Involvement of the vacuolar H(+)-ATPases in the secretory pathway of HepG2 cells.

The macrolide antibiotic concanamycin B is a highly selective inhibitor (IC50 = 5 nM) of the H(+)-ATPases of the vacuolar system. We have examined the effects of concanamycin B on the constitutive secretory pathway of the human hepatoma cell line, HepG2. In cells exposed to 10 nM concanamycin B, transport from the endoplasmic reticulum to the Golgi occurs at normal rates, as determined by pulse-chase analysis of endoglycosidase H-sensitive product in conjunction with subcellular fractionation experiments. However, intra-Golgi trafficking or Golgi to plasma membrane delivery is significantly impaired. A delay in the onset of secretion of the major secretory proteins, albumin, alpha 1-antitrypsin and transferrin is observed. Processing of N-linked glycans by sialyltransferases is inhibited, resulting in secreted glycoproteins which are modified less extensively. In view of the acidic pH of the trans-Golgi and the trans-Golgi network, these studies suggest that acidification by vacuolar ATPases is critical to achieving timely secretion and correct N-linked glycan modifications of proteins which follow the constitutive secretory pathway.