MYO19 interacts weakly with Miro2 GTPases on the mitochondrial outer membrane

ABSTRACT: MYO19 interacts with mitochondria through a unique C-terminal mitochondrial association domain (MyMOMA). The specific molecular mechanisms for localization of MYO19 to mitochondria are poorly understood. Using promiscuous biotinylation, we have identified ~100 candidate MYO19 interacting proteins, a subset of which were also identified in via affinity-capture experiments. 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 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 (PARF) analysis. MyMOMA constructs containing a putative membrane insertion motif but lacking the complete Miro2 interacting region, MYO19853-935-GFP, displayed slow exchange kinetics. MYO19898-970-GFP, which does not include the membrane-insertion motif, displayed rapid exchange kinetics, suggesting that the MYO19 and Miro2 interaction is weaker than between MYO19 and 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 a role for Miro2 in regulation and integration of actin- and microtubule-based mitochondrial activities.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Omar Quintero  

LAB HEAD: Omar A. Quintero

PROVIDER: PXD015004 | Pride | 2019-09-10

REPOSITORIES: Pride