Subcellular localization of the Galphai3 protein and G alpha interacting protein, two proteins involved in the control of macroautophagy in human colon cancer HT-29 cells

Abstract

Autophagic sequestration is controlled by the Galphai3 protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the Galphai3 protein is preferentially associated with Golgi membranes but co-localization was also observed with the endoplasmic reticulum (ER) membrane. The Galphai2 protein, which is not involved in the control of autophagic sequestration, is associated with the plasma membrane. Transfection of chimaeric Galphai proteins (Galphai3/2, Galphai2/3) containing the N- and C-terminal parts of the relevant Galphai demonstrated that the C-terminal part of the Galphai3 protein, by governing its membrane localization [de Almeida, Holtzman, Peters, Ercolani, Ausiello and Stow (1994) J. Cell Sci. 107, 507-515], is important in the control of macroautophagic sequestration. G alpha interacting protein (GAIP),which stimulates the GTPase activity of the Galphai3 protein and favours macroautophagic sequestration in HT-29 cells,was shown, by immunofluorescence studies using confocal microscopy, to be confined to the cytoplasm. The cytoplasmic distribution of GAIP only partially overlaps with that of the Galphai3 protein. However, the presence of the two proteins on Golgi and ER membranes was confirmed by subcellular fractionation. These results point to the importance of the cytoplasmic localization of the Galphai3 protein and GAIP in controlling autophagic sequestration in HT-29 cells.