Figure 4
From: Interaction of MDM33 with mitochondrial inner membrane homeostasis pathways in yeast
Mdm33 affects mitochondrial phospholipid homeostasis.
(A) Phospholipidome of mitochondria isolated from cells transformed with pYX223-MDM33 for expression of MDM33 under control of the GAL1/10 promoter (blue) or the corresponding empty vector (orange) and grown in synthetic complete medium containing galactose as carbon source. Phospholipid analysis was done by quantitative mass spectrometry. Bars represent the mean values of two independent mitochondrial preparations. The mean values of two technical replicates for each of the mitochondrial preparations are indicated by black dots. (B) Wild type cells expressing mitochondrial matrix targeted GFP (mtGFP) and ERFP-tagged Mmm1 (pRS316-MMM1-ERFP) were transformed with pYX223-MDM33 or the empty vector, grown to the logarithmic growth phase in synthetic complete medium containing galactose as carbon source and analyzed by fluorescence microscopy. Bar, 5 μm. (C) Western blot analysis of whole cell extracts of wild type cells carrying an empty vector or pYX223-MDM33. Cells were grown overnight in synthetic complete medium containing galactose as carbon source and diluted to logarithmic growth phase. Protein was extracted from the cells by boiling in sample buffer after alkaline treatment. (D) In vitro Psd1 activity assay. Mitochondria were isolated from wild type cells carrying an empty vector or overexpressing Mdm33 (pYX223-MDM33) or a FLAG-tagged version of Mdm33 (pYX223-F-MDM33) and incubated with liposomes containing NBD-PS for 30 minutes at 30 °C. Total lipids were isolated and separated by TLC. Shown is the NBD-fluorescence. The same samples were analyzed by Western blotting. (E) Quantifications are the ratio of the NBD-PE and NBD-PS signals normalized to the wild type-ratio. Shown are mean values and standard deviation obtained from three independent experiments.
