Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking*

Katie M. O'Callaghan, Veronica Ayllon, Jean O'Keeffe, Yanru Wang, Orla T. Cox, Gary Loughran, Michael Forgac, Rosemary O'Connor

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H(+)-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.