title: Positively charged residues within the MYO19 MyMOMA domain are essential for proper localization of MYO19 to the mitochondrial outer membrane.
abstract: Myosins are well characterized molecular motors essential for intracellular transport. MYO19 copurifies with mitochondria, and can be released from mitochondrial membranes by high pH buffer, suggesting that positively-charged residues participate in interactions between MYO19 and mitochondria. The MYO19-specific mitochondria outer membrane association (MyMOMA) domain contains approximately 150 amino acids with a pI approximately 9 and is sufficient for localization to the mitochondrial outer membrane. The minimal sequence and specific residues involved in mitochondrial binding have not been identified. To address this, we generated GFP-MyMOMA truncations, establishing the boundaries for truncations based on sequence homology. We identified an 83-amino acid minimal binding region enriched with basic residues (pI ∼ 10.5). We sequentially replaced basic residues in this region with alanine, identifying residues R882 and K883 as essential for mitochondrial localization. Constructs containing the RK882-883AA mutation primarily localized with the endoplasmic reticulum (ER). To determine if ER-associated mutant MyMOMA domain and mitochondria-associated wild type MyMOMA display differences in kinetics of membrane interaction, we paired FRAP analysis with permeabilization activated reduction in fluorescence (PARF) analysis. Mitochondria-bound and ER-bound MYO19 constructs displayed slow dissociation from their target membrane when assayed by PARF; both constructs displayed exchange within their respective organelle networks. However, ER-bound mutant MYO19 displayed more rapid exchange within the ER network than did mitochondria-bound MYO19. Taken together these data indicate that the MyMOMA domain contains strong membrane-binding activity, and membrane targeting is mediated by a specific, basic region of the MYO19 tail with slow dissociation kinetics appropriate for its role(s) in mitochondrial network dynamics. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Biology, University of Richmond, VA 23173.
authors.forename: Jenci L
authors.initials: JL
authors.lastname: Hawthorne
authors.affiliation: Department of Biology, University of Richmond, VA 23173.
authors.forename: Prachi R
authors.initials: PR
authors.lastname: Mehta
authors.affiliation: Department of Biology, University of Richmond, VA 23173.
authors.forename: Pali P
authors.initials: PP
authors.lastname: Singh
authors.affiliation: Department of Biology, University of Richmond, VA 23173.
authors.forename: Nathan Q
authors.initials: NQ
authors.lastname: Wong
authors.affiliation: Department of Biology, University of Richmond, VA 23173.
authors.forename: Omar A
authors.initials: OA
authors.lastname: Quintero
mesh_terms: D019943:Amino Acid Substitution; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051336:Mitochondrial Membranes / Q000378:metabolism*; D020125:Mutation, Missense; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D021381:Protein Transport / Q000502:physiology
chemical_list: C548150:Myo19 protein, human; D009218:Myosins
keywords: mitochondria; myosin; organelle transport; outer membrane