Mdm35p interacts with Ups proteins. (A) (Left panel) MDM35 (−) and MDM35FLAG (+) mitochondria expressing Ups1pMyc, Ups2pMyc, or Ups3pMyc were incubated with increasing amounts of DSG. Proteins were analysed by immunoblotting using anti-Myc antibodies. Arrowheads show crosslinked products. (Right panel) After incubation with DSG, mitochondria were lysed and subjected immunoprecipitation using anti-FLAG antibodies. (B) MDM35 (−) and MDM35FLAG (+) mitochondria expressing Ups2pGFP and Ups3pMyc were solubilized with digitonin and subjected to immunoprecipitation using anti-FLAG M2-agarose. The mitochondrial lysate (Control), the flow-through fraction (FT), and the eluate (Elute) are shown. Proteins were analysed by immunoblotting using the indicated antibodies. An asterisk indicates a non-specific band. (C) MDM35FLAG mitochondria expressing Ups2pGFP and Ups3pMyc were solubilized and analysed by glycerol density gradient centrifugation in the presence (+FLAG) or absence (−FLAG) of anti-FLAG antibodies. (D) GST and GST-Mdm35p were incubated with radiolabelled Ups proteins and pelleted with glutathione-agarose beads; 10% of the input (control) and 100% of the bound fraction (GST and GST-Mdm35p) were resolved by SDS–PAGE, and analysed by coomassie brilliant blue staining (left panel) and autoradiography (right panel).

mdm35Δ mitochondria contain decreased amounts of Ups proteins. (A) Whole cell extracts prepared from wild-type (+) and mdm35Δ (−) cells expressing Ups1pFLAG, Ups2pFLAG, or Ups3pFLAG were subjected to immunoblotting using the indicated antibodies. (B) Isolated mitochondria were analysed by immunoblotting. Interestingly, Ups2p exhibited a doublet in mdm35Δ mitochondria. As shown in Supplementary Figure S4, this was independent of phosphorylation or an altered redox state of cysteine residues. (C) Mitochondria were visualized in wild-type, mdm35Δ, and ups1Δups2Δups3Δ cells using matrix-targeted Su9-GFP (Sesaki et al, 2006). Cells were grown to log phase in YPD, fixed in 3.7% formaldehyde, and then examined by differential interference contrast (DIC) and fluorescence (Su9-GFP) microscopy. Bars, 3 μm. (D) Quantitation of mitochondrial morphology. Cells containing tubular mitochondria were scored. Values are mean±s.e.m. (n=3). At least 300 cells were examined in each experiment.

Ups protein import requires TOM. (A) After incubation at 37°C for 10 min, wild-type and tom40-4 mitochondria were mixed with radiolabelled Ups proteins for the indicated times then treated with proteinase K followed by SDS–PAGE and autoradiography. Import into wild-type mitochondria after 10 min was set as 100%. C, control (10% of precursor proteins). A bar graph shows amounts of imported proteins at 4 min. Values are mean±s.e.m. (n=3). (B) Radiolabelled Ups2p was added to MDM35 (WT), MDM35FLAG, and mdm35Δ mitochondria. After incubation in the presence (+) or absence (−) of 300 μM DSG, mitochondria were subjected to SDS–PAGE and autoradiography. (C) mdm35Δ mitochondria were incubated with radiolabelled Ups2p and treated with 300 μM DSG. Mitochondria were solubilized and subjected immunoprecipitation using the indicated antibodies. The immunoprecipitates were analysed by SDS–PAGE and autoradiography.

Mdm35p and Ups2p are required for phospholipid metabolism. (A) UPS2FLAG (WT Ups2p), GAL1-UPS2FLAG (WT Ups2p↑), and mdm35ΔGAL1-UPS2FLAG (mdm35Δ Ups2p↑) cells were grown in YPLac containing 0.4% galactose in the presence of ³²Pi. Phospholipids were extracted from crude mitochondrial fractions and separated on thin-layer chromatography (left panel). Amounts PE relative to total phospholipids were determined, and those detected in wild-type (WT) mitochondria were set to 100%. Values are mean±s.e.m. (n=3). Expression of Ups2pFLAG was confirmed by immunoblotting of isolated mitochondria, with (+) and without (−) proteinase K treatments (PK) (upper right panels). The OM protein Tom22p and the IM protein Tim50p are shown as controls. (B) Wild-type, ups1Δ and ups2Δ cells carrying MDM35FLAG (Mdm35p) or GAL1-MDM35FLAG (Mdm35p↑) were grown in YPLac containing 0.4% galactose in the presence of ³²Pi. Phospholipids were extracted from crude mitochondrial fractions and separated on thin-layer chromatography (upper left panel). Amounts of CL and PE were quantitated as described in (A). Values are mean±s.e.m. (n=3). Overexpression of MDM35FLAG was confirmed by immunoblotting of whole cell extracts (lower left panels). Tom40p is shown as a control. (C) A model for Ups protein import.

Loss of Mdm35p rescues defects in cell growth, mitochondrial protein import, and cardiolipin abundance in ups1Δ cells. (A) Serial dilutions of yeast cells were spotted onto YPD and YPGE plates and grown at 30°C for 2 and 4 days, respectively. (B) Whole cell extracts prepared from wild-type (MDM35) and mdm35Δ cells lacking UPS1 (1Δ) and/or UPS2 (2Δ) were analysed by immunoblotting (p, precursor form; m, mature form). (C) Analysis of mitochondrial phospholipids. Cells were grown in YPLac containing 0.4% sucrose in the presence of ³²Pi. Phospholipids were extracted from crude mitochondrial fractions and separated on thin-layer chromatography. CL, cardiolipin; PA, phosphatidic acid; PG, phosphatidylglycerol; PI, phosphatidylinositol; PC, phosphatidylcholine. Amounts of each lipid relative to total phospholipids were determined, and those detected in wild-type mitochondria were set to 100%. Values are mean±s.e.m. (n=3).

Mdm35p facilitates efficient import of Ups proteins into mitochondria. (A) Cells carrying MDM35FLAG (WT) or GAL1-MDM35FLAG (Mdm35↓) were grown in YPLac containing 0.4% glucose for 12 h. Mitochondria were isolated from these cells and analysed by immunoblotting using the indicated antibodies. (B) Phospholipids were extracted from wild-type, Mdm35↓, and psd1Δ mitochondria, and separated on thin-layer chromatography. Phospholipids were visualized by molybdenum blue (Sigma). psd1Δ mitochondria were used as a control. Psd1p is a mitochondrial phosphatidylserine decarboxylase that is required for the production of PE in this organelle (Clancey et al, 1993). (C) Mitochondria were incubated with radiolabelled precursors for the indicated times then treated with proteinase K followed by SDS–PAGE and autoradiography. Import into wild-type mitochondria after 10 min was set as 100%. C, control (10% of precursor proteins). A bar graph shows amounts of imported proteins at 4 min. Values are mean±s.e.m. (n=3). (D) In vitro import assays using wild-type and Mdm35p-overexpressing mitochondria were performed and analysed as described in (C). Immunoblotting of mitochondria using the indicated antibodies are shown.

Ups protein import does not require TIM23 and MIA. (A) In vitro import assays were performed using wild-type and tim50-ts mitochondria. m, mature form. Import into wild-type mitochondria after 10 min was set as 100%. A bar graph shows amounts of imported proteins at 4 min. Values are mean±s.e.m. (n=3). (B) Wild-type and cys-null Ups2p were imported into wild-type mitochondria in vitro. Amounts relative to the input are determined. The input is set as 100%. (C) ups2Δ cells carrying a control vector, wild-type UPS2FLAG, and cysteine-null UPS2FLAG were grown in SCD-Trp in the presence of ³²Pi. Mitochondrial phospholipids were analysed on thin-layer chromatography (upper left panel) and quantiated as described in Figure 2C. Whole cell extracts were subjected to immunoblotting using antibodies to FLAG and Tim23p (lower left panels). (D, E) In vitro import assays were performed using wild-type and Tim10p-depleted mitochondria (D), and wild-type and tim8Δtim13Δ mitochondria (E). Import into wild-type mitochondria after 10 min was set as 100%. C, control (10% of precursor proteins). Bar graphs show amounts of imported proteins at 4 min. Values are mean±s.e.m. (n=3).