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

Abstract

The macrolide antibiotic concanamycin B is a highly selective inhibitor (IC6,, = 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 exp d to 10 n M concanamycin B, transport from the endoplasmic reticulum to the Golgi occurs at normal ratee, 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, al-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 trum-Golgi and the trum-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.The vectorial transport of proteins from the endoplasmic reticulum to the cell surface for which no specific regulatory sorting signals have been identified is often referred to as the constitutive secretory pathway (Palade, 1975).It is widely held that such proteins move by default to the plasma membrane, unless specifically sorted to other compartments (Pfeffer and Rothman, 1987; Mellman and Simons, 1992).Cellular sorting and targeting processes appear to rely on the internal ionic environment of components of the endocytic and exocytic pathways (Mellman et aL, 1986;Anderson and Orci, 1988; Mellman and Simons, 1992).The plasma membrane, mitochondria, and the vacuolar membrane system each possess unique electrogenic or non-electrogenic proton pumping ATPases which lower intralumenal pH (Bowman et al., 1988; Nelson, 1992a).Genetic experiments in the yeast Sacchuro- myces cereuisiae have provided strong evidence implicating