Interaction of Cardiolipin with LC3/GABARAP Family Members in Cargo Recognition during Mitophagy

Abstract

Removal of damaged mitochondria through selective autophagy (mitophagy) is critical for maintaining cellular function. However, how cell recognizes dysfunctional mitochondria which should be degraded is still unclear. To date extensive focus has been placed upon protein-protein interactions between autophagy receptor proteins, and there is less knowledge regarding the role of lipid-protein interactions in the selective recognition of damaged mitochondria. The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial degradation, and the externalization of CL to the outer mitochondrial membrane (OMM) has been identified as a mitophagic signal recognized by the autophagic protein LC3B in neuronal cells. This protein belongs to LC3/GABARAP family which consists of six members divided into the LC3 and GABARAP subfamilies. As with their yeast ortholog Atg8, these proteins are conjugated to the autophagosomal membranes where they drive autophagosome formation and selective recognition of autophagic cargo. The aim of our work is to analyze the role of each member of the LC3 / GABARAP family in the recognition of externalized cardiolipin and activation of mitophagy. With this purpose, we have analyzed the interaction of LC3 / GABARAP family members with CL using model membranes and compared these results with the translocation of the GFP tagged-LC3 / GABARAP to the mitochondria in SH-SY5Y cells treated with rotenone. Our observations indicate that the presence of CL increases the interaction of any protein of this family, particularly the LC3 subfamily. In addition, mutational studies strengthen the idea that LC3s would bind mitochondria using their N-terminal region while the C-terminal region would target the autophagosomal membrane. Our data support the notion that LC3 proteins might be the ones in charge of cargo recognition.