MYO19 interacts with mitochondria through a C-terminal membrane association domain (MyMOMA). Specific mechanisms for localization of MYO19 to mitochondria are poorly understood. Using promiscuous biotinylation data in combination with existing affinity-capture databases, we have identified a number of putative MYO19-interacting proteins. We chose to explore the interaction between MYO19 and the mitochondrial GTPase Miro2 by expressing mchr-Miro2 in combination with GFP-tagged fragments of the MyMOMA domain and assaying for recruitment of MYO19-GFP to mitochondria. Coexpression of MYO19898-970 -GFP with mchr-Miro2 enhanced MYO19898-970 -GFP localization to mitochondria. Mislocalizing Miro2 to filopodial tips or the cytosolic face of the nuclear envelope did not recruit MYO19898-970 -GFP to either location. To address the kinetics of the Miro2/MYO19 interaction, we used FRAP analysis and permeabilization-activated reduction in fluorescence analysis. MyMOMA constructs containing a putative membrane-insertion motif but lacking the Miro2-interacting region displayed slow exchange kinetics. MYO19898-970 -GFP, which does not include the membrane-insertion motif, displayed rapid exchange kinetics, suggesting that MYO19 interacting with Miro2 has higher mobility than MYO19 inserted into the mitochondrial outer membrane. Mutation of well-conserved, charged residues within MYO19 or within the switch I and II regions of Miro2 abolished the enhancement of MYO19898-970 -GFP localization in cells ectopically expressing mchr-Miro2. Additionally, expressing mutant versions of Miro2 thought to represent particular nucleotide states indicated that the enhancement of MYO19898-970 -GFP localization is dependent on Miro2 nucleotide state. Taken together, these data suggest that membrane-inserted MYO19 is part of a larger complex, and that Miro2 plays a role in integration of actin- and microtubule-based mitochondrial activities.
Keywords: GTPase; Miro; mitochondria; myosin; outer membrane.
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