MYO19 interacts weakly with Miro2 GTPases on the mitochondrial outer membrane
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.
Sample Processing Protocol
Streptavadin pulldown MYO19824-970-BioID2 stable cells were plated in 150mm cell culture dishes. Once they reached approximately 80% confluency, they were grown in the presence of 50M biotin overnight. Biotinylated proteins were purified as previously described [Mehus et al. 2016]. Briefly, cells were lifted from the plate using 0.25% trypsin EDTA, and washed with PBS via centrifugation (700xg, 4˚C, 10 minutes). Cells were resuspended in lysis buffer (50mM Tris HCl, 500mM NaCl, 0.2% SDS, 1mM DTT, 10 g/ml aprotinin, 10g/ml leupeptin, 1mM PMSF, pH 7.4). TritonX-100 was added to a final concentration of 2%, and the lysate was mixed by inversion. Samples were then placed on ice and sonicated twice (Branson Sonifer 450 Digital, 40% amplitude, 30% duty cycle, 30, 0.5s pulses), with a 2-minute incubation on ice between sonications. Lysates were diluted ~4-fold with 50mM Tris HCl, pH7.4 prior to a third round of sonication. Insoluble material was removed from the lysate via centrifugation (16,5000g, 4˚C, 10 minutes). Magnetic streptavidin beads were washed in 50mM Tris HCl, pH7.4 prior to incubation with the lysate supernatant at 4˚C with gentle rocking overnight. The sample were then collected to the side of the tube using a magnetic stand. The beads were washed (8 minute incubation time) twice with wash buffer 1 (2% SDS), once with wash buffer 2 (50mM Hepes, 500mM NaCl, 1mM EDTA, 1% TritonX-100, 0.1% deoxycholic acid, pH 7.5), and once with wash buffer 3 (10mM Tris HCl, 250mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% deoxycholic acid, pH 7.4). The beads were then resuspended in 50mM Tris HCl, pH7.4, with 10% saved for western blot analysis. The remaining sample was resuspended in 50mM ammonium bicarbonate in preparation for mass spectrometry analysis. Mass spectrometry sample preparation, analysis, data filtering, and bioinformatics Samples were subjected to in-solution Trypsin digestion following a RapiGest SF Surfactant (Waters, Part# 186001861, Waters Corporation, 34 Mapel St., Milford, MA 01757) elution. Trypsinzation was achieved by incubating eluted protein with excess trypsin (Promega V5111) at 37˚C overnight (16-18 hours). Subsequently, eluted peptides were desalted using a Pierce C-18 spin column (Thermo 89870) and followed by an ethyl acetate cleanup step to remove detergents. Peptides were dried down and then re-suspended in 2% Formic Acid LC-MS grade water solution for mass spec analysis. Peptides were separated using reverse-phase nano-HPLC by a nanoACQUITY UPLC system (Waters Corporation). Peptides were trapped on a 2 cm column (Pepmap 100, 3μM particle size, 100 Å pore size), and separated on a 25cm EASYspray analytical column (75μM ID, 2.0μm C18 particle size, 100 Å pore size) at 45˚C. The mobile phases were 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). A 180-minute gradient of 2-25% buffer B was used with a flow rate of 300nl/min. Mass spectral analysis was performed by a Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). The ion source was operated at 2.4kV and the ion transfer tube was set to 300˚C. Full MS scans (350-2000 m/z) were analyzed in the Orbitrap at a resolution of 120,000 and 1e6 AGC target. The MS2 spectra were collected using a 1.6 m/z isolation width and were analyzed either by the Orbitrap or the linear ion trap depending on peak charge and intensity using a 3s TopSpeed CHOPIN method [Davis et al. 2017]. Orbitrap MS2 scans were acquired at 7500 resolution, with a 5e4 AGC, and 22ms maximum injection time after HCD fragmentation with a normalized energy of 30%. Rapid linear ion trap MS2 scans were acquired using an 4e3 AGC, 250ms maximum injection time after CID 30 fragmentation. Precursor ions were chosen based on intensity thresholds (>1e3) from the full scan as well as on charge states (2-7) with a 30-s dynamic exclusion window. Polysiloxane 371.10124 was used as the lock mass.
Data Processing Protocol
Raw mass spectrometry data were searched against the Swiss-Prot human sequence database (released 2/2017) using MaxQuant version 126.96.36.199. The parameters for the search were as follows: specific tryptic digestion with up to two missed cleavages, static carbamidomethyl cysteine modification, variable protein N-terminal acetylation and methionine oxidation, Label Free Quantification (LFQ) and match between runs were enabled. Protein identifications were filtered for a false discovery rate (FDR) of 1%, and potential contaminants and decoys were removed. To score candidate protein-protein interactions, SAINTq version 0.0.4 using LFQ values was used and then filtered for a 10% FDR.
Bocanegra JL, Fujita BM, Melton NR, Cowan JM, Schinski EL, Tamir TY, Major MB, Quintero OA. The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes. Cytoskeleton (Hoboken). 2019 PubMed: 31479585