Mutations in the proximal half of the Myo2 cargo binding domain produce mitochondrial distribution and morphology defects. (A) Ribbon (top) and space-filling (middle and bottom) diagrams of the Myo2 tail structure were generated with PyMOL software (PyMOL Molecular Graphics System, version 1.3; Schrödinger). The locations of mutated amino acid residues are indicated. Residues L1301 and Q1233 mutated in the myo2(LQ) allele are highlighted in red. Residue V1236 is not indicated, as it is hidden in the interior of the domain. Red, >20% buds devoid of mitochondria; orange, 10–20% buds devoid of mitochondria; yellow, <10% buds devoid of mitochondria. (B) Cells were grown in yeast extract/peptone/dextrose (YPD) medium to the logarithmic growth phase and incubated for 3 h at the indicated temperature. Mitochondrial morphology (top) and buds devoid of mitochondria (bottom) were quantified at ambient temperature by fluorescence microscopy of mtGFP-expressing cells. Cells containing branched tubular mitochondria in the absence of any mitochondrial aggregation or fragmentation were classified as WT-like mitochondria. Quantifications are mean values from three independent experiments (n = 100), and error bars indicate standard deviations. (C) Cells expressing mtGFP were grown to the logarithmic growth phase in glucose-containing minimal medium, fixed, stained with rhodamine-phalloidin, and observed by confocal fluorescence microscopy. (left to right) Bright field image, maximum intensity projection of GFP fluorescence, maximum intensity projection of rhodamine fluorescence, and merged image generated with bioView3D software (The Center for Bio-Informatics, University of California, Santa Barbara, CA). Bars, 5 µm.