The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

Abstract

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.

Miro: A molecular switch at the center of mitochondrial regulation

“…Originally, Myo19 was thought to act as a peripheral membrane protein, interacting with lipids directly. However, recent work discovered that Miro1/2 are receptors for recruiting and stabilizing Myo19 to the mitochondria based on knockout studies of Miro1 and Miro2, in addition to proximity labeling approaches (Figure c,d). Biochemical studies demonstrated that the Myo19 tail contains both Miro1/2‐independent and Miro1/2‐dependent mitochondrial targeting sites .…”

“…However, recent work discovered that Miro1/2 are receptors for recruiting and stabilizing Myo19 to the mitochondria based on knockout studies of Miro1 and Miro2, in addition to proximity labeling approaches (Figure c,d). Biochemical studies demonstrated that the Myo19 tail contains both Miro1/2‐independent and Miro1/2‐dependent mitochondrial targeting sites . Interestingly, Miro1/2‐mediated recruitment is regulated by the nucleotide state of Miro1/2 N‐terminal GTPase domain .…”

Developmental regulation of microtubule‐based trafficking and anchoring of axonal mitochondria in health and diseases

“…While stable subplasmalemmal actin rings form periodic structures that wrap around axons to support the axonal membrane, dynamic intra‐axonal filaments help to enrich actin at presynaptic terminals (Ganguly et al., 2015; Xu, Zhong, & Zhuang, 2013). It has been reported that mitochondrial receptors Miro1/2 regulate recruitment and stability of myosin‐19, thus likely coordinating MT‐ and actin‐based mitochondrial trafficking in mouse embryonic fibroblasts (MEFs) (Bocanegra et al., 2020; López‐Doménech et al., 2018; Oeding et al., 2018; Quintero et al., 2009). However, the role of myosin‐19 in axonal mitochondrial trafficking is not clear.…”

Structural assembly of the human Miro1/2 GTPases based on the crystal structure of the N-terminal GTPase domain

“…Differential binding localization studies suggest that Miro1 and Miro2 interact differently with the motor adapters TRAK1 and TRAK2 (van Spronsen et al, 2013). The nGTPase domains of HsMiro1 and HsMiro2, which are believed to play the key role in the interactions with TRAK1/2 (Bocanegra et al, 2019;Oeding et al, 2018), are structurally quite similar, as they exhibit 73% sequence identity and 88% sequence similarity. Consequently, the set of 21 nonhomologous amino acid substitutions between them presumably differentiate the functions of the two proteins.…”