Identification of the interaction networks of Mitofusin-1 and Mitofusin-2

Abstract

[eng] Mitofusins (MFN1/2) have been demonstrated to play a role in several mitochondrial functions such as mitochondrial dynamics and bioenergetics, interorganelle communication, immune response, autophagy, cell cycle progression, cellular senescence and apoptosis. However, the role of MFN1/2 in the molecular mechanisms underlying these processes remains poorly understood. Here, we have studied MFN1/2 functions and regulation from an interactome perspective in order to gain insight into the accurate mechanisms by which they are involved in those cellular events. To this end, we have generated two HeLa cell lines expressing an HA-tag fused to the C-terminal domain of MFN1 and MFN2 applying CRISPR/Cas9 technology. This has facilitated MFN1/2 pulldown under endogenous expression levels in basal conditions and in conditions of nutrient deprivation. The fact that MFN1/2 expression levels remain endogenous is key since most of their binding partners known so far have been studied under overexpression conditions. Subsequently, we have performed mass spectrometry to identify interactors of MFN1 and MFN2 in basal or starvation conditions. We have observed that RNA-binding proteins are enriched in basal conditions among MFN1 and MFN2 binding candidates and that they are absent in starvation conditions. We have validated that FXR1, AGO2, RAB5C and NDFIP2 interact with MFN1 and MFN2, and that S100A8, S100A9 and SLC27A2 interact with MFN2. We have found that RAB5C is necessary for mitochondrial morphology maintenance. Our data have also shown that SLC27A2 is a protein important for the process of autophagy.