Analysis of the mitochondria-associated degradation pathway (MAD) in yeast cells

Abstract

Mitochondria are critical for cellular function in health, disease and aging. Mitochondria-associated degradation (MAD), a pathway for quality control of the organelle, recognizes and ubiquitinates unfolded mitochondrial proteins, removes them from the organelle using a conserved segregase complex, which contains an AAA-ATPase Cdc48 and its cofactors, and degrades them using the ubiquitin-proteasome system (UPS). Here, we describe an approach to 1) study the turnover and ubiquitination of candidate MAD substrates, 2) assay retrotranslocation and export of MAD substrates from the mitochondrial matrix in vitro, and 3) study interactions between MAD substrates and Cdc48 using the budding yeast, Saccharomyces cerevisiae, as a model organism.

INTRODUCTION

Mitochondria are essential organelles through their roles in processes including oxidative phosphorylation, the Krebs cycle, metabolite production, calcium and reactive oxygen species (ROS) homeostasis, and the regulation of apoptosis (Sharma, Smith, Yao, & Mair, 2019). Not surprisingly, there are several pathways for mitochondrial quality control, which repair the organelle or degrade mitochondria that are damaged beyond repair. Mitochondrial chaperones and proteases promote re-folding and degradation, respectively, of misfolded mitochondrial proteins. The mitochondria-associated degradation pathway (MAD) mediates degradation of mitochondrial proteins by the ubiquitin-proteasome system (UPS). Finally, damaged portions of mitochondria can be released from the organelle in mitochondria-derived vesicles (MDVs). However, mitochondria that are damaged beyond repair are degraded in lysosomes (the vacuole in yeast) by mitophagy, a specific form of macroautophagy. These quality control pathways, in turn, are regulated by stress-induced signaling pathways including the mitochondrial unfolded protein response (UPRmt) and the mitochondrial stress-induced nuclear transcriptional response pathway (Ng, Wai, & Simonsen, 2021; Sugiura, McLelland, Fon, & McBride, 2014).

MAD is a member of a family of organelle quality control pathways, including ER-associated degradation (ERAD), and the endosome and Golgi-associated degradation (EGAD) (Hirsch, Gauss, Horn, Neuber, & Sommer, 2009; Schmidt et al., 2019) (Fig. 1). In these pathways, unfolded proteins are identified, marked by post-translational modification with ubiquitin and removed from organelles by a segregase protein complex. The segregase complex contains the conserved AAA-ATPase Cdc48 (VCP/p97 in mammals, Ter94 in Drosophila) and its cofactors, and transfers ubiquitinated, unfolded proteins to the proteasome for degradation (Azuma et al., 2014; Heo et al., 2010; Tanaka et al., 2010; Wu, Li, & Jiang, 2016; Xu, Peng, Wang, Fang, & Karbowski, 2011). Although the core components of the segregase complex are similar in MAD, ERAD and EGAD, the segregase complex is targeted to specific organelles in all three pathways by segregase-associated, organelle-specific substrate-recruiting factors. In MAD, Doa1 (phospholipase A2-activating protein or PLAP/PLAA in Drosophila and humans) is a substrate recruitment factor that localizes the segregase complex to mitochondria in yeast (Wu et al., 2016) (Qiu et al., 2010).

Figure 1.

The mitochondria-associated degradation pathway (MAD) (Liao, Wolken, Serrano, Srivastava, & Pon, 2020)

Previous studies in our laboratory and others revealed that MAD is critical for cellular fitness and mitochondrial quality under basal and stressed conditions, and that MAD is required for normal chronological lifespan in yeast (Liao et al., 2020; Wu et al., 2016). However, a comprehensive understanding of mitochondrial proteins that are targets for MAD, and the mechanism of MAD, remain elusive. For example, most evidence indicates that MAD substrates localize to the mitochondrial outer membrane. Indeed, the outer membrane proteins Fzo1, Mdm34, Msp1, and Tom70 have been identified as MAD substrates in yeast (Cohen, Leboucher, Livnat-Levanon, Glickman, & Weissman, 2008; Heo et al., 2010; Wu et al., 2016). Moreover, mitofusins and Mcl-1 have been identified as MAD substrates in mammalian cells (Tanaka et al., 2010; Xu et al., 2011). However, our recent study revealed that mitochondrial matrix proteins, including Kgd1p (a subunit of the α-ketoglutarate dehydrogenase TCA cycle complex) and Pim1 (Lon proteases), are MAD targets, and that 70% of the MAD substrates identified localize to the mitochondrial inner membrane or matrix (Liao et al., 2020). Thus, MAD function in mitochondrial protein quality control extends beyond the mitochondrial outer membrane to mitochondrial matrix and inner membrane proteins.

These studies also raise a question: how are MAD substrates that are in the matrix or inner membrane of the organelle targeted to the cytosol for proteasomal degradation in MAD? A crucial step in investigating this question was the development of an assay for retrotranslocation: the delivery of putative substrates from the interior of mitochondria to the cytosol where they are accessible to cytosolic proteasomes. Here, we describe a method to assess retrotranslocation of MAD substrates from the mitochondrial matrix to the surface of the organelle using the budding yeast, Saccharomyces cerevisiae, as a model system (Liao et al., 2024). This assay was first developed to study release of unfolded nuclear-encoded mitochondrial proteins from the mitochondrial intermembrane space (Bragoszewski et al., 2015). We also describe methods to inhibit MAD without affecting related quality control pathways in other organelles, to characterize MAD substrates and to study their interactions with the Cdc48 subunit of the segregase complex.

BEFORE YOU BEGIN

Exposure of yeast to mitochondrial stressors, such as paraquat-induced mitochondrial ROS, results in accumulation of damaged MAD substrates in mitochondria, as well as increases in their ubiquitination and interaction with Cdc48 (Liao et al., 2020). Moreover, inhibition of MAD by deletion of DOA1, which targets the segregase complex to mitochondria in MAD (Fig. 1), accentuates these stress-induced phenotypes. Therefore, the methods described here for analysis of MAD substrates are carried out using wild-type yeast strains as well as strains that bear a deletion in DOA1, to test for MAD-specific effects. Since Doa1 is also required for the maintenance of free ubiquitin levels in yeast (Johnson, Ma, Ota, & Varshavsky, 1995), mono-ubiquitin is maintained at endogenous levels in doa1Δ cells by ectopic expression of plasmid borne or integrated ubiquitin in lieu of endogenous ubiquitin genes (e.g. strains PLY054 and PLY119, see Yeast Strains Table). In this way, the role of the MAD pathway can be investigated, largely uncoupled from the free ubiquitin concentration.

Elevated mitochondrial stress also results in increased retrotranslocation of MAD substrates from the mitochondrial matrix to the surface of the organelle. Here, we describe a method to assess retrotranslocation of MAD substrates using mitochondria that are isolated from wild-type or doa1Δ yeast. In this assay, MAD substrates are tagged with an epitope or fluorescent protein, expressed at wild-type levels and detected using western blot analysis. The yeast strains used for these studies are described in the Yeast Strains Table.

MATERIALS AND EQUIPMENT

Materials for preparation of crude mitochondria

• SC (synthetic complete) media for yeast growth: 6.7 g/L DIFCO^™ yeast nitrogen base w/o amino acids (BD, Franklin Lakes, NJ), 20 g/L glucose, 10 mL/L 100x stock solutions of amino acids/bases, pH 5.5 (adjust pH with NaHCO₃ or HCl). The 100x stock solutions of amino acids/bases are listed below.

  Amino acids/bases (MilliporeSigma catalog numbers)	Stock concentration*
  Adenine (A9126-25G)	2 mg/mL in 0.05 M HCl
  Uracil (U1128-25G)	2 mg/mL in 0.5% NaHCO3
  L-arginine (A8094-25G)	1 mg/mL
  L-histidine (H5659-25G)	1 mg/mL
  L-leucine (L8912-25G)	10 mg/mL
  L-lysine (L8662-25G)	3 mg/mL
  L-methionine (M9625-25G)	2 mg/mL
  L-phenylalanine (P5482-25G)	5 mg/mL
  L-tryptophan (T8941-25G)	2 mg/mL
  L-tyrosine (added as solid) (T8566-25G)	Added as a solid to 30 mg/L

  ^*

  All solutions are prepared in H₂O, with the exception of adenine and uracil solutions, which are solubilized as described. All solutions are autoclaved and stored at 4°C (except uracil which should be stored at room temperature, RT). Uracil can precipitate at 4°C but remains in solution at RT.

  To prepare media for yeast cell growth and isolation of mitochondria, add 500 mL of SC media to a 2 L Erlenmeyer flask, insert gauze or cotton plug into the mouth of the Erlenmeyer flask, cover the gauze plug with aluminium foil, autoclave and store at RT.

  Note: Typically, yeast are grown in Lactate medium (3 g/L Yeast extract, 0.5 g/L Glucose, 0.5 g/L CaCl₂•2H₂O, 0.5 g/L NaCl, 0.6 g/L MgCl₂•6H₂O, 1 g/L KH₂PO₄, 1g/L NH₄Cl, 22 mL/L of 90% Lactic acid, 7.5 g/L NaOH, adjust pH to 5.5 with NaOH pellets) for mitochondrial isolation. Here, we use glucose-based media (SC) for propagation of cells for isolation of mitochondria because MAD functions are analyzed using yeast cells that are propagated in glucose-based media (Liao et al., 2020).

• Zymolyase 20T (AMSBIO, Cambridge, MA)

• Complete^™, EDTA-free Protease Inhibitor Cocktail (Roche, MilliporeSigma, St. Louis, MO). 1 tablet per 50 mL solution.

Stock solutions

• 1 M Tris–SO₄, pH 9.4. Autoclave and store at room temperature (RT).

• 1 M DTT. Dissolve in water and store in aliquots at −20°C.

• 1 M MgCl₂. Autoclave and store at RT.

• 2.4 M Sorbitol. Autoclave and store at RT.

• 200 mM Phenylmethylsulfonylfluoride (PMSF) (MilliporeSigma, St. Louis, MO). Dissolve in 100% ethanol, aliquot and store at −20°C.

• 1 M HEPES–KOH, pH 7.4. Adjust pH with KOH, autoclave and store at RT.

• 1 M KPi, pH 7.4. Autoclave and store at RT.

Working solutions

• Tris–DTT buffer: 0.1M Tris–SO₄, pH 9.4, 10 mM DTT

• SP buffer: 1.2 M Sorbitol, 20 mM KPi, pH 7.4

• SEH buffer: 0.6 M Sorbitol, 20 mM HEPES–KOH, pH 7.4, 2 mM MgCl₂,

  Add Protease Inhibitor Cocktail (1 tablet of Protease inhibitor cocktail/50 mL), PMSF (1 mM) and deubiquitinase inhibitor NEM (10 mM) immediately before use.

Materials for measurement of steady-state and ubiquitinated protein levels in crude mitochondria

• HisPur^™ Ni-NTA Magnetic Beads (ThermoFisher SCIENTIFIC, Waltham, MA)

• Cell Disruption Media 0.5mm Glass Beads (Scientific Industries, Bohemia, New York)

• Cell Lysis Buffer 1 (CLB1): 50 mM Tris pH 8.0, 300 mM NaCl, 0.1% Tween 20. Add Protease Inhibitor Cocktail (1 tablet of Protease inhibitor cocktail/50 mL), PMSF (1 mM) and deubiquitinase inhibitor NEM (10 mM) immediately before use.

• Wash Buffer 1 (WB1): 50 mM Tris pH 8.0, 300 mM NaCl, 10 mM Imidazole. Add Protease Inhibitor Cocktail (1 tablet of Protease inhibitor cocktail/50 mL) and PMSF (1 mM) immediately before use.

• Elution Buffer: 50 mM Tris pH 8.0, 300 mM NaCl, 500 mM Imidazole, 0.1% Tween 20.

  Add Protease Inhibitor Cocktail (1 tablet of Protease inhibitor cocktail/50 mL) and PMSF (1 mM) immediately before use.

Materials for retrotranslocation of MAD from mitochondria

• Release buffer: 0.6 M Sorbitol, 20 mM HEPES-KOH, pH 7.4, 2 mM MgCl₂, 5 mM Methionine, 10 mM KPi pH 7.4. Add Protease Inhibitor Cocktail (1 tablet of Protease inhibitor cocktail/50 mL) and PMSF (1 mM) immediately before use.

• 100% (w/v) TCA: The solubility of TCA is 1 g/10 mL in H₂O. Store at 4°C

• Acetone: Store at −20°C

• 4x SDS Sample buffer: 0.25M Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 4 mg/mL bromophenol blue. Aliquot and store at −20°C. Add 2-mercaptoethanol to 20% (v/v) immediately before use.

Materials for co-immunoprecipitation of MAD substrates and Cdc48

• Protein A-coupled Magnetic Beads (ThermoFisher SCIENTIFIC, Waltham, MA)

• Cell Disruption Media 0.5mm Glass Beads (Scientific Industries, Bohemia, New York)

• Cell Wash Buffer (CWB): 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA.

  Add Protease Inhibitor Cocktail (1 tablet of Protease inhibitor cocktail/50 mL) and PMSF (1 mM) immediately before use.

• Cell Lysis Buffer 2 (CLB2): 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol. Add Protease Inhibitor Cocktail (1 tablet of Protease inhibitor cocktail/50 mL), PMSF (1 mM) and deubiquitinase inhibitor NEM (10 mM) immediately before use.

• Wash Buffer 2 (WB2): 50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 10% glycerol. Add Protease Inhibitor Cocktail (1 tablet of Protease inhibitor cocktail/50 mL) and PMSF (1 mM) immediately before use.

• 4x SDS Sample buffer: 0.25M Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 4 mg/mL Bromophenol blue. Aliquot and store at −20°C. Add 2-mercaptoethanol to 20% (v/v) immediately before use.

Equipment

• Preparative Refrigerated Centrifuge (Sorvall RC5C, ThermoFisher SCIENTIFIC, Waltham, MA)

• Fixed angle rotors for preparative refrigerated centrifuge (e.g. GS-3 and SS34 rotors for the Sorvall RC5C centrifuge, ThermoFisher SCIENTIFIC, Waltham, MA)

• 500-mL Polycarbonate Oak Ridge Centrifuge Bottles for fixed angle (Sorvall GS-3) rotor (ThermoFisher SCIENTIFIC, Waltham, MA)

• 50-mL Polycarbonate Oak Ridge Centrifuge Tubes for fixed angle (Sorvall SS34) rotor (ThermoFisher SCIENTIFIC, Waltham, MA)

• FRESCO 21 Microcentrifuge tubes (1.5 mL, ThermoFisher SCIENTIFIC, Waltham, MA)

• Mini-Centrifuge for microcentrifuge tubes (ThermoFisher SCIENTIFIC, Waltham, MA)

• Oribital Shaking Incubator, New Brunswick Innova 4330 Refrigerated Incubator Shaker (Eppendorf, Enfield, CT)

• 40-mL Dounce Tissue Grinders (DURAN WHEATON KIMBLE, Millville, NJ)

• Vortex-Genie 2 with TurboMix Attachment (Scientific Industries, Bohemia, NY)

• Magnetic Separation Rack DynaMag-2 (ThermoFisher SCIENTIFIC, Waltham, MA)

• Multipurpose tube rotator (Fisher Scientific, Hampton, NH)

• Protein and DNA gel imaging system, ChemiDoc MP Imaging System (Bio-Rad, Hercules CA)

STEP-BY-STEP METHOD DETAILS

For many experiments, mitochondria are isolated from yeast cells under control and test conditions. For example, mitochondria are isolated from doa1Δ cells after incubation in the presence or absence of a mitochondrial stressor (e.g. paraquat) to assess the effect of elevated proteostatic stress on mitochondria under conditions where MAD is disabled. The protocol for preparation of mitochondria from strains or conditions of interest that are propagated in glucose-based media is described below.

Preparation of crude mitochondria

Timing: 3–4 hr for subcellular fractionation

• 1Prepare media for yeast preculture: Add 25 mL of SC media to a 125 mL Erlenmeyer flask, insert gauze or cotton plug into the mouth of the Erlenmeyer flask, cover gauze plug with aluminium foil, autoclave and store at RT.
• 2Prepare yeast preculture 1 day prior to subcellular fractionation: Inoculate 2–3 colonies of yeast expressing His-tagged ubiquitin (e.g. SUB592) into 25 mL of SC in a 125-mL Erlenmeyer flask. Incubate at 30°C with shaking at 200 rpm in an orbital shaking incubator (New Brunswick Innova 4330) overnight.
• 3Inoculate the yeast cell culture for preparation of mitochondria the night before subcellular fractionation and confirm that yeast are in mid-log phase after growth overnight. Measure the optical density of 1 mL of preculture prepared in step 2 at 600 nm. Inoculate a 2 L Erlenmeyer flask containing 500 mL of SC media and grow at 30°C with shaking at 200 rpm, overnight to mid-log phase. For WT strains, inoculate with 1 OD₆₀₀ of cells. For doa1Δ cells, inoculate with 1.5 OD₆₀₀ of cells.
• 4To harvest yeast for subcellular fractionation, tare 500-mL polycarbonate centrifuge bottles. Transfer cell cultures from 2 L Erlenmeyer flasks to tared centrifuge bottles and concentrate cells by centrifugation at 1500 × g for 5 min at RT using a fixed angle rotor (e.g. Sorvall GS-3 rotor) in a preparative refrigerated centrifuge (e.g. Sorvall RC5C).
• 5Discard the supernatant. Resuspend the cell pellet in each polycarbonate centrifuge tube with 200 mL water and concentrate resuspended cells by centrifugation at 1500 × g at RT for 5 min.
• 6Discard the supernatant and determine the weight of the wet cell pellet in the tared centrifuge bottles (typically 6–8 g/L of SC medium).
• 7Resuspend cell pellet in a total of 20 mL Tris-DTT buffer. Transfer the suspension to a fresh 125 mL Erlenmeyer flask and incubate at 30°C with shaking at 200 rpm for 15 min in an orbital shaking incubator (e.g. New Brunswick Innova 4330).
• 8Transfer the cell suspension to 50-mL polycarbonate centrifuge tubes. Concentrate cells by centrifugation at 1500 × g for 5 min at RT using a fixed angle rotor in a preparative refrigerated centrifuge (e.g. Sorvall SS34 rotor in RC5C centrifuge).
• 9Discard the supernatant and resuspend the cell pellet with 20 mL of SP buffer. Concentrate cells by centrifugation at 1500 × g for 5 min at RT.
• 10Resuspend cells in SP buffer containing Zymolyase 20T (7.5 mg/g wet cell), transfer suspension to a fresh 125 mL Erlenmeyer flask, and incubate at 30°C with shaking at 200 rpm for 40 min in an orbital shaking incubator.

  Note: Zymolyase treatment removes yeast cell wall (generating spheroplasts).

Critical: All further steps should be carried out on ice using reagents that are pre-chilled to 4°C.

• 11Transfer spheroplast suspension to a fresh 50-mL polycarbonate centrifuge tubes. Concentrate spheroplasts by centrifugation at 4500 × g for 5 min at 4°C using a preparative refrigerated centrifuge.
• 12Discard the supernatant and resuspend the pellet with 20 mL ice-cold SEH buffer. Concentrate spheroplasts by centrifugation at 4500 × g for 5 min at 4°C.
• 13Discard the supernatant and resuspend the pellet with 20 mL ice-cold SEH buffer. Transfer the suspension to a pre-chilled 40-mL glass Dounce homogenizer and homogenize spheroplasts with 18 forceful strokes on ice.
• 14Transfer the homogenate to a fresh, pre-chilled 50-mL polycarbonate centrifuge tubes. Centrifuge the homogenate at 1500 × g for 5 min at 4°C using a preparative refrigerated centrifuge. The pellet obtained contains intact cells, cellular debris and nuclei.
• 15Transfer the supernatant to a fresh pre-chilled 50-mL polycarbonate centrifuge tube and centrifuge at 12,000 × g for 10 min at 4°C using a preparative refrigerated centrifuge. The pellet obtained contains crude mitochondria.
• 16Discard the supernatant. Resuspend the crude mitochondrial pellet with 1 mL ice-cold SEH buffer and transfer the mitochondrial suspension into 1.5 mL microcentrifuge tubes. Centrifuge at 700 × g for 5 min at 4°C using a mini-centrifuge. The pellet contains residual cell debris, intact cells and nuclei.
• 17Transfer the supernatant to fresh microcentrifuge tubes and centrifuge at 1500 × g for 5 min at 4°C using a microcentrifuge. The pellet contains residual cell debris, intact cells and nuclei.
• 18Transfer the supernatant to fresh microcentrifuge tubes and centrifuge at 12,000 × g for 5 min at 4°C using a mini-centrifuge. Discard the supernatant and resuspend the pellet in 200 μL of ice-cold SEH buffer. This fraction is referred as “crude mitochondria.”
• 19Determine protein recovery in the crude mitochondria fraction (e.g. BCA protein assay).
• 20Note: Typical yields are 200–400 μg of protein in the mitochondria fraction isolated from a mid-log phase yeast culture propagated in 500 mL of SC media.
• 21Store the mitochondria preparation in aliquots of >10 mg/ml protein by quick freezing in liquid nitrogen and storage at −80°C.

Pause Point: Isolated mitochondria can be stored in aliquots of >10 mg/ml protein by quick freezing in liquid nitrogen at −80°C for weeks. However, we use freshly prepared mitochondria for analysis of ubiquitin in MAD substrates in isolated mitochondria and retrotranslocation of MAD from mitochondria (see below).

Measurement of steady-state levels of MAD substrates in isolated mitochondria

Studies on specific MAD substrates are carried out using mitochondria isolated from yeast strains that express tagged mitochondrial proteins of interest. Tags are inserted into genes of interest at their chromosomal loci, and strains bearing tags are validated to confirm that 1) genes are tagged, 2) the tag does not affect the localization of the protein or mitochondrial function, and 3) tagged proteins are quantitative targeted to mitochondria. Finally, the steady-state level of tagged proteins in mitochondria is determined by western blot analysis.

To control for equal protein loading in western blot analysis, 2,2,2-trichloroethanol (TCE) is used for fluorescent visualization of proteins following electrophoresis (Ladner, Yang, Turner, & Edwards, 2004). TCE is incorporated into the acrylamide gels during preparation of the gels, and exposure of SDS gels to ultraviolet light (300 nm) after electrophoresis results in a covalent modification of the tryptophan indole ring on proteins with TCE. The fluorescence of TCE-modified proteins is detected and quantitated using a gel imaging system.

Timing: 10 min for sample preparation

1. For each condition under consideration, place 40 μg of crude mitochondria in a 1.5 mL microcentrifuge tube and concentrate mitochondria by centrifugation at 12000 × g for 5 min at 4°C using a mini-centrifuge.

2. Resuspend the mitochondrial pellet 45 μL SEH buffer with 15 μL 4x SDS sample buffer.

3. Place the samples in a heating block at 70°C for 5 min.

4. Perform Western blot analysis to detect steady-state levels of MAD substrates in these samples.

5. To visualize total loading proteins as loading control, prepare SDS-PAGE gels containing 0.5% TCE.

6. Before transfer of proteins from the SDS gel to the membrane for western blot analysis, expose the SDS gel to ultraviolet light at 300 nm for 2.5 min to activate the protein cross-linking by TCE and detect fluorescent protein bands using ChemiDoc MP Imaging System. Typical exposure time for detection of TCE-modified proteins is 3 sec.

7. For quantitation of the steady-state levels of tagged mitochondrial proteins of interest, normalize the intensity of the fluorescence of tagged proteins detected in the western blot to the intensity of TCE-fluorescence of bands in the corresponding lane in the SDS gel detected in Step 6.

Analysis of ubiquitin in MAD substrates in isolated mitochondria

These studies are carried out using mitochondria isolated from yeast strains that express tags on all endogenous or ectopically expressed ubiquitin genes. To inhibit MAD, cells bearing a deletion of DOA1, the substrate-specific co-factor that targets the segregase complex to mitochondria, are used. To promote unfolding of mitochondrial proteins, cells are exposed to defined mitochondrial stressors (e.g. paraquat). Here, tags are inserted into genes of interest at their chromosomal loci and strains bearing tags are validated to confirm that the tag does not affect ubiquitin function and ubiquitin is expressed at levels found in wild-type cells.

Timing: 1–2 hr

• 1Add 250 μL 0.5-mm glass beads to each microcentrifuge tube. Resuspend the glass beads with 500 μL of ice-cold Cell Lysis Buffer 1 (CLB1). Quickly concentrate glass beads by centrifugation in a mini-centrifuge at maximum speed for 5 sec. Discard the supernatant.
• 2Transfer 400 μg of crude mitochondria from samples of interest to a fresh microcentrifuge tube. Concentration the mitochondria by centrifugation at 12,000 × g for 5 min at 4°C using a mini-centrifuge.
• 3Resuspend the mitochondrial pellet with 500 μL of ice-cold CLB1 and transfer the suspension to a microcentrifuge tube containing 0.5-mm glass beads (Step 1).
• 4Vortex the sample at max speed for 5 min at 4°C using Vortex-Genie 2 with TurboMix Attachment.
• 5Centrifuge the lysate at 16000 × g for 5 min at 4°C using a mini-centrifuge. Transfer the supernatant to a fresh microcentrifuge tube.
• 6Resuspend the pellet and glass beads from Step 5 with ice-cold 500 μL of CLB1 and vortex the sample at max speed for 5 min at 4°C.
• 7Concentrate glass beads by centrifugation of the lysate at 16000 × g for 5 min at 4°C.
• 8Transfer the supernatant to the microcentrifuge tube containing the supernatant (Step 5).
• 9Save 45 μL of supernatant in a fresh microcentrifuge tube as “Input.” For western blot analysis of the input sample, solubilize by adding 15 μL of 4x SDS Sample Buffer and heating at 70°C for 5 min using a heating block.
• 10
  Prepare HisPur Ni²⁺ NTA beads.

  1. For each sample, transfer 50 μL of the beads with buffer to a microcentrifuge tube.
  2. Place the mixture in Magnetic Separation Rack for 1 min to separate the beads from the buffer. Remove and discard the buffer.
  3. Resuspend the beads with 500 μL of CLB1.
• 11Place the mixture in Magnetic Separation Rack for 1 min to separate the beads from the buffer. Remove and discard the buffer.
• 12Add supernatant from Step 7 to the prepared HisPur Ni²⁺ NTA beads and mix gently.
• 13Incubate for 30 min at 4°C with rotation using a multipurpose tube rotator.

  Note: His-tagged ubiquitinated proteins bind to the beads in this step.

• 14Place the sample in Magnetic Separation Rack for 1 min to separate the protein-bound beads from the reaction mixture.
• 15Transfer 45 μL of the buffer to a fresh microcentrifuge tube and save this sample as “Flowthrough” for the affinity purification. For western blot analysis, solubilize by adding 15 μL of 4x SDS Sample Buffer and heating at 70°C for 5 min.
• 16Discard the remaining buffer in samples in the Magnetic Separation Rack and gently resuspend the beads by inverting the tube to mix with 500 μL of Wash Buffer 1 (WB1).
• 17Subject the mixture to centrifugation at 2000 × g for 5 sec to transfer beads that adhere to the walls or top of the microcentrifuge tube to the bottom of the tube. Place the mixture in the Magnetic Separation Rack for 1 min. Remove and discard the buffer.
• 18Wash beads twice with 500 μL of ice-cold WB1 and collect separate beads from the wash as described in Step 16–17.

Critical: Resuspend beads carefully and use a quick centrifugation (Step 17) to collect beads that adhere to the tube walls and top.

• 19To elute the sample, resuspend the ubiquitinated protein-bound beads with 45 μL of Elution Buffer (EB) and incubate for 5 min at RT with rotating using a tube rotator.
• 20Place samples in the Magnetic Separation Rack and let stand for 1 min at RT. Transfer the supernatant, which contains eluted, ubiquitinated proteins, to a fresh microfuge tube. This material is the “Eluate” for the affinity purification and will be used for protein determinations and western blot analysis.
• 21Add 15 μL of 4x SDS sample buffer to the eluate and solubilize by heating to 70°C for 5 min. Store the solubilized eluate at −80°C.

Retrotranslocation of MAD substrates in isolated mitochondria

This protocol measures the time course for retrotranslocation of substrates from the mitochondrial matrix to the surface of the organelle. Here, mitochondria are isolated from wild-type or doa1Δ cells that express a tagged MAD substrate (e.g. Kgd1, a mitochondrial matrix protein and MAD substrate). Isolated mitochondria are incubated in “release buffer” for 10, 30 and 60 min at 30°C, and the reaction is stopped by transfer of samples to ice. Thereafter, mitochondria are separated from the reaction mixture by low-speed centrifugation, and proteins that are recovered in the mitochondrial pellet or released from the organelle following retrotranslocation are analyzed by western blot. To confirm that the release is not due to rupture of the mitochondrial outer membrane, cytochrome b2, a protein that is loosely associated with the outer leaflet of the inner mitochondrial membrane, is also monitored in western blots. Here, proteins that are released from mitochondria under conditions where cytochrome b2 is quantitatively retained within the organelle are interpreted as proteins that undergo retrotranslocation.

Timing: 2–3 hr

• 1Transfer 400 μg of crude mitochondria from yeast strain of interest to a microcentrifuge tube and concentrate mitochondria by centrifugation at 12000 × g for 5 min at 4°C using a mini-centrifuge.
• 2Discard the supernatant and resuspend the mitochondrial pellet in 600 μL of release buffer. Transfer 200 μL aliquots of the resuspended mitochondria to 3 fresh microcentrifuge tubes.
• 3Incubate these 3 tubes at 30°C. After 10, 30 or 60 min of incubation, transfer samples from 30°C to storage in ice.
• 4Concentrate mitochondria by centrifugation at 12000 × g for 5 min at 4°C using a mini-centrifuge.
• 5Carefully remove and transfer the supernatants to fresh microcentrifuge tubes using a micropipette.

Critical: Use care to avoid disrupting the mitochondrial pellet when removing the supernatant.

• 6Solubilize the mitochondrial pellets by addition of 50 μL of 2x SDS sample buffer and heating at 70°C for 5 min.
• 7Add 20 μL of 100% TCA to the microfuge tubes containing the supernatants in Step 5. Let samples stand at 4°C for 1 hr.
• 8Collect TCA-precipitated proteins by centrifugation at 12,000 × g for 10 min at 4°C. Discard the supernatants.
• 9Add 500 μL of acetone prechilled at −20°C to the microcentrifuge tubes containing TCA-precipitated proteins and let them stand at −20°C for 30 min.
• 10Concentrate precipitated proteins by centrifugation at 12,000 × g for 10 min at 4°C. Discard the supernatants.
• 11Dry the protein pellets at RT for 5 min.
• 12Add 50 μL of 2x SDS sample buffer to the protein pellets and solubilize by heating to 70°C for 5 min.

Co-immunoprecipitation of MAD substrates and Cdc48 from whole-cell extracts

Association of the MAD substrate of interest with Cdc48 is then assessed by co-immunoprecipitation of the two proteins from whole-cell extracts. Alternatively, the MAD substrate can be tagged with 6xHis and the association of the substrate of interest with Cdc48 is assessed by affinity purification using the HisPur Ni²⁺ NTA beads described above.

Timing:

Preparation of yeast cell cultures: 2 days

Co-immunoprecipitation from yeast cells: 2 days

• 1Prepare yeast preculture 2 days prior to co-immunoprecipitation: Inoculate 2–3 colonies of yeast expressing Kgd1-13xMyc into 5 mL of SC in a 50 mL Falcon tube. Loosely cap the Falcon tube and incubate at 30°C with shaking at 200 rpm in an orbital shaking incubator (New Brunswick Innova 4330) overnight.
• 2Measure the OD₆₀₀ of 1 mL of the preculture.
• 3On the day before the co-immunoprecipitation, transfer 0.02 OD₆₀₀ of cells to 20 mL of SC in a sterile 125 mL Erlenmeyer flask and incubate at 30°C with shaking at 200 rpm in an orbital shaking incubator (New Brunswick Innova 4330) overnight.
• 4Measure the OD₆₀₀ of the overnight culture. For mid-log phase cells, the OD₆₀₀ should be 0.3/mL. Typical yields: 5 OD₆₀₀/20mL culture.

  Note: Typically, the doubling time of yeast growing in SC is 2 hr.

• 5Prepare glass beads for solubilization of yeast cells: For each sample analyzed, add 250 μL 0.5-mm glass beads to a microcentrifuge tube. Resuspend the glass beads with 500 μL of ice-cold Cell Lysis Buffer 2 (CLB2). Concentrate the glass beads by centrifugation for 5 sec using a mini-centrifuge. Remove and discard the supernatant.
• 6Transfer mid-log phase yeast cultures prepared in Step 3 to 50-mL polycarbonate centrifuge tubes and concentrate cells by centrifugation at 1500 × g for 5 min at 4°C using a preparative refrigerated centrifuge (e.g. Sorvall RC5C Centrifuge with a SS34 rotor).
• 7Resuspend the cells with 1 mL ice-cold Cell Wash Buffer (CWB), transfer the suspension to a fresh microcentrifuge tube and measure the OD₆₀₀ of cells.
• 8Transfer 5 OD₆₀₀ of the cells to a fresh microcentrifuge tube and concentrate cells by centrifugation at 3500 × g for 5 min at 4°C using a mini-centrifuge.
• 9Discard the supernatant and resuspend the cell pellet with 500 μL of ice-cold Cell Lysis Buffer 2 (CLB2).
• 10Transfer the resuspended pellet to a microcentrifuge tube containing 250 μL 0.5-mm washed glass beads.
• 11Vortex the sample at max speed for 5 min at 4°C using Vortex-Genie 2 with TurboMix Attachment. Centrifuge the cell lysates at 16000 × g for 5 min using a mini-centrifuge.
• 12Transfer the supernatant to a fresh microcentrifuge tube.
• 13Resuspend the pellet and glass beads from Step 12 with ice-cold 500 μL of CLB2 and vortex the sample at max speed for 5 min at 4°C using Vortex-Genie 2 with TurboMix Attachment. Centrifuge the lysate at 16000 × g for 5 min using a mini-centrifuge.
• 14Pool the supernatants from Steps 12 and 13. Save 45 μL of the pooled supernatants as “Input.” For western blot analysis, add 15 μL of 4x SDS Sample Buffer and solubilize samples by heating to 70°C for 5 min in a heat block.
• 15Add 10 μg antibody (eg. anti-Myc) to the pooled supernatants and incubate overnight (16 hr) at 4°C with mixing using a tube rotator.
• 16
  Next day, prepare Protein A-coupled magnetic beads.

  1. For each sample analyzed, transfer 50 μL of the protein A bead suspension to a microcentrifuge tube.
  2. Place the mixture in Magnetic Separation Rack and let stand for 1 min. Remove and discard the buffer.
  3. Resuspend the protein A-beads with 500 μL of CWB.
  4. Place the mixture in Magnetic Separation Rack and let stand for 1 min. Remove and discard the buffer.
  5. Add 500 μL of CWB to protein A-beads.
• 17Place the mixture in Magnetic Separation Rack for 1 min to separate the beads from the CWB. Discard CWB and resuspend the beads with supernatant/antibody mixture prepared in Step 16.
• 18Incubate for 1 hr at 4°C with rotating using a tube rotator.

  Note: Antibody-bound proteins bind to the beads in this step.

• 19Place the sample in Magnetic Separation Rack for 1 min to separate the proteins bound to protein A-beads. Transfer 45 μL of the buffer to a fresh microfuge tube and save this sample as “Flowthrough” for the affinity purification. For western blot analysis, add 15 μL of 4x SDS Sample Buffer and place to a heat block at 70°C for 5 min.
• 20Discard the remaining buffer in samples from Step 20 in the Magnetic Separation Rack. Add 500 μL of Wash Buffer 2 (WB2) to the bead and gently mix by inverting the tube.

  Displace beads that adhere to the tube walls and top by centrifugation art 2000 × g for 5 sec at 2000 × g.

• 21Place samples in the Magnetic Separation Rack for 1 min. Remove and discard the buffer.
• 22Repeat the steps 20–21 twice to wash the beads.

Critical: Resuspend beads slowly and carefully by inverting the tube and collect any beads that adhere to the tube walls or top by centrifugation.

• 23To elute the sample, resuspend the protein A-beads with 60 μL 1x SDS Sample Buffer and heat at 70°C for 5 min using a heating block.
• 24Place the sample in Magnetic Separation Rack for 1 min. Save the supernatant as “Eluate” for western blot analysis.

EXPECTED OUTCOMES

Analysis of steady-state protein levels, ubiquitination and association with Cdc48.

In MAD, proteins that are targeted for degradation are ubiquitinated, and these proteins are transferred from the mitochondrial surface to the proteasome for degradation by the conserved AAA-ATPase Cdc48 (VCP/p97 in mammals and Ter94 in Drosophila) and its cofactor Doa1 (PLAA or PLAP in mammals and Drosophila, Phospholipase A-2-activating protein). Thus, inhibition of MAD by deletion of DOA1 results in an increase in total ubiquitinated proteins in isolated mitochondria (Fig. 2). Moreover, if a protein is a MAD target, the steady-state and ubiquitinated levels of the protein are expected to increase in isolated mitochondria (Fig. 2A–B), and Cdc48 should co-immunoprecipitate with the MAD target in whole-cell lysates (Fig. 2C–D). Finally, since Doa1 is the main cofactor in recognizing MAD targets, the deletion of DOA1 is expected to decrease the association between Cdc48 and the target (Fig. 2C–D). Our previous study revealed that MAD is the major pathway of mitochondrial quality control under paraquat-induced chronic, low-level oxidative stress (Liao et al., 2020). Thus, the association between Cdc48 and the MAD target is expected to increase in cells treated with paraquat (Fig. 2C–D).

Figure 2.

Analysis of the MAD pathway. (A-B) Steady-state and ubiquitinated levels of the MAD substrate Kgd1. Western blot (A) and quantification (B) of crude mitochondria (input) and total ubiquitinated proteins affinity purified with Ni²⁺-NTA magnetic beads from isolated mitochondria (IP). Blots were probed with antibodies against GFP to detect GFP-tagged Kgd1 and against 6xHis for ubiquitin detection. Total protein load was assessed using trichloroethanol (TCE). N = 5, one-way ANOVA with Sidak’s multiple comparison test. (C-D) Co-immunoprecipitation of the MAD substrate Kgd1 and Cdc48. Western blot (C) and quantification (D) of whole-cell lysates (input) and proteins immunoprecipitated with an anti-Myc antibody (IP) from whole-cell lysates of cells expressing Kgd1-13xMyc. Blots were probed with antibodies against Myc to detect Myc-tagged Kgd1 and against Cdc48. Total protein load was assessed using trichloroethanol (TCE). N = 7, one-way ANOVA with Sidak’s multiple comparison test. Mean + SEMs; *p < 0.05; **p < 0.01; ****p < 0.0001. (Adapted from (Liao et al., 2020))

Retrotranslocation analysis.

If matrix proteins like Kgd1 are MAD substrates, they are expected to be retrotranslocated from the mitochondrial matrix across the mitochondrial inner and outer membrane (MIM and MOM) to the cytosol for degradation by the proteasome. Thus, the matrix MAD substrate Kgd1 is expected to be released from mitochondria isolated from WT and doa1Δ in a time-dependent manner: Kgd1 should be recovered in the release fraction after 10-min incubation, and an increase in Kgd1 release is expected upon 30–60 min incubation. Moreover, to determine if the observed retrotranslocation and release is specific for MAD and not due to defects in the integrity of mitochondria, proteins that are not MAD substrates (Tom40, cytochrome b2 and Cit1, which localize to the outer membrane, intermembrane space and matrix, respectively) should not be released from isolated mitochondria (Fig. 3).

Figure 3.

Retrotranslocation of MAD substrates from the mitochondrial matrix. (A, B) Representative western blots of the mitochondrial pellet (Pellet) and released proteins (Released) from isolated mitochondria from WT (+) and doa1Δ (−) cells after incubation in release buffer for 10, 30, and 60 min. The blot was probed with antibodies against GFP for detection of Kgd1, and antibodies against Tom40, Cyb2 (A) and Cit1 (B). Total protein load was assessed using TCE. (C) Quantification of Kgd1p released from isolated mitochondria in (A). Kgd1p signals were normalized to TCE and then normalized to Kgd1p at 10 min in WT (+) or doa1Δ (−) in the released fraction (n=3, 1-way ANOVA with Sidak’s multiple comparison test, *p<0.05; **p<0.01; ***p<0.001) Adapted from (Liao et al., 2024)

ADVANTAGES

• The conditions used (deletion of DOA1 and expression of ubiquitin at endogenous levels) inhibit MAD without affecting MAD-related pathways or cellular levels of mono-ubiquitin.

• Analysis of steady-state protein levels and ubiquitination in isolated mitochondria from doa1Δ cells ensures that changes observed are MAD-specific and not due to defects in import of proteins into the organelle.

• Analysis of interactions with proteins of interest with Cdc48 provides additional evidence that proteins are MAD substrates.

• This is a novel method to detect the retrotranslocation of matrix MAD substrates from isolated mitochondria.

LIMITATIONS

• Additional methods, such as analyzing mRNA levels, are essential for determining whether any observed increase in protein levels results from reduced degradation or increased biogenesis.

OPTIMIZATION AND TROUBLESHOOTING

Problem 1

Low signal intensity of input in the measurement of steady-state and ubiquitinated levels.

Potential Solution 1

Increase the amount of mitochondria analyzed.

Problem 2

Low signal intensity of 6xHis-tagged ubiquitin protein signals in the eluate.

Potential Solution to 2

Check the Flowthrough. If most 6xHis-tagged ubiquitin signals are recovered in the Flowthrough fraction, the efficiency of binding of 6xHis-tagged ubiquitinated proteins and Ni²⁺-NTA beads is low. Increase the amount of Ni²⁺-NTA beads used or the incubation time for Ni²⁺-NTA beads with mitochondrial lysates.

Problem 3

Mitochondrial proteins that are not MAD substrates in the matrix are detected in the release fraction.

Potential Solution 3

Parts of the mitochondrial pellet are also removed with the supernatant. Use care to avoid disrupting the mitochondrial pellet when removing the supernatant.

Problem 4

Low recovery of Cdc48 in co-immunoprecipitation experiments.

Potential Solution 4

Increase amounts of cells or primary antibody used. Since the Cdc48-bound MAD targets are eventually degraded by the proteasome, the signals of Cdc48 co-immunoprecipitated with MAD targets are typically low.

Problem 5

High background or non-specific bands of Cdc48 signals in elution in co-immunoprecipitation of MAD substrates and Cdc48

Potential Solution 5

Increase the times of wash steps (Steps 21–23). Perform the negative controls using cells that do not express tagged proteins. Compare the patterns of non-specific bands in negative controls and samples to exclude the non-specific bands.

CONCLUSION/SUMMARY

The MAD pathway is critical for mitochondrial quality control and normal yeast lifespan. While MAD has been characterized primarily in budding yeast, critical MAD components (e.g. Cdc48 and Doa1) are conserved and targets for mutation in human disease. Thus, MAD may also be critical for mitochondrial and cellular fitness in other eukaryotes. Here, we describe methods to modulate MAD specifically without affecting related organelle quality control pathways and to characterize MAD targets by studying the levels and ubiquitination of MAD targets in isolated mitochondria, and interactions with Cdc48 using the budding yeast, Saccharomyces cerevisiae. We also describe methods to investigate the retrotranslocation of MAD substrates from the mitochondrial matrix to the surface of the organelle.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-GFP (Roche)	MilliporeSigma, St. Louis, MO	Cat. #11814460001; PRID: AB_390913
Mouse monoclonal anti-6xHis (HIS.H8) (Invitrogen)	ThermoFisher SCIENTIFIC, Waltham, MA	Cat. #MA1-21315; PRID: AB_557403
c-Myc Monoclonal Antibody (9E10) (Invitrogen)	ThermoFisher SCIENTIFIC, Waltham, MA	Cat. #13-2500 RRID:AB_2533008
Rabbit polyclonal anti-Cdc48 (S. cerevisiae)	As One International Inc, Santa Clara, CA	Cat. #62-303
Chemicals, Peptides, and Recombinant Proteins
Zymolyase 20T	AMSBIO, Cambridge, MA	Cat. #120491-1
Phenylmethylsulfonylfluoride (PMSF)	MilliporeSigma, St. Louis, MO	Cat.#52332
2,2,2-Trichloroethanol (TCE)	MilliporeSigma, St. Louis, MO	Cat.#T54801
N-ethylmaleimide (NEM)	MilliporeSigma, St. Louis, MO	Cat.#E3876
cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche)	MilliporeSigma, St. Louis, MO	Cat. #11873580001
Trichloroacetic acid (TCA)	MilliporeSigma, St. Louis, MO	Cat. #T6399
Critical Commercial Assays
HisPur™ Ni-NTA Magnetic Beads	ThermoFisher SCIENTIFIC, Waltham, MA	Cat. #88831
Pierce™ Protein A Magnetic Beads	ThermoFisher SCIENTIFIC, Waltham, MA	Cat. #88845
Pierce™ BCA Protein Assay Kits	ThermoFisher SCIENTIFIC, Waltham, MA	Cat. #23225
Cell Disruption Media 0.5mm Glass Beads	Scientific Industries, Bohemia, New York	Cat. #SI-BG05
Experimental Models: Yeast Strains
BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0)	Open Biosystems
PLY054 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 doa1Δ::LEU2 ho::TEF-UBI4-kanMX4)	(Liao et al., 2020)
SUB592 (MATa lys2-810 leu2-3,−112 ura3-52 his3Δ200 trp1-1[am] ubi1-Δ1::TRP1 ubi2-Δ2::ura3 ubi3-Δub-2 ubi4-Δ2::LEU2 + pUB221 [YEp pCUP16xHis-Myc-Ub URA3], pUB100 [YEp pGPDUBI1-tail HIS3])	(Spence et al., 2000)
PLY119 ((MATa lys2-810 leu2-3,−112 ura3-52 his3Δ200 trp1-1[am] ubi1-Δ1::TRP1 ubi2-Δ2::ura3 ubi3-Δub-2 ubi4-Δ2::LEU2 + pUB221 [YEp pCUP16xHis-Myc-Ub URA3], pUB100 [YEp pGPDUBI1-tail HIS3] doa1Δ::kanMX6)	(Liao et al., 2020)
PLY132 ((MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 TOM70-6xHis-HIS3 KGD1-13xMyc-URA3)	(Liao et al., 2020)
PLY134 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 doa1Δ::LEU2 ho::TEF-UBI4-kanMX4 TOM70-6xHis-HIS3 KGD1-13xMyc-URA3)	(Liao et al., 2020)
PLY144 ((MATa lys2-810 leu2-3,−112 ura3-52 his3Δ200 trp1-1[am] ubi1-Δ1::TRP1 ubi2-Δ2::ura3 ubi3-Δub-2 ubi4-Δ2::LEU2 + pUB221 [YEp pCUP16xHis-Myc-Ub URA3], pUB100 [YEp pGPDUBI1-tail HIS3] KGD1-GFP-bleMX6)	(Liao et al., 2020)
PLY145 (MATa lys2-810 leu2-3,−112 ura3-52 his3Δ200 trp1-1[am] ubi1-Δ1::TRP1 ubi2-Δ2::ura3 ubi3-Δub-2 ubi4-Δ2::LEU2 + pUB221 [YEp pCUP16xHis-Myc-Ub URA3], pUB100 [YEp pGPDUBI1-tail HIS3] doa1Δ::kanMX6 KGD1-GFP::bleMX6	(Liao et al., 2020)
Recombinant DNA (Plasmids)
pFA6-GFP-bleMX6	(Gadaleta, Iwasaki, Noguchi, Noma, & Noguchi, 2013)	Addgene plasmid # 33141
pFA6-13Myc-Ura3	(Liao et al., 2020)
Software and Algorithms
GraphPad Prism 9	GraphPad: https://www.graphpad.com/scientific-software/prism/
Image Lab	Bio-Rad, Hercules CA: https://www.bio-rad.com/en-tw/product/image-lab-software?ID=KRE6P5E8Z