Figure 5

Mdm33 is required for mitochondrial fission.

(A) Cells expressing mitochondrial matrix targeted GFP (mtGFP) were grown to logarithmic growth phase in YPD, either mock-treated or incubated for 40 min with 0.5 mM sodium azide, fixed in 3.7% formaldehyde and analyzed by fluorescence microscopy. For each strain cells containing fragmented mitochondria were scored. Error bars indicate standard deviations of 3 independent experiments with 150 cells per experiment. Images are merges of DIC and GFP fluorescence. Bar, 5 μm. (B) Time lapse series of cells grown to logarithmic growth phase in synthetic complete medium and expressing mitochondrial matrix targeted ERFP (mtERFP) and Dnm1-GFP. Images represent maximum intensity projections of fluorescence image z stacks. Arrows highlight mitochondrial division events. Bar, 5 μm. (C) Cells expressing Dnm1-GFP, Mmm1-ERFP and mitochondria targeted BFP (mtBFP) were grown to logarithmic growth phase in synthetic complete medium and analyzed by fluorescence microscopy. For both strains cells were scored for association of Mmm1-ERFP with Dnm1-GFP patches. Bar, 5 μm. (D) Strains were transformed with a multicopy plasmid for overexpression of MDM33 from the inducible GAL1/10 promoter (pYX223-MDM33) or an empty vector. 10-fold serial dilutions were spotted on synthetic complete medium containing glucose (repression of the GAL promoter) or galactose (induction of the GAL promoter) as carbon source and incubated at 30 °C for 2-4 days. (E) Control cells (empty vector) and cells overexpressing MDM33 from the GAL1/10 promoter (pYX223-MDM33) and mtGFP were grown overnight in synthetic complete medium containing galactose as carbon source, diluted to logarithmic growth phase and analyzed by fluorescence microscopy. Bar, 5 μm. (F) Electron micrographs of ultrathin sections of cells grown as in (E). Bars, 200 nm.