The role of Rabconnectin-3β regulation in mTORC1 and autophagy

• Autor(a) principal: Moreira, Joana Brandão
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/29010
• topic: Rabconnectin-3β; V-ATPases; Autophagy; mTORC1

Rabconnectin-3β is a protein, encoded by the WDR7 gene. It is constituted by 9 WD repeats, which consist in conserved regions of around 40 amino acids that often assume a 7-8 bladed beta-propeller form. This protein forms a complex with Rabconnectin-3α which is presumably involved in the regulation of the V-ATPase pump. The V-ATPase pump is a complex that acidifies several intracellular organelles, including lysosomes and synaptic vesicles. The function of Rabconnectin-3 appears to be conserved from yeast, in which its orthologue RAVE, plays an important role in the regulation of the V-ATPase complex. The V-ATPase activity is a key determinant of lysosomal acidification, which impacts additional cellular pathways, such as the nutrient sensor mTORC1, and the autophagy pathway. Thus, the goal of our study was to characterize the role of Rabconnectin-3 complex, and particularly Rabconnectin-3β, in mTORC1 and autophagy. Using mouse fibroblasts with overexpression or silencing of Rabconnectin-3β, several key markers of the mTORC1 and autophagy pathways were characterized with techniques such as Western blot, qPCR and multicolour confocal microscopy. It was observed that knockdown of Rabconnectin-3β resulted in up regulation of autophagic flux and lysosomal biogenesis, whereas mTORC1 was downregulated. Reciprocally, overexpression of Rabconnectin-3β resulted in increased mTORC1 activity and decreased lysosomal biogenesis and autophagy. Furthermore, colocalization experiments revealed that Rabconnectin-3β is located in the lysosomes, both in full medium and under nutrient starvation. The overall results offer novel insights on the role of Rabconnectin-3β on the regulation of lysosomal V-ATPases, and through it, on autophagy and mTORC1 pathways.