Guanine nucleotide exchange on heterotrimeric Gi3 protein controls autophagic sequestration in HT-29 cells.
Recent results have shown that autophagic sequestration in the human colon cancer cell line HT-29 is controlled by the pertussis toxin-sensitive heterotrimeric Gi3 protein. Here we show that transfection of an antisense oligodeoxynucleotide to the alphai3-subunit markedly inhibits autophagic sequestration, whereas transfection of an antisense oligodeoxynucleotide to the alphai2-subunit does not change the rate of autophagy in HT-29 cells. Autophagic sequestration was arrested in cells transfected with a mutant of the alphai3-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 betagamma dimer from the GTP-bound alphai3-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 alphai3-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 alphai3-subunit (C351S) or S47N alphai3-subunit (S47N/C351S) exhibit a high rate of autophagy.
INSERM U410 Neuroendocrinologie et Biologie Cellulaire Digestives, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France.
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Ogier-Denis
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JJ
Houri
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Bauvy
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Codogno
D001343:Autophagy / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D008954:Models, Biological; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D037342:Pertussis Toxin; D011506:Proteins / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D020662:Guanine Nucleotide Exchange Factors; D016376:Oligonucleotides, Antisense; D011506:Proteins; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins