The factors contributing to the establishment of the steady state Golgi pH (pH G ) were studied in intact and permeabilized mammalian cells by fluorescence ratio imaging. Retrograde transport of the nontoxic B subunit of verotoxin 1 was used to deliver pH-sensitive probes to the Golgi complex. To evaluate whether counter-ion permeability limited the activity of the electrogenic V-ATPase, we determined the concentration of K ؉ in the lumen of the Golgi using a null point titration method. The [K ؉ ] inside the Golgi was found to be close to that of the cytosol, and increasing its permeability had no effect on pH G . Moreover, the capacity of the endogenous counter-ion permeability exceeded the rate of H ؉ pumping, implying that the potential across the Golgi membrane is negligible and has little influence on pH G . The V-ATPase does not reach thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pH G . In fact, active H ؉ pumping was detectable even below the resting pH G . A steady state pH was attained when the rate of pumping was matched by the passive backflux of H ؉ (equivalents) or "leak." The nature of this leak pathway was investigated in detail. Neither vesicular traffic nor H ؉ /cation antiporters or symporters were found to contribute to the net loss of H ؉ from the Golgi. Instead, the leak was sensitive to voltage changes and was inhibited by Zn 2؉ , resembling the H ؉ conductive pathway of the plasma membrane. We conclude that a balance between an endogenous leak, which includes a conductive component, and the H ؉ pump determines the pH at which the Golgi lumen attains a steady state.Stringent regulation of the internal pH of endomembrane compartments is required for their optimal function. In mitochondria, alkalinization of the matrix contributes to the generation of the transmembrane proton-motive force used to generate ATP (1). By contrast, luminal acidification is essential for the distribution and degradation of internalized ligands in the endocytic pathway (2-4), whereas it regulates post-translational modification and sorting of proteins along the secretory pathway (5-7). The pH varies in different subcompartments of the endocytic pathway, with acidification increasing progressively from the endocytic vesicles and early endosomes to late endosomes and ultimately lysosomes (8). Conversely, the pH of the secretory pathway becomes more acidic as the cargo travels toward the cell surface. Although the pH of the endoplasmic reticulum is thought to be near neutral (9, 10), acidification develops along the Golgi complex and is maximal at the transGolgi network (11-13). The development of such gradients appears to be important in targeting and retrieving components to and from individual subcompartments (6, 15). However, little is known about the determinants of pH in each subcompartment and particularly about the source of their differential acidification. In the case of the secretory pathway, this paucity of information is attributable, in part, to methodolo...

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H+-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pHG) was measured by ratio imaging. Whereas pHG of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH ∼7.0). The Golgi buffering power and the rate of H+ leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pHG. We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.

A small fraction of the molecules internalized by endocytosis reaches the Golgi complex through a retrograde pathway that is poorly understood. In the present work, we used bacterial toxins to study the retrograde pathway in Vero cells. The recombinant B subunit of verotoxin 1B (VT1B) was labeled with fluorescein to monitor its progress within the cell by confocal microscopy. This toxin, which binds specifically to the glycolipid globotriaosyl ceramide, entered endosomes by both clathrin-dependent and -independent pathways, reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics of internalization and the subcellular distribution of VT1B were virtually identical to those of another glycolipid-binding toxin, the B subunit of cholera toxin (CTB). Retrograde transport of VT1B and CTB was unaffected by addition of weak bases in combination with concanamycin, a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that these agents neutralized the luminal pH of the compartments where the toxin was located. Therefore, the retrograde transport of glycolipids differs from that of proteins like furin and TGN38, which require an acidic luminal pH. Additional experiments indicated that the glycolipid receptors of VT1B and CTB are internalized independently and not as part of lipid “rafts” and that internalization is cytochalasin insensitive. We conclude that glycolipids utilize a unique, pH-independent retrograde pathway to reach compartments of the secretory system and that assembly of F-actin is not required for this process.