Subcellular localization of the G(alpha 13) 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(alpha i3) protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the G(alpha i3) protein is preferentially associated wi th Golgi membranes but co-localization was also observed with the endoplasm ic reticulum (ER) membrane. The G(alpha i2) protein, which is not involved in the control of autophagic sequestration, is associated with the plasma m embrane. Transfection of chimaeric G(alpha i) proteins (G(alpha i3/2), G(al pha i2/3)) containing the N- and C-terminal parts of the relevant G(alpha i ) demonstrated that the C-terminal part of the G(alpha i3) protein, by gove rning its membrane localization [de Almeida, Holtzman, Peters, Ercolani, Au siello and Stow (1994) J. Cell Sci. 107, 507-515], is important in the cont rol of macroautophagic sequestration. G alpha interacting protein (GAIP),wh ich stimulates the GTPase activity of the G(alpha i3) protein and favours m acroautophagic sequestration in HT-29 cells,was shown, by immunofluorescenc e studies using confocal microscopy, to be confined to the cytoplasm. The c ytoplasmic distribution of GAIP only partially. overlaps with that of the G (alpha i3) protein. However, the presence of the two proteins on Golgi and ER membranes was confirmed by subcellular fractionation. These results poin t to the importance of the cytoplasmic localization of the G(alpha i3) prot ein and GAIP in controlling autophagic sequestration in HT-29 cells.