Subcellular localization of the Gαi3 protein and G alpha interacting protein, two proteins involved in the control of macroautophagy in human colon cancer HT-29 cells

Autophagic sequestration is controlled by the G_αi3 protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the G_αi3 protein is preferentially associated with Golgi membranes but co-localization was also observed with the endoplasmic reticulum (ER) membrane. The G_αi2 protein, which is not involved in the control of autophagic sequestration, is associated with the plasma membrane. Transfection of chimaeric G_αi proteins (G_αi3/2, G_αi2/3) containing the N- and C-terminal parts of the relevant G_αi demonstrated that the C-terminal part of the G_αi3 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 G_αi3 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 G_αi3 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 G_αi3 protein and GAIP in controlling autophagic sequestration in HT-29 cells.