Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast

Mdm31 and Mdm32 are located in the mitochondrial inner membrane. (A) Subfractionation of yeast cells. Wild-type cells were subfractionated by differential centrifugation into mitochondria, microsomes, and cytosol. Mitochondria were further purified on a sucrose gradient, microsomes were purified on a percoll gradient. 50 μg of protein of each fraction was analyzed by Western blotting. The ADP/ATP carrier (AAC) served as a marker for mitochondria, Erp1 for ER, and Bmh2 for soluble cytosolic proteins. White lines indicate that intervening lanes have been spliced out. (B) Subfractionation of mitochondria. Isolated wild-type mitochondria were subfractionated, proteins were precipitated with TCA, and 50 μg of each fraction was analyzed by Western blotting. Lane 1, intact mitochondria; lane 2, intact mitochondria treated with proteinase K (PK); lane 3, mitoplasts generated by hypotonic swelling and treated with PK; lane 4, mitochondria solubilized with Triton X-100 and treated with PK. Markers used were Dld1 as an inner membrane protein exposed to the intermembrane space and Mge1 as a soluble matrix protein. (C) Carbonate fractionation of mitochondria. Isolated wild-type mitochondria were extracted with carbonate, proteins were precipitated with TCA, and 50 μg of each fraction was analyzed by Western blotting. Lanes 1 and 3, soluble protein–containing fraction after carbonate extraction (supernatant, SN); lanes 2 and 4, membrane protein–containing fraction after carbonate extraction (P, pellet). Markers used were AAC as an integral inner membrane protein and Mge1 as a soluble matrix protein.

Cells lacking Mdm31 and Mdm32 harbor aberrant mitochondria. (A) Mitochondrial morphology. Wild-type (a), Δmdm31 (c and d), Δmdm32 (e and f), and Δmdm31/Δmdm32 (b) cells expressing mtGFP were grown to log phase in YPD (yeast extract, peptone, and glucose) medium and analyzed by confocal fluorescence microscopy. (left) Maximum intensity projections of several optical planes covering the entire cell, merged with a bright field transmission image. (right) A representative single optical plane. (B) Morphology of microfilaments, vacuole, and the ER. Cells were grown to log phase in glucose-containing medium. Then, they were either fixed and stained with rhodamine-phalloidin (for actin), or living cells were stained with 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (for vacuole), or cells expressing ER-targeted GFP were examined directly. Left, phase-contrast image; right, fluorescence microscopy. (C) Mitochondrial fusion. Cells of opposite mating type preloaded with mtGFP or mitochondria-targeted DsRed (mtDsRed) were mated, and zygotes were analyzed by phase-contrast and fluorescence microscopy.

Ultrastructure of mitochondria in cells lacking Mdm31 and Mdm32. (A) Cross section of mitochondria in wild-type cells. (C) Cross section of a giant mitochondrion in a Δmdm31 cell. (B and D) Cross sections of mitochondria in Δmdm32 cells. (E–H) Cross sections of mitochondria in Δmdm31/Δmdm32 cells. (A–F) mtGFP was labeled with immunogold to identify the matrix compartment. White arrows in A and E point to inner membrane cristae; black arrows in G point to membrane bridges between the inner membrane und circular inclusions. All images are displayed at the same magnification with the exception of H, which is an enlargement of G.

Movement of mitochondria is compromised in cells lacking Mdm31 and Mdm32. (A) Wild-type cells expressing mtGFP were grown to log phase in glucose-containing medium, transferred to a microscope chamber that was continuously flushed with fresh medium, and analyzed by confocal time-lapse microscopy. Left, bright field image; right, representative time points of the remodelling process shown as maximum intensity projections of several optical planes. Δmdm31 (B), Δmdm32 (C), and Δmdm31/Δmdm32 (D) cells were analyzed as in A. Arrows point to shape changes of aberrant organelles. (E) Interaction of mitochondria with actin filaments in vitro. Isolated mitochondria of wild-type (WT), Δmdm31, and Δmdm32 cells were incubated without or with isolated actin filaments in the presence or absence of ATP. After centrifugation of mitochondria through a sucrose cushion, bound actin was detected by immunoblotting. The mitochondrial protein AAC served as a loading control.

Mdm31 and Mdm32 are required for maintenance of normal mtDNA nucleoids. (A) Growth phenotypes. Wild-type, Δmdm31, Δmdm32, and Δmdm31/Δmdm32 cells were grown overnight in glucose-containing medium. Then, 10-fold serial dilutions were spotted onto plates containing glucose (YPD) or glycerol (YPG) as carbon source. YPD plates were incubated for 2 d and YPG plates for 3 d at the indicated temperatures. (B) Nucleoid structure and localization of Mmm1-containing complexes. Wild-type, Δmdm31, and Δmdm32 cells expressing an Mmm1-DsRed fusion protein under control of the MMM1 promoter were grown to log phase in glucose-containing medium. For staining of mtDNA nucleoids, expression of an Abf2-GFP fusion protein under control of a GAL-promoter was induced by shifting the cells to galactose-containing medium for 1 h (top), or cells were incubated in the presence of 1 μg/ml DAPI for 15 min. It should be noted that nuclear DNA is not stained under these conditions (Aiken Hobbs et al., 2001). Cells were washed in glucose-containing medium and analyzed by differential interference contrast (DIC) and fluorescence microscopy. Bars, 2 μm. (C) Steady-state levels of mitochondrial proteins. Mitochondria were isolated from wild-type (WT), Δmdm31, Δmdm32, and Δmdm31/Δmdm32 cells, and equal amounts of mitochondrial protein were analyzed by immunoblotting using the indicated antisera. The mitochondrial matrix protein Mge1 served as a loading control.

Mdm31 and Mdm32 are parts of two separate complexes in the inner membrane. (A) Gel filtration analysis of wild-type mitochondria. Isolated mitochondria were solubilized in digitonin-containing buffer and loaded onto a gel filtration column. After chromatography, proteins were precipitated with TCA, and fractions from the same column run were analyzed by Western blotting using affinity-purified antisera against Mdm31 (closed squares), Mdm32 (closed circles), and Mmm1 (open squares). Signals were quantified by densitometry and plotted as a percentage of total Mdm31, Mdm32, and Mmm1 protein in the extract. Arrows indicate molecular masses of calibration standards. (B) Δmdm31, Δmdm32, Δmdm31/Δmdm32, and Δmmm1 mitochondria were analyzed as in A. (C) Coimmunoprecipitation of Mdm32 with Mdm31. Radiolabeled Mdm32 (lane 1) was imported into wild-type mitochondria (lane 2). Mitochondria were lysed with Triton X-100 and subjected to coimmunoprecipitation with affinity-purified antibodies against Mdm31 (lane 3). In control reactions, coimmunoprecipitation was analyzed in Δmdm31 mitochondria (lanes 4 and 5), and preimmuneserum was used after import of Mdm32 into wild-type mitochondria (lanes 6 and 7). Signals were analyzed by SDS-PAGE and autoradiography. The amount of precursor protein in lane 1 corresponds to 10% of the material that was used for the import reactions; the amount of import reactions loaded in lanes 2, 4, and 6 corresponds to 10% of the material that was used for coimmunoprecipitation. p, precursor form of Mdm32; m, mature form of Mdm32. White lines indicate that intervening lanes have been spliced out.