Guanine Nucleotide Exchange on Heterotrimeric G_i3 Protein Controls Autophagic Sequestration in HT-29 Cells*

Abstract

Recent results have shown that autophagic sequestration in the human colon cancer cell line HT-29 is controlled by the pertussis toxin-sensitive heterotrimeric G_i3 protein. Here we show that transfection of an antisense oligodeoxynucleotide to the α_i3-subunit markedly inhibits autophagic sequestration, whereas transfection of an antisense oligodeoxynucleotide to the α_i2-subunit does not change the rate of autophagy in HT-29 cells. Autophagic sequestration was arrested in cells transfected with a mutant of the α_i3-subunit (Q204L) that is restricted to the GTP-bound form. In Q204L-expressing cells, 3-methyladenine-sensitive degradation of long lived [¹⁴C]valine-labeled proteins was severely impaired and could not be stimulated by nutrient deprivation. Autophagy was also reduced when dissociation of the βγ dimer from the GTP-bound α_i3-subunit was impaired in cells transfected with the G203A mutant. In contrast, a high rate of pertussis toxin-sensitive autophagy was observed in cells transfected with an α_i3-subunit mutant (S47N) which has an increased guanine nucleotide exchange rate and increased preference for GDP over GTP. Cells that express pertussis toxin-insensitive mutants of either wild-type α_i3-subunit (C351S) or S47N α_i3-subunit (S47N/C351S) exhibit a high rate of autophagy.