Mdm35 interacts with Ups1 and Ups2. (A) Coimmunoprecipitation of Mdm35 with Ups1^MYCand Ups2^MYC. Mitochondria isolated from wild-type cells (WT, CG214) or cells overexpressing either Ups1^MYC (Ups1^MYC↑, CG630) or Ups2^MYC (Ups2^MYC↑, CG626) were solubilized and subjected to coimmunoprecipitation using MYC-specific antibodies. Eluates were analysed by Tris/Tricine SDS–PAGE and subsequent silver staining. Ups1, Ups2 and Mdm35 were identified by peptide mass finger printing. The asterisk (^*) marks a protein binding unspecifically to the beads. (B, C) Affinity purification of Mdm35 with endogenous levels of Ups1^TAP and Ups2^TAP. Mitochondria isolated from wild-type cells (WT,CG214) or cells expressing either (B) Ups2^TAP (CG591) or (C) Ups1^TAP (CG593) from the endogenous gene locus were solubilized and proteins were affinity purified using immunoglobulin G-coupled beads. Total (T), pellet (P), supernatant (S) flow-through (Ft) fractions (1%) and the eluate (E) fraction (100%) were analysed by Tris/Tricine SDS–PAGE, followed by Ponceau S staining (PoS) and immunoblotting. The asterisk (^*) marks a protein band cross-reacting with Mdm35 antibodies.

Mdm35 controls the accumulation of Ups2 in mitochondria. (A) Steady-state levels of Ups2^MYC in cells containing different amounts of Mdm35. Wild-type (WT) and Δmdm35 cells expressing genomically tagged Ups2^MYC were transformed with either YCplac111∷ADH (PY127, PY129) or YCplac111∷ADH encoding MDM35 ([MDM35]) (PY128, PY130). Cells were grown to mid-log phase in selective synthetic glucose media. Proteins were extracted from mid-log phase cells by alkaline lysis, analysed by SDS–PAGE and immunoblotting using Yme1-, MYC- and affinity-purified Mdm35-specific antibodies (upper panel). Five-fold serial dilutions of these cells were spotted on selective, glucose-containing plates and incubated at 16°C (lower panel). (B, C) Ups2 overexpression does not restore normal PE levels and cell growth in the absence of Mdm35. (B) TLC analysis of mitochondrial lipids in wild-type (WT, CG214), Δmdm35 (CG524), Ups2^MYC↑ (CG626) and Δmdm35 Ups2^MYC↑ (PY64) cells (upper panel). The asterisk (^*) indicates an unidentified lipid species. Mitochondria purified by sucrose-gradient centrifugation were used for determination of the phospholipid profile and were analysed by SDS–PAGE and immunoblotting using porin- (Por1) and MYC-specific antibodies (lower panel). (C) Five-fold serial dilutions of mid-log phase cultures of the cells were spotted on YP plates containing galactose and incubated at 16 or 30°C.

Mdm35 controls cell growth and the lipid composition of mitochondrial membranes in an Yme1-dependent manner. (A) Genetic interaction of MDM35 and YME1. Diploid Δmdm35Δyme1 cells that were obtained by mating of Δmdm35 and Δyme1 cells (CG324 × VIA5, CG560 × CG113) were sporulated and meiotic spores were grown on glucose-containing medium. White arrows indicate double-mutant progenies in the W303 strain background or inviable double-mutant spores in the strain S288c. (B) Reduced PE and CL levels in Δmdm35Δyme1 mitochondria. Phospholipid profiles of wild-type (WT, CG1) and Δyme1Δmdm35 (CW414) mitochondria were analysed by TLC. The asterisk (^*) indicates an unidentified lipid species. (C) Ups2 accumulates in Δmdm35Δyme1 cells. Mitochondria were isolated from wild-type (WT, CG1), Δmdm35 (CG323), Δyme1 (VIA4), and Δmdm35Δyme1 (CW414) cells grown on galactose-containing media and analysed by Tris/Tricine gradient SDS–PAGE and immunoblotting using porin (Por1)- and affinity-purified Ups2-specific antibodies. (D) Accumulation of Ups2 in mitochondria upon import in vitro depends on Mdm35. Radiolabelled Ups1, Ups2 and, for control, Tim9, were imported into wild-type (WT, CG1), Δmdm35 (CG323) and Mdm35-overexpressing (CW343) mitochondria for the indicated time. The analysis of the samples by Tris/Tricine gradient SDS–PAGE and autoradiography is shown in the upper panels. A quantification of imported Ups1, Ups2 and Tim9 is shown in the lower panels. Protein imported into wild-type (WT) mitochondria after 4 min was set to 100%. Data represent±standard deviation of six independent experiments. ^***P<0.0005.

A conserved regulatory pathway determines the accumulation of Ups1 and Ups2 in the intermembrane space, which regulate the phospholipid composition of mitochondrial membranes. Ups1 and Ups2 are unstable proteins that are degraded by the i-AAA protease Yme1 and in the case of Ups1, also by Atp23. Binding of Mdm35 stabilizes Ups1 and Ups2 against proteolysis and ensures accumulation of both proteins in the intermembrane space. Both Ups1 and Ups2 have been found to be membrane associated (Tamura et al, 2009; Osman et al, 2009a) but are shown as soluble proteins in the IMS for simplicity. See text for details. IM, mitochondrial inner membrane; IMS, mitochondrial intermembrane space; OM, mitochondrial outer membrane.

Phenotypic similarities between Mdm35- and Ups2-deficient cells. (A) Steady-state levels of mitochondrial proteins in Δmdm35Δphb1 [PHB1] cells. Mitochondria were isolated from Δphb1 [PHB1] (CG278) and Δmdm35Δphb1 [PHB1] (CG287) cells grown on galactose-containing media in the presence of doxycycline (Dox, 2 μg/ml) for the times indicated. Mitochondrial proteins were analysed by SDS–PAGE and immunoblotting. (B) Dissipation of the mitochondrial membrane potential in cells lacking Mdm35 and Phb1. Mitochondria were isolated from Δphb1 [PHB1] (CG278) and Δmdm35Δphb1 [PHB1] (CG278) cells grown on galactose-containing media in the presence or absence of Dox for the indicated times. Membrane potential was measured using the membrane-potential sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃ (5)). Data represent ±s.d. of three independent experiments. (C) Psd1 activity in the absence of Mdm35. Mitoplasts derived from wild-type (WT, CG1) or Δmdm35 (CG323) cells were generated by osmotic swelling. To monitor the disruption of the outer membrane, mitochondria were resuspended in SHKCl buffer (0.1 μg/μl) and subjected to trypsin treatment (0.25 μg/μl, 30 min, 4°C). Samples were further analysed by SDS–PAGE and immunoblotting using antibodies directed against the intermembrane space protein Yme1 and the matrix protein Mge1 (upper panel). Mitoplasts were incubated with NBD-PS for the indicated time periods. Phospholipids were separated by TLC and fluorescent NBD-PE was quantified by fluorescence imaging (lower panel). NBD-PE accumulating in wild-type (WT) mitochondria after 20 min was set to 100%. Data represent ±s.d. of three independent experiments. ^*P<0.05, ^**P<0.005. (D) Effects of MDM35 deletion in Δups1 cells. Five-fold serial dilutions of wild-type (WT, CG1), Δmdm35 (CG323), Δmdm35Δups2 (CW128), Δups2 (CW130), Δups1 (CW143), Δmdm35Δups1 (CW144) cells were spotted on YPD and incubated at the indicated temperature (upper panel). The mitochondrial lipid profile was analysed by TLC (lower panel). The asterisk (^*) indicates an unidentified lipid species.

Ups2 is degraded by the i-AAA protease Yme1. (A) Accumulation of Ups2 in Δyme1 cells. Wild-type cells (WT, CG214) and cells lacking different mitochondrial peptidases [Δimp1 (Euroscarf 732), Δcym1 (Euroscarf 4266), Δoma1 (Euroscarf 6003), Δyme1(CG113), Δatp23 (CW1), Δpcp1 (Euroscarf 4731), Δprd1(Euroscarf 3464)] were grown to mid-log phase on glucose-containing media. Mitochondria-enriched membrane fractions were analysed by SDS–PAGE and immunoblotting using Yme1- and affinity-purified Ups2-specific antibodies. Asterisks (^*) indicate protein bands cross-reacting with the Ups2-antibodies. (B) Ups1 and Ups2 accumulate in mitochondria lacking proteolytically active Yme1. Mitochondria isolated from wild-type cells (WT, CG1), Δyme1(VIA4) cells or Δyme1 cells expressing proteolytically inactive Yme1^E541Q (YTE26) were analysed by SDS–PAGE and immunoblotting using Yme1-, Tom40- and affinity-purified Ups1- and Ups2-specific antibodies. (C) Binding of Ups2 to proteolytically inactive Yme1^E541Q. Mitochondria isolated from yme1 cells expressing either Yme1^E541Q (YTE26) or a variant thereof carrying an N-terminal hexahistidine tag (His Yme1^E541Q) (YTE89) were solubilized and extracts subjected to Ni-NTA affinity chromatography. Eluate fractions were analysed by SDS–PAGE and Coomassie staining. Ups2 was detected by peptide mass finger printing in the eluate only when mitochondria harbouring His-Yme1^E541Q were analysed. (D) Degradation of Ups2 by Yme1 following import into mitochondria in vitro. Radiolabelled Ups2 was imported into mitochondria derived from either wild-type (WT, CG1), Δyme1 (VIA4), Δatp23 (CW3) or Δyme1Δatp23 (CW79) cells. The stability of newly imported Ups2 upon further incubation at 37°C was assessed by SDS–PAGE and autoradiography. Quantification of Ups2 accumulation in mitochondria is represented in the lower panel. Newly imported Ups2 was set to 100%. Data represent ±s.d. of four independent experiments. ^***P<0.0005.

Mdm35 controls degradation of Ups1 by Yme1 and Atp23. (A) Ups1 is less abundant than Ups2. Wild-type (WT) and Δmdm35 cells expressing genomically tagged Ups2^MYC (PY102, PY117) or Ups1^MYC (PY103, CW385) were grown to mid-log phase. Proteins were extracted and analysed by SDS–PAGE and immunoblotting using MYC- and porin (Por1)-specific antibodies. (B) Steady-state levels of Ups1^MYC in the absence of Mdm35. Wild-type (WT) and Δmdm35 cells expressing genomically modified Ups1^MYC were transformed with either YCplac111∷ADH (CW394, CW398) or YCplac111∷ADH encoding MDM35 ([MDM35]) (CW396). Mitochondria-enriched membrane fractions were analysed by SDS–PAGE and immunoblotting using MYC-, Yme1- and affinity-purified Mdm35-specific antisera. (C) Ups1 is stabilized in mitochondria lacking YME1 and ATP23. Radiolabelled Ups1 was imported into mitochondria isolated from WT (CG1), Δyme1 (VIA4), Δatp23 (CW3) or Δyme1Δatp23 (CW79) cells and the stability of newly imported Ups1 upon further incubation at 37°C was assessed by SDS–PAGE and autoradiography. Quantification of Ups1 accumulation in mitochondria is shown in the lower panel. Newly imported Ups1 was set to 100%. Data represent ±s.d. of four independent experiments. ^*P<0.05, ^**P<0.005, ^***P<0.0005. (D) Steady-state levels of Ups1 and Ups2 in mitochondria lacking Yme1 and Atp23. Mitochondria were isolated from WT (CG), Δatp23 (CW3), Δyme1 (VIA4), Δatp23Δyme1 (CW79), Δyme1Δmdm35 (CW414) and Δmdm35 (CG323) cells and analysed by SDS–PAGE and immunoblotting using Yme1-, Tom40- and affinity-purified Mdm35-, Ups1- and Ups2-specific antibodies. (E) Quantitative assembly of Ups1 and Ups2 with Mdm35 in Δyme1 mitochondria. Sucrose-gradient purified mitochondria isolated from Δyme1 cells were solubilized in 1% (w/v) dodecylmaltoside (DDM) at a protein concentration of 5 mg/ml. After a clarifying spin, extracts were subjected to sizing chromatography using a Superose 12 column (GE Healthcare), which had been equilibrated with 0.1% (w/v) DDM, 25 mM Tris/HCl pH 7.4, 150 mM NaCl, 10% (v/v) glycerol. Eluate fractions were TCA-precipitated and analysed by gradient Tris/Tricine gradient SDS–PAGE and immunoblotting using Ups1-, Ups2- and Mdm35-specific antisera. (a) alcoholdehydrogenase (150 kDa), (b) ovalbumine (44.3 kDa), (c) cytochrome c (12.4 kDa). The asterisk (^*) refers to a band unspecifically cross-reacting with the respective antiserum (D, E).