Fig. 4: Effects of amino-acid substitutions of phosphorylation sites of Dnm1 by Aurora kinase in Dnm1 on mitochondrial division.
a In vitro kinase assay of recombinant CmAUR and site-directed Dnm1 mutants and those with mutations of 4, 7, 8, and 9 residues (see Supplementary Table 3). b Quantification of in vitro kinase assays of CmDnm1 single amino-acid variants. Signals from different membranes were normalized against the luminosity of wild-type CmDnm1. The relative value of wild-type CmDnm1 was 1. n = 3 for T139A, n = 4 for S726A. The error bars indicate standard error of the mean. c Frequency of transformants expressing CmDnm1 variants. Alanine and phosphomimetic glutamic acid mutants of CmDnm1T139 and CmDnm1S726 were overexpressed in C. merolae cells. According to classification Fig. 2d, transfectants were counted. Transfectants with an aberrant number of plastids (three or more) were not counted. d Normal phenotype of dividing mitochondrion in cell overexpressing wild-type CmDnm1. e Phenotype of single mitochondrion without division in cell overexpressing CmDnm1S570E. f Phenotype of multiple chloroplasts in CmDnm1T139A overexpression mutant. g Frequency of cells with multiple chloroplasts among Dnm1-mutant transfectants. Total countable transfectants were counted. h In vitro kinase assay of human Aurora kinases with recombinant GST-human Drp1. Bars: 1 μm.
