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

Jennifer L. Bocanegra; Barbara M. Fujita; Natalie R. Melton; James M. Cowan; Elizabeth L. Schinski; Tigist Y. Tamir; Michael B. Major; Omar A. Quintero

doi:10.1002/cm.21560

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

Jennifer L. Bocanegra
, Barbara M. Fujita
, Natalie R. Melton
, James M. Cowan
, Elizabeth L. Schinski
, Tigist Y. Tamir
, Michael B. Major
, Omar A. Quintero

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

Abstract

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 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 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 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 microtubule-based mitochondrial activities.

Original language	English
Pages (from-to)	149-166
Number of pages	18
Journal	Cytoskeleton
Volume	77
Issue number	3-4
DOIs	https://doi.org/10.1002/cm.21560
State	Published - Mar 1 2020

Keywords

GTPase
Miro
mitochondria
myosin
outer membrane

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10.1002/cm.21560

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@article{008887b0cef047b6ae1ac47dadf0743f,

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

abstract = "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",

author = "Bocanegra, \{Jennifer L.\} and Fujita, \{Barbara M.\} and Melton, \{Natalie R.\} and Cowan, \{James M.\} and Schinski, \{Elizabeth L.\} and Tamir, \{Tigist Y.\} and Major, \{Michael B.\} and Quintero, \{Omar A.\}",

note = "Funding Information: OAQ was supported by a grant from the National Institute of General Medical Sciences at the NIH (R15 GM119077) and by funding from the University of Richmond School of Arts \& Sciences. JLB and JMC were supported by the NIGMS grant to OAQ. BMF was supported by the Robert F. Smart Award from the Biology Department, and funding from the School of Arts \& Sciences at the University of Richmond. NRM and ELS were supported by funding from the University of Richmond School of Arts \& Sciences. TYT was supported by an HHMI Gilliam Fellowship for Advanced Study. We would like to thank Uri Manor, Anna Hatch, Jenci Hawthorne, Rebecca Adikes, Sarah Rice, Susan Walsh, and Andrew Moore for critical reading and discussions related to this manuscript. 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. Experiments requested by the reviewers were completed during the 10-week 2019 summer research session at University of Richmond. We would also like to that Edward Salmon for his example and inspiration. Publisher Copyright: {\textcopyright} 2019 Wiley Periodicals, Inc.",

year = "2020",

month = mar,

day = "1",

doi = "10.1002/cm.21560",

language = "English",

volume = "77",

pages = "149--166",

journal = "Cytoskeleton",

issn = "1949-3584",

number = "3-4",

}

TY - JOUR

T1 - The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

AU - Bocanegra, Jennifer L.

AU - Fujita, Barbara M.

AU - Melton, Natalie R.

AU - Cowan, James M.

AU - Schinski, Elizabeth L.

AU - Tamir, Tigist Y.

AU - Major, Michael B.

AU - Quintero, Omar A.

N1 - Funding Information: OAQ was supported by a grant from the National Institute of General Medical Sciences at the NIH (R15 GM119077) and by funding from the University of Richmond School of Arts & Sciences. JLB and JMC were supported by the NIGMS grant to OAQ. BMF was supported by the Robert F. Smart Award from the Biology Department, and funding from the School of Arts & Sciences at the University of Richmond. NRM and ELS were supported by funding from the University of Richmond School of Arts & Sciences. TYT was supported by an HHMI Gilliam Fellowship for Advanced Study. We would like to thank Uri Manor, Anna Hatch, Jenci Hawthorne, Rebecca Adikes, Sarah Rice, Susan Walsh, and Andrew Moore for critical reading and discussions related to this manuscript. 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. Experiments requested by the reviewers were completed during the 10-week 2019 summer research session at University of Richmond. We would also like to that Edward Salmon for his example and inspiration. Publisher Copyright: © 2019 Wiley Periodicals, Inc.

PY - 2020/3/1

Y1 - 2020/3/1

N2 - 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.

AB - 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.

KW - GTPase

KW - Miro

KW - mitochondria

KW - myosin

KW - outer membrane

UR - https://www.scopus.com/pages/publications/85073771972

U2 - 10.1002/cm.21560

DO - 10.1002/cm.21560

M3 - Article

C2 - 31479585

AN - SCOPUS:85073771972

SN - 1949-3584

VL - 77

SP - 149

EP - 166

JO - Cytoskeleton

JF - Cytoskeleton

IS - 3-4

ER -

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

Abstract

Keywords

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