Effect of Rab GTPases on the autophagic degradation of long-lived proteins

ZOPPINO, MARTÍN C.F.; GUTIERREZ, MAXIMILIANO G.; MUNAFÓ DANIELA B. AND COLOMBO MARÍA ISABEL

Bariloche, Argentina

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular (SAIB).; 2003

SAIB

            Autophagy is a degradative pathway by which cells sequester cytosolic proteins and organelles into double membrane vesicles (autophagosomes) that deliver their content to the lysosomes. This process plays an important role in cell maintenance and development and it is regulated by nutrient deprivation. Little is known about the proteins that regulate the autophagic pathway in mammalian cells. MAP-LC3 (microtubule-associated protein 1 light chain 3) was the first mammalian protein identified that, after processing, specifically associates with autophagosome membranes. Rab GTPases are small GTP-binding proteins that control specific intracellular traffic events. We have shown that Rab24 colocalizes with LC3 after induction of autophagy. Surprisingly, our results indicate that in CHO cells, overexpression of LC3 and Rab24 wild type proteins reduce the autophagy-induced long-lived protein degradation. In contrast, overexpression of disable membrane associated mutants (LC3 G120A, LC3 G120A,DC22 and Rab24S67L) have no effect on autophagic degradation. On the other hand, Rab7 wild type and the active mutant Rab7Q67L which control the aggregation and fusion of late endosomes/lysosomes induced protein degradation; while the inactive mutant Rab7T22N hampered the fusion of autophagosomes with lysosomes. These results indicate that Rab proteins are key molecular components for the normal progression of autophagy             Autophagy is a degradative pathway by which cells sequester cytosolic proteins and organelles into double membrane vesicles (autophagosomes) that deliver their content to the lysosomes. This process plays an important role in cell maintenance and development and it is regulated by nutrient deprivation. Little is known about the proteins that regulate the autophagic pathway in mammalian cells. MAP-LC3 (microtubule-associated protein 1 light chain 3) was the first mammalian protein identified that, after processing, specifically associates with autophagosome membranes. Rab GTPases are small GTP-binding proteins that control specific intracellular traffic events. We have shown that Rab24 colocalizes with LC3 after induction of autophagy. Surprisingly, our results indicate that in CHO cells, overexpression of LC3 and Rab24 wild type proteins reduce the autophagy-induced long-lived protein degradation. In contrast, overexpression of disable membrane associated mutants (LC3 G120A, LC3 G120A,DC22 and Rab24S67L) have no effect on autophagic degradation. On the other hand, Rab7 wild type and the active mutant Rab7Q67L which control the aggregation and fusion of late endosomes/lysosomes induced protein degradation; while the inactive mutant Rab7T22N hampered the fusion of autophagosomes with lysosomes. These results indicate that Rab proteins are key molecular components for the normal progression of autophagy             Autophagy is a degradative pathway by which cells sequester cytosolic proteins and organelles into double membrane vesicles (autophagosomes) that deliver their content to the lysosomes. This process plays an important role in cell maintenance and development and it is regulated by nutrient deprivation. Little is known about the proteins that regulate the autophagic pathway in mammalian cells. MAP-LC3 (microtubule-associated protein 1 light chain 3) was the first mammalian protein identified that, after processing, specifically associates with autophagosome membranes. Rab GTPases are small GTP-binding proteins that control specific intracellular traffic events. We have shown that Rab24 colocalizes with LC3 after induction of autophagy. Surprisingly, our results indicate that in CHO cells, overexpression of LC3 and Rab24 wild type proteins reduce the autophagy-induced long-lived protein degradation. In contrast, overexpression of disable membrane associated mutants (LC3 G120A, LC3 G120A,DC22 and Rab24S67L) have no effect on autophagic degradation. On the other hand, Rab7 wild type and the active mutant Rab7Q67L which control the aggregation and fusion of late endosomes/lysosomes induced protein degradation; while the inactive mutant Rab7T22N hampered the fusion of autophagosomes with lysosomes. These results indicate that Rab proteins are key molecular components for the normal progression of autophagy