GUANINE-NUCLEOTIDE EXCHANGE ON HETEROTRIMERIC G(I3) PROTEIN CONTROLS AUTOPHAGIC SEQUESTRATION IN HT-29 CELLS

Recent results have shown that autophagic sequestration in the human c olon cancer cell line HT-29 is controlled by the pertussis toxin-sensi tive heterotrimeric G(i3) protein. Here we show that transfection of a n antisense oligodeoxynucleotide to the alpha(i3)-subunit markedly inh ibits autophagic sequestration, whereas transfection of an antisense o ligodeoxynucleotide to the alpha(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 alpha(i3)-subunit (Q204L) that is restricted to the GTP-bound form. In Q204L expressing cells, 3-meth yladenine-sensitive degradation of long lived [C-14]valine-labeled pro teins was severely impaired and could not be stimulated by nutrient de privation. Autophagy was also reduced when dissociation of the beta ga mma dimer from the GTP-bound alpha(i3)-subunit was impaired in cells t ransfected with the G203A mutant. In contrast, a high rate of pertussi s toxin-sensitive autophagy was observed in cells transfected with an alpha(i3)-subunit mutant (S47N) which has an increased guanine nucleot ide exchange rate and increased preference for GDP over GTP. Cells tha t express pertussis toxin-insensitive mutants of either wild-type alph a(i3)-subunit (C351S) or S47N alpha(i3)-subunit (S47N/C351S) exhibit a high rate of autophagy.