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.

for critical reading and discussions related to this manuscript. Hypotheses and experiments in this study were conceived by OAQ. Experiments were performed by JLB, BMF, NRM, JMC, ELS, TYT, and OAQ.Data analysis and manuscript preparation were completed by JLB, BMF, NRM, JMC, TYT, MBM, and OAQ. Except for the proteomics analysis, all experiments for the initial submission were completed during the 10-week 2018 summer research session at University of Richmond. We would also like to that Edward Salmon for his example and inspiration.3 Abstract MYO19 interacts with mitochondria through a C-terminal membrane association domain (MyMOMA).The specific mechanisms for localization of MYO19 to mitochondria are poorly understood. Using new promiscuous biotinylation data in combination with existing affinity-capture databases, we have identified a number of putative MYO19-interacting proteins. We chose to further 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. Co-expression of MYO19 898-970 -GFP with mchr-Miro2 enhanced MYO19 898-970 -GFP localization to mitochondria. Mislocalizing Miro2 to filopodial tips or the cytosolic face of the nuclear envelope did not recruit MYO19 898-970 -GFP to either location. To address the kinetics of the Miro2/MYO19 interaction, we used FRAP analysis and permeabilization-activated reduction in fluorescence (PARF) analysis. MyMOMA constructs containing a putative membrane insertion motif but lacking the Miro2-interacting region displayed slow exchange kinetics. MYO19 898-970 -GFP, which does not include the membrane-insertion motif, displayed rapid exchange kinetics, suggesting that the 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 MYO19 898-970 -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 MYO19 898-970 -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 microtubulebased mitochondrial activities.

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