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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Cytoskeleton (Hoboken). 2016 May 24;73(6):286–299. doi: 10.1002/cm.21305

Figure 3. Elimination of positively charged residues in the T2 region of human MYO19 alters mitochondrial association.

Figure 3

A) Mutation of 8 positively charged residues within the MYO19 T2 results in the loss of mitochondrial localization. Mutant versions of GFP-MYO19 T2 (R855A, R915A, K923A, RK927-928AA, and R932A) colocalize with Mitotracker-labeled mitochondria in HeLa cells while GFP-T2 RK882-883AA fails to localize to mitochondria. Introducing the RK882-883AA mutation into GFP-MyMOMA domain or full-length GFP-MYO19 also results in disrupted mitochondrial localization. Scale bar is 10μm. B) The RK882-883AA version of GFP T2 colocalizes with DsRed2-ER labeled tubules. C) Quantification of mitochondrial localization by calculation of the mito/cyto ratio indicates that the RK882-883AA mutant version of GFP-T2 has a decreased mito/cyto ratio when compared to wild-type GFP-T2. (*p<0.0001, t-test, nT2=24 cells and nRK882-882AA=26). Error bars represent 90th and 10th percentiles; the box top, middle, and bottom indicate the 75th, 50th, and 25th, percentile respectively. The symbol (∎) indicates the sample mean. D) Binding of purified GFP-T2 and GFP-T2 RK882-883AA mutant protein to membrane lipid strips indicates that both constructs binds to a subset of negatively charged membrane lipids (acidic lipids shown in red). Neither construct bound the mitochondria-specific acidic lipid, cardiolipin. Lipid strips were processed at the same time and developed on the same piece of film and are representative of three experimental trials. Blots included the following lipids: triglyceride (trigly), diacylglycerol (DAG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (cardio), phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-triphosphate (PIP3), cholesterol (chol), sphingomyelin (sphng), and 3-sulfogalactosylceramide (sulfa).