Dynamic Proximity Networks of Myosin-19 (Myo19) and its Mitochondrial Receptors Miro2 and Metaxin-3

Myosin-19 (Myo19) plays a crucial role in mitochondrial dynamics, cristae organization and ER-mitochondria contact sites. It regulates cytokinesis and inheritance of mitochondria to daughter cells. To better understand the dynamic molecular network of Myo19 during the cell cycle, we determined the in vivo proximity protein interaction networks of Myo19 in interphase and prometaphase using proximity-based TurboID biotinylation followed by mass spectrometry. We further determined the proximity networks of its known mitochondrial binding partners Miro2 and metaxin-3. The outer mitochondrial membrane protein Miro2 not only binds but also stabilizes Myo19. This interaction depends on the nucleotide state of the N-terminal GTPase domain of Miro2. Therefore, we analysed both the proximity networks of Miro2 and its GTP-binding mutant Miro2 T18N. We were able to show a differential association of Myo19 during the cell cycle with functional protein clusters and a participation of Myo19 in mitochondrial trafficking, ER-mitochondria contact sites and mitochondria intermembrane space bridging complex/cristae organizing system. The proximity network of Myo19 showed more overlap with Miro2 than metaxin-3. Abolishing GTP-binding to the N-terminal GTPase domain of Miro2 reduced the number of proteins in proximity of Miro2 considerably. In conclusion, we discovered a comprehensive dynamic in vivo protein proximity network of Myo19 and its mitochondrial receptors Miro2 and metaxin-3.