The Role Of ATAD3A In Regulating Mitochondria And Endoplasmic Reticulum Contacts

Eukaryotic cells contain many organelles,including mitochondria,endoplasmic reticulum and lysosomes.Various organelles can get close to each other through multiple proteins on the membrane surface,which can form contact sites in specific areas and produce new specific functions.Mitochondrion are the main sites of cell energy generation,and endoplasmic reticulum is an important organelle that can regulate intracellular Ca²⁺signaling and mediate the synthesis of proteins and lipids.The complex membrane structure of endoplasmic reticulum can form dynamic contact sites with other organelles and structure,such as mitochondria and cytoplasmic membrane（PM）.The contact sites of endoplasmic reticulum and mitochondria（The mitochondria-associated membrane sites,MAMs）has been focus of the study of interactions between organelles in recent years.MAMs directly mediate interactions between mitochondria and endoplasmic reticulum,the lipid synthesis and transport,Ca²⁺uptake and release between the mitochondrion and endoplasmic reticulum,signal transduction,and also play a very important role in mitochondrial dynamics.In this paper,the role of mitochondrial membrane protein ATAD3A encoded by nuclear genome in MAMs was preliminarily investigated.Firstly,we found that the shape of most mitochondrion in the ATAD3A-deficient He La and HCT116 cells were large spherical.Immunofluorescence and electron microscopy analysis showed that ATAD3A play a very important role in the maintenance of normal mitochondrial morphology.In addition,the loss of ATAD3A leads to the decrease of ATP level in cells,indicating that ATAD3A affects the mitochondrial function.Since ATAD3A is a mitochondria membrane protein locating at the outer membrane,we explored whether ATAD3A could mediate contact between mitochondrial and endoplasmic reticulum.We found that ATAD3A depletion resulted in a significant reduction of the interaction between mitochondria and endoplasmic reticulum.On the contrary,the interaction between mitochondrion and endoplasmic reticulum increased significantly in the cells overexpressing ATAD3A.Further,several proteins that may interact with ATAD3A,including endoplasmic reticulum protein Calnexin,were initially screened by co-immunoprecipitation and mass spectrometry analysis.Western boltting analysis showed that ATAD3A depletion resulted in a significant decrease of Calnexin in the protein level,indicating that ATAD3A regulates the stability of Calnexin by interacting with Calnexin.Therefore,Calnexin-ATAD3A axis may mediate the interaction between mitochondria and endoplasmic reticulum by N-terminal of ATAD3A located in the outer membrane of mitochondria interacting with Calnexin to establish the mitochondria-endoplasmic reticulum contacts.In summary,ATAD3A regulates interaction between mitochondrial and endoplasmic reticulum,then affecting mitochondrial functions.Our findings may provide a new molecular mechanism of mitochondrial and endoplasmic reticulum interaction.