Positively charged residues within the MYO19 MyMOMA domain are essential for proper localization of MYO19 to the mitochondrial outer membrane.

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.