io fluorescence imaging, The resting pH, calibrated using either ionop hores or the ''null point'' technique, averaged 5.95 in Chinese hamste r ovary cells and 5.91 in HeLa cells, The acidification was dissipated upon addition of concanamycin, a selective blocker of vacuolar-type A TPases, The counterion conductance was found to be much greater than t he rate of H+ pumping at the steady state, suggesting that the acidifi cation is not limited by an electrogenic potential, Both Cl- and K+ we re found to contribute to the overall counterion permeability of the T GN, 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 fluores cent probes to specific organelles, The technique provides real-time, noninvasive, and quantitative determinations of the pH, allowing the s tudy 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 intri nsic H+ permeability and a high conductance to monovalent ions.