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Aug 2020
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Protein Import Assay into Mitochondria Isolated from Human Cells
从人细胞中分离的线粒体蛋白质输入测定   

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Abstract

Mitochondria are essential organelles containing approximately 1,500 proteins. Only approximately 1% of these proteins are synthesized inside mitochondria, whereas the remaining 99% are synthesized as precursors on cytosolic ribosomes and imported into the organelle. Various tools and techniques to analyze the import process have been developed. Among them, in vitro reconstituted import systems are of importance to study these processes in detail. These experiments monitor the import reaction of mitochondrial precursors that were previously radiolabeled in a cell-free environment. However, the methods described have been mostly performed in mitochondria isolated from S. cerevisiae. Here, we describe the adaptation of this powerful assay to import proteins into crude mitochondria isolated from human tissue culture cells.


Graphic abstract:



Overview of the assay to monitor protein import into mitochondria isolated from human cells


Keywords: In organello import (细胞器中导入), Isolated mitochondria (分离线粒体), Radiolabeled proteins (放射性同位素标记蛋白), Cell-free protein synthesis (无细胞蛋白质合成), Human tissue culture cells (人体组织培养细胞)

Background

Mitochondrial proteins perform critical functions in energy conversation, heat production, lipid and iron metabolism, signaling, and cell death pathways (Vafai and Mootha, 2012; Spinelli and Haigis, 2018; Habich et al., 2019b; Pfanner et al., 2019). Human mitochondria contain approximately 1500 different proteins, but only 13 are translated in the matrix on mitochondrial ribosomes. Thus, the vast majority of proteins are synthesized as precursors on cytosolic ribosomes and then imported into mitochondria. Different import pathways have been identified that direct proteins to their correct submitochondrial localization (Hartl et al., 1989; Chacinska et al., 2009; Schmidt et al., 2010; MacPherson and Tokatlidis, 2017; Endo and Tamura, 2018; Hansen and Herrmann, 2019). Most of these pathways have been characterized using a handful of model substrates. However, it becomes increasingly clear that many proteins have different dependencies on import machinery components and that the intrinsic protein properties that drive import can vary. In vitro reconstituted systems using isolated mitochondria are powerful tools that allow rapid analysis of mitochondrial protein import. These assays allow rapid and parallel analysis of protein import and its determinants. In contrast to assays in intact cells, cytosolic processes – including proteasomal degradation, differing protein transcription/translation rates, competition with other precursors, and contributions of specific chaperone systems – can be excluded in import assays using isolated mitochondria.


Most described import experiments have been performed in mitochondria isolated from S. cerevisiae (Mokranjac and Neupert, 2007; Weckbecker and Herrmann, 2013; Tang and Tang, 2015), mainly because yeast genetics allowed detailed mechanistic analysis and insight into import pathways. We adapted the protocol for monitoring protein import into mitochondria enriched from human tissue culture cells. The combination with CRISPR-Cas9 mediated gene editing (knock-outs and knock-ins), siRNA-mediated protein depletion, and stable cell lines overexpressing components of the human import machinery allow a detailed investigation of human mitochondrial protein import. This enables the investigation of the differences between human and yeast mitochondrial import as well as the analysis of human patient mutations.


Materials and Reagents

  1. Cultivation of HEK293 cells

    1. Cell culture dishes 100 × 20 mm (Sarstedt, catalog number: 83.3902)

    2. Cell culture dishes 150 × 20 mm (Sarstedt, catalog number: 83.3903)

    3. Autoclaved disposable glass Pasteur pipets without cotton pad (VWR, catalog number: HECH40567001)

    4. Serological pipettes: Sterile individually packed,

      2 ml pipettes (Sarstedt, catalog number: 83.3903)

      5 ml pipettes (Sarstedt, catalog number: 86.1252.001)

      10 ml pipettes (Sarstedt, catalog number: 86.1254.001)

      25 ml pipettes (Sarstedt, catalog number: 86.1685.001)

    5. Flp-In T-REx HEK293 cells (Human embryonic kidney 293, Invitrogen, catalog number: R78007)

    6. Dulbecco’s modified Eagle medium (DMEM) medium-complete (see Recipes)

      10% Fetal calf serum (FCS, Sigma-Aldrich, catalog number: F0804) and

      1% penicillin/streptomycin (P/S, Sigma-Aldrich, catalog number: P0781-100ML)

      DMEM high glucose (Thermo Fisher, catalog number: 41965-062)

    7. Dulbecco's Phosphate Buffered Saline (PBS) (see Recipes)

      PBS powder (Sigma-Aldrich, catalog number: D5652)

    8. Trypsin-EDTA solution (see Recipes)

      10× Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T4174)


  2. Isolation of mitochondria from HEK293 cells

    1. Cell scraper 16 mm (Sarstedt, catalog number: 831832)

    2. 15 ml Falcon tubes (VWR, catalog number: 734-0451)

    3. 3 × 150 mm confluent dishes plated with HEK293 cells

      Note: Cells are seeded 3-4 days prior to the mitochondrial isolation. Cells are grown to 90% confluency. See Note 2.

    4. CompleteTM Protease Inhibitor Cocktail EDTA-free tablets (Roche, catalog number: 11873580001). Add freshly before use. Aliquots can be stored at -20°C

    5. PBS, store at 4°C before use

    6. Bradford Reagent ROTI® Quant (Roth, catalog number: K015.1)

    7. 1× buffer M (see Recipes)

      Mannitol (Merck, catalog number: 1.05983)

      Sucrose (Sigma-Aldrich, catalog number: S0389)

      HEPES-KOH, pH 7.4 (VWR, catalog number: 441487)

      EGTA-KOH, pH 7.4 (Roth, catalog number: 3054.1)


  3. Synthesis of [35S]-labeled precursor

    1. TNT® Quick Coupled Transcription/Translation System SP6 Promoter (Promega, catalog number: L2080). See Note 3.

    2. EasyTagTM EXPRESS 35S Protein Labeling Mix, [35S]-, 7 mCi, 1175 Ci/mmol (PerkinElmer, catalog number: NEG772007MC). See Note 4.

    3. DNA template (1 µg ml-1) containing the gene of the mitochondrial precursor inserted downstream of the SP6 promoter. Plasmid vector: pGEM-3/4 (Promega). Store at -20°C.

    4. Nuclease-free double-distilled water

    5. 200 mM L-methionine (Merck, catalog number: 1057070100) in nuclease-free double-distilled water. Store at 4°C

    6. 50 mM ethylene diamine tetra acidic acid (EDTA) disodium salt 2-hydrate, pH 8 (AppliChem, catalog number: A2937) in nuclease-free double-distilled water

    7. 1× sample buffer (see Recipes)

      Tris(hydroxymethyl)aminomethane (Tris, VWR, catalog number: 0497)

      Glycerol (Sigma-Aldrich, catalog number: G7757)

      Bromophenol blue (Roth, catalog number: A512.1)

      Dithiothreitol (DTT, AppliChem, catalog number: A1101)


  4. Import of [35S]-labeled precursors into isolated mitochondria

    1. Blotting membrane, nitrocellulose, AmershamTM Protran® (VWR, catalog number: 10600001), 0.2 µm pore size

    2. 20 mM Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich, catalog number: C2759)

    3. 200 mM phenylmethylsulfonyl fluoride (PMSF, AppliChem, catalog number: A0999.0100)

    4. Crushed ice

    5. Triton X-100 (AppliChem, catalog number: A1388)

    6. 1× buffer M (see Recipes)

    7. 20 µg/import reaction mitochondria (see: Recipes)

    8. 20 µg ml-1 peqGOLD proteinase K (see Recipes)

    9. 1× sample buffer (see Recipes)

    10. SEH buffer (see Recipes)

Equipment

  1. Cultivation of HEK293 cells

    1. Laminar flow hood class II (ENVAIR eco)

    2. CO2 incubator

    3. Vacuum pump

    4. Microscope

    5. Centrifuge (e.g., Eppendorf, model: 5702R)

    Equipped cell culture laboratory containing, e.g., a laminar flow hood class II (ENVAIR eco), a CO2 incubator for cell cultivation set at 37°C and 5% CO2, a vacuum pump for removal of medium using sterile glass Pasteur pipets, a microscope, and a cell counter and a cooling centrifuge.


  2. Isolation of mitochondria from HEK293 cells

    1. Electric stirrer (Heidolph RZR 2020)

    2. Potter S homogenizer (15 ml) with tight-fitting PTFE (polytetrafluorethylen) plungers (Sartorius, catalog number: 8542406)

      The range of clearance (i.e., the average distance between the pestle and the cylinder walls) optimal for mitochondria isolation is thereby from 0.045-0.065 mm.

    3. Centrifuge (Eppendorf, model: 5702R)

    4. Centrifuge (Eppendorf, model: 5417R)

    5. Vacuum pump (KNF laboport minipump) for removal of medium using sterile glass Pasteur pipets.


  3. [35S]-labeled precursor synthesis

    Hypoxic Glove Box and Cabinet (Coy Laboratory Products, “Coy 1 Person O2 Control Glove Box”, catalog number: 031615); not necessary if precursor protein is not prone to air oxidation (e.g., does not contain or contains only few cysteine residues).


  4. Import of [35S]-labeled precursors into isolated mitochondria

    1. ThermoMixer Eppendorf (F1.5, #EP5384000012)

    2. Centrifuge (Eppendorf, model: 5417R)

    3. System for SDS-PAGE electrophoresis and (semidry) blotting (Bio-Rad, MiniPROTEAN Tetra Cell, catalog number: 1658000)

    4. Imaging System Typhoon FLA 9500 (GE Healthcare, catalog number: 9351515)

    5. Image Eraser FLA (GE Healthcare, catalog number: 12999750)

Software

  1. ImageQuant TL 8.1 (GE Healthcare Life Science).

Procedure

An overview of the procedure is depicted in Figure 1.



Figure 1. Schematic representation of the steps in the ”in organello import assay” protocol

  1. Cultivation of HEK293 cells

    HEK293 cells are cultured in DMEM medium complete containing high glucose, FCS, and a penicillin/streptomycin antibiotic mixture. For passaging, HEK293 cells are cultured on 100 mm dishes in 10 ml DMEM medium complete until 90% confluency. For the described experiment, HEK293 cells are plated on 150 mm dishes in 30 ml DMEM medium complete and cultivated until they reach confluency. Special care must be taken for some cells in which mitochondrial proteins (e.g., of the mitochondrial import machinery) have been either depleted or overexpressed; different cultivating conditions might be required.

    1. Cultivate HEK293 cells on 100 mm dishes in 10 ml DMEM complete medium in an incubator at 37°C and 5% CO2 until they reach 90% confluency.

    2. Transfer the HEK293 dishes into a surface-sterilized laminar flow hood class II.

    3. Transfer the sterile DMEM medium complete, sterile PBS, and sterile Trypsin-EDTA into the laminar flow hood. All solutions should be preheated to 37°C.

    4. Remove the medium using an autoclaved disposable glass Pasteur pipet and a vacuum pump.

    5. Rinse the cells carefully by adding 10 ml of sterile PBS at the edge of the dish using a serological pipette and a pipette boy.

    6. Remove the PBS using an autoclaved disposable glass Pasteur pipet and a vacuum pump.

    7. Add 1 ml of sterile Trypsin-EDTA dropwise onto the cells using a serological pipette and a pipette boy.

    8. Incubate the cells for 5 min at 37°C and 5% CO2 until cells detach from the dish. Flick the dish with your finger.

    9. Transfer the dish back into the surface-sterilized laminar flow hood and add 9 ml of DMEM medium complete.

    10. Singularize the cells using a 10 ml serological pipette and a pipette boy. Press the pipette tip softly onto the dish bottom.

    11. Transfer three times 2 ml of the cell suspension into a 150 mm dish containing 28 ml DMEM medium complete. After this step, you should have three dishes with the same number of cells.

    12. Add 9 ml of DMEM medium complete to the “old” 100 mm dish for further cultivation.

    13. Transfer all plates into the incubator at 37°C and 5% CO2 and cultivate the HEK293 cells plated onto the three 150 mm dishes until they reach confluency.


  2. Isolation of mitochondria from HEK293 cells

    This protocol does not yield highly pure mitochondria but a fraction enriched in mitochondria. This is sufficient to monitor protein import into mitochondria. For an efficient isolation procedure, make sure that all buffers, materials, and reagents are precooled at 4°C. Always work on crushed ice. Isolated mitochondria are delicate (the mitochondrial membranes can easily be disrupted); be careful when pipetting solutions containing mitochondria and use cut pipette tips.

    1. Rinse the three confluent 150 mm dishes plated with HEK293 cells (approximately 30 × 106 cells per 150 mm dish) with 10 ml of ice-cold PBS.

    2. Add 10 ml of ice-cold PBS and gently scrape the cells off using a cell scraper. Transfer the cell solutions into 3 × 15 ml Falcon tubes.

    3. Pellet the cells at 500 × g for 5 min at 4°C in 15 ml Falcon tubes using a centrifuge (Eppendorf 5702R). Remove PBS using a vacuum pump.

    4. Resuspend all three cell pellets together in a total of 5 ml 1× M buffer containing 1× CompleteTM Protease Inhibitor Cocktail using a 10 ml serological pipette and a pipette boy. See Note 6.

    5. Transfer cells to precooled Potter S homogenizer cylinder. Homogenize cells using the precooled potter homogenizer on crushed ice (1,000 rpm). Move the cylinder (the pestle/plunger is fixed in the stirrer) up and slowly down (1x up and down counts as one stroke). Perform 15 strokes. In this step, the plasma membrane is opened, and organelles and the cytosol are released. See Note 7.

    6. Cell lysis can be monitored by pipetting 20 µl of suspension onto a glass slide and placing a coverslip on top before looking at cell integrity under the light microscope using Trypan Blue staining.

    7. Transfer the cell homogenate to a 15 ml Falcon tube. Pellet the homogenized cells at 600 × g for 5 min at 4°C using a centrifuge (Eppendorf 5702R).

    8. Collect the supernatant and store at 4°C.

    9. If you suspect that cell lysis might have been not efficient, perform Steps B10-B12; otherwise, proceed with Step B13.

    10. Resuspend the pellet from Step B7, which still might contain intact cells, in 5 ml 1× M buffer and repeat the potter step (15 strokes, 1,000 rpm).

    11. Pellet the homogenized cells at 600 × g for 5 min at 4°C in 15 ml Falcon tubes using a centrifuge (Eppendorf 5702R).

    12. Collect the supernatant and pool with the supernatant from Step B7.

    13. Distribute the supernatant in 2 ml aliquots into 2 ml tubes using cut 1 ml-pipette tips. You should have three completely filled 2 ml tubes at this step. Spin at 600 × g for 5 min at 4°C using a centrifuge (Eppendorf 5417R) to pellet nuclei.

    14. Carefully collect the supernatant from step 13 using cut 1 ml-pipette tips (avoid touching the pellet with the pipet tip) and transfer to fresh 2 ml tubes. Spin supernatant at 8,000 × g for 10 min at 4°C using a centrifuge (Eppendorf 5417R).

      Note: The pellet from this step contains the crude mitochondria.

    15. Carefully collect the supernatant and discard it. Avoid touching the pellet with the pipette tip but still remove the supernatant completely.

    16. Following this protocol will yield 3 × 2 ml tubes containing pellet. With a cut 200 µl-pipette tip, gently resuspend these pellets, which contain crude mitochondria in a total of 1 ml ice-cold 1× M buffer (without complete protease inhibitor cocktail). Combine pellets into one 2 ml tube. Add another 1 ml ice-cold 1× M buffer (without the complete protease inhibitor cocktail). See Note 8.

    17. Spin the 2 ml tube at 6,000 × g for 10 min at 4°C using a centrifuge (Eppendorf 5417R) and carefully remove the supernatant.

      Note: The pellet from this step contains the mitochondria.

    18. Resuspend the mitochondrial fraction using a cut 200 µl-pipette tip in 400 µl of ice-cold 1× M buffer.

    19. Store on ice until the protein content has been determined. Afterward, proceed immediately with the import experiments.

    20. Quantify the concentration of the isolated mitochondria using the Bradford Reagent ROTI® Quant Assay according to the manufacturer’s instructions. Use a range of different dilutions of the mitochondria solution for protein determination to ensure that at least one sample falls within the standard range. For wild-type HEK293 cells, a good start is protein determination on a 1:10 dilution of the mitochondria solution.


  3. [35S]-labeled precursor synthesis

    Cytosolic ribosomes synthesize precursors in their reduced states (i.e., cysteine residues are present as thiols). Cytosolic enzymes maintain these precursors in their reduced state to achieve efficient translocation into mitochondria ( Durigon et al., 2012 ; Banci et al., 2013 ; Habich et al., 2019a ). To generate reduced radiolabeled precursors, perform the following steps in a hypoxic glove box and cabinet with 0.5% residual O2. Sulfur-35 [35S] is a low energy beta emitter. Refer to the general safety precautions when working with radioactivity.

    1. Plan the synthesis of radioactively labeled precursors well. In general, synthesis should take place at least on the day before mitochondria isolation and protein import.

    2. To synthesize 50 µl of the [35S]-labeled precursor lysate, mix 40 µl of TNT® Quick Coupled Transcription/Translation Mix and 2 µl of EasyTagTM EXPRESS 35S Protein Labeling Mix with 1 µg of DNA template. Fill the mixture up to 50 µl with nuclease-free double-distilled water.

    3. Incubate for 90 min at 30°C in the hypoxic glove box cabinet under low O2 concentration.

    4. To stop the reaction, add 1 µl of 200 mM L-methionine.

    5. Incubate for 5 min at 37°C.

    6. Add 1 µl 50 mM of EDTA, which initiates the dissociation of ribosomes.

    7. Store the lysates at -80°C. See Note 9.

    8. To test the efficiency of the quick transcription/translation reaction before the mitochondrial import process, resuspend 0.5 µl of the radiolabeled lysate in 20 µl of 1× sample buffer containing 50 mM DTT.

    9. Boil all samples for 5 min at 95°C.

    10. Analyze the samples by SDS-PAGE.

    11. Transfer the proteins onto a nitrocellulose blotting membrane and dry it.

    12. Expose the nitrocellulose blotting membrane to an autoradiography film/screen.

    13. After 16 h, develop the autoradiography film using an imaging system (e.g., Typhoon). The protein signal should be well visible at the correct molecular weight. If the signal is very strong after 16 h, then dilute the lysate accordingly. If the signal is weak, the synthesis might not have worked properly, or the protein of interest contains too few methionine residues. See Note 10.


  4. In vitro import of [35S]-labeled precursors into isolated mitochondria

    In the import experiment, the translocation efficiency of mitochondrial precursors across the outer mitochondrial membrane is monitored. Non-imported pre-proteins are accessible to degradation by treatment with the serine protease, proteinase K, whereas translocated precursors are protected inside the organelle. Different variations of the protocol test the impact of the membrane potential or specific components of the import machinery on precursor import (see Note 11). For example, the impact of the membrane potential can be tested by using the proton gradient uncoupler CCCP. The import of proteins to the correct intramitochondrial localization can be tested by mitochondrial fractionation after the import.

    Isolated mitochondria from HEK293 cells are very delicate. Be careful when pipetting mitochondria and perform the import reaction on the same day of the mitochondrial isolation.

    1. Plan the layout of the experiment for determining the import kinetics, e.g., how many time points are required to assess mitochondrial import. Good time points for an initial experiment are, for example, 1, 10, and 30 min. Additionally, controls are required. The “input” control allows calculating the import efficiency and comparing biological replicates. The “Triton X-100” control allows assessing whether unfolded/folded precursors are susceptible to protease treatment. This is important as resistance to degradation will prevent distinguishing between imported and non-imported protein (= degraded by protease). The “no membrane potential” control is important to test whether the import of the specific precursor of interest depends on the membrane potential. All these samples and controls add up to the total number of import reactions necessary.

    2. Distribute 20 µg of mitochondria per import in reaction tube (import can be performed independently for each time point, i.e., one reaction tube per time point or using a master mix from which aliquots are drawn at each time point). Centrifuge at 8,000 × g for 5 min at 4°C using a centrifuge (Eppendorf 5417R). Remove supernatant. The pellet contains mitochondria. For the “normal” import kinetics assessment, mix carefully 20 µg pelleted crude mitochondria with 4 µl [35S]-labeled precursor lysate (potentially diluted after the lysate test, see Step B13) using a cut 10 µl pipette tip. Avoid vortexing since this breaks the mitochondrial membranes. For each time point, prepare an individual reaction tube on ice. See Note 12.

    3. For the “no membrane potential” control: mix carefully 20 µg of pelleted crude mitochondria with 1 μl of 20 mM CCCP. Allow 5 minutes incubation time. Add 4 µl of [35S]-labeled precursor lysate.

    4. The import reaction starts as soon as mitochondria and lysate are mixed. During the import reaction, place the samples at 30°C while shaking at 600 rpm in a ThermoMixer.

    5. Stop the reaction at desired time points by placing the samples on ice. Directly centrifuge 5 min at 8,000 × g. Remove supernatant. The pellet contains mitochondria with imported protein.

    6. For proteinase K treatment, resuspend mitochondria in 400 µl of 1× M buffer containing 20 µg of ml-1 proteinase K.

    7. Incubate for 20 min on ice.

    8. For the “Triton X-100” control, resuspend mitochondria in step 6 in 400 µl of 1× M buffer containing 20 µg ml-1 of proteinase K and 0.5% Triton X-100.

    9. Stop the digest by adding 2 µl of 200 mM PMSF (1 mM final).

    10. Centrifuge at 10,000 × g for 5 min at 4°C using a centrifuge (Eppendorf 5417R). Remove supernatant. The pellet contains mitochondria with imported protein.

    11. Add 400 µl of buffer M containing 1 mM PMSF and spin at 10,000 × g for 5 min at 4°C using a centrifuge (Eppendorf 5417R). Remove supernatant. The pellet contains mitochondria with imported protein. Centrifuge at 10,000 × g for 1 min at 4°C using a centrifuge (Eppendorf 5417R). Remove residual supernatant. The pellet contains mitochondria with imported protein.

    12. Resuspend the pellet in 40 µl of 1× sample buffer containing 50 mM DTT. See Note 13.

    13. Boil all samples for 5 min at 95°C.

    14. Analyze the samples by SDS-PAGE.

    15. Transfer the proteins onto a nitrocellulose blotting membrane and dry it.

    16. Expose the nitrocellulose blotting membrane to an autoradiography film/screen.

    17. After one day up to several days, develop the autoradiography film/screen using an imaging system (e.g., Typhoon). An example of such an experiment is shown in Figure 2.



      Figure 2. In organello import assay of a mitochondrial matrix protein SOD2 (containing a mitochondrial targeting signal, MTS) and an IMS protein, Cox19 (no MTS). A. SOD2 and COX19 are localized in matrix and IMS, respectively. The mitochondrial membrane potential is important for the import of matrix proteins and also for IMS proteins without MTS in human cells. B. Import kinetic of radiolabelled SOD2 into isolated HEK293 mitochondria. The SOD2 precursor is processed upon reaching the matrix (mature form). Depletion of the membrane potential (CCCP) prevents import. Mitochondria-localized SOD2 is not resistant to proteinase K (PK) treatment as its signal is lost upon Triton X-100 treatment. Imported SOD2 was visualized by autoradiography (18 h exposure). C. Import of Cox19 into HEK293 mitochondria. COX19 does not contain an MTS. Thus, imported and non-imported COX19 migrate at the same height. COX19 import is dependent on the membrane potential (lower signal in CCCP control).

Data analysis

The protein signals were quantified using ImageQuant TL 8.1 (GE Healthcare Life Science) and plotted using Microsoft Excel. Experiments are usually repeated three times and presented as averages with standard deviation.

Notes

  1. Cells with impaired import machinery (e.g., obtained through CRISPR-Cas9-mediated deletion of import machinery components) might have difficulties growing in normal medium. In this case, uridine (50 µg/ml final, Sigma-Aldrich, #U3003), sodium pyruvate (Sigma-Aldrich, #S8636), and non-essential amino acids (Sigma-Aldrich, #M7145) can be added.

  2. From 3 × 150 mm plates of confluent HEK293 cells, a total of 0.5-1.0 mg crude mitochondria can be obtained. Different tissue culture cells can give quite different yields. For example, for HeLa cells, a total of 0.2-0.3 mg crude mitochondria can be obtained.

  3. The TNT® Quick Coupled Transcription/Translation System can also be ordered to accommodate a T7 promotor depending on your starting plasmid.

  4. The EasyTagTM EXPRESS 35S Protein Labeling Mix, [35S]-, 7mCi, 1175 Ci/mmol from Perkin Elmer works best for the in organello import assay.

  5. Keep Proteinase K in single-use aliquots and avoid multiple freeze-thaw cycles as this abolishes the enzymatic activity. Aliquots can be stored at -20°C for several months.

  6. The volume of the 1× M buffer used for suspending cells before potter homogenization depends on the cell amount used. For 3 × 150 mm plates, use 5 ml of 1× M buffer. For more cells, multiply this amount accordingly. For fewer cells, still use 5 ml of 1× M buffer.

  7. Incubation at this step leads to cell swelling, which is beneficial for cell lysis during potter homogenization. While swelling, the potter homogenizer can be assembled by screwing the PTFE plunger to the electric stirrer. Set the electric stirrer to 1,000 rpm. Before you start to homogenize, test if the plunger is tightly assembled and does not show any horizontal movement. Move the cylinder containing the cell suspension only when the plunger is rotating to avoid vacuum and air bubbles. Also, avoid creating a vacuum with the up-stroke to keep the mitochondria intact during homogenization. This is done by performing the up-stroke movement very slowly.

  8. From this point on, the 1× M buffer does not contain protease inhibitor cocktail to avoid inhibition of proteinase K in the import assay.

  9. Note that the half-life of radioactive decay of 35S is 87 days. Radioactive lysates should be used as fresh as possible to obtain maximum signal intensities. Moreover, a longer lysate storage time will lead to the oxidation of cysteine residues, which might prevent import.

  10. If the (mature) protein does not contain enough methionine residues (and less critical, enough cysteines), additional methionine residues might be added to increase signal strength (e.g., addition of 4 methionine residues at the C-terminus). Alternatively, precursors might also be translated non-radioactively (or expressed and purified from E. coli and unfolded) and then detected by other methods, including through immunoblot against an additional tag.

  11. Variation of the described import reaction might be implemented to analyze import pathways in mechanistic detail.

    1. Analyzing the submitochondrial localization of the imported protein: Perform a submitochondrial fractionation after import, either by digitonin titration or hypoosmotic swelling combined with PK treatment, to remove the outer membrane and IMS proteins.

    2. Interaction of precursor with import machinery components: Perform immunoprecipitation experiments after import, possibly using crosslinking approaches to stabilize the interactions.

    3. Analysis of redox processes during import: Perform imports in the presence of different amounts of membrane-permeable (DTT) or impermeable (TCEP, glutathione) reducing agents. A dependency on these agents indicates the occurrence of redox processes during import (e.g., because of oxidative folding of the precursor).

    4. Analysis of import pathways: Numerous cell lines lacking components of the import machinery have been generated ( Chiusolo et al., 2017 ; Habich et al., 2019a ; Richter et al., 2019 ); performing import experiments into mitochondria isolated from these cells allows mapping the specific import pathway in mechanistic detail.

  12. In the import reaction with freshly isolated mitochondria, the addition of energy-regenerating components (like ADP, malate, NADH, etc.) appears unnecessary. Still, for some precursor proteins, this might be considered to further boost import efficiency.

  13. For SDS-PAGE, 20 µl of the sample is sufficient for a good detection with a short exposure time.

Recipes

  1. Dulbecco’s modified Eagle medium (DMEM) medium-complete

    Add 10% fetal calf serum and 1% penicillin/streptomycin to a fresh bottle of DMEM high glucose. Store at 4°C and prewarm to 37°C before use. See Note 1.

  2. Dulbecco's Phosphate Buffered Saline (PBS)

    1. Dissolve one bottle PBS powder in double-distilled water according to the manufacturer’s description

    2. Sterilize by autoclaving

    3. Store at 4°C and prewarm to 37°C before use

  3. Trypsin-EDTA solution

    1. Dilute 10× Trypsin-EDTA solution 1:10 with PBS

    2. Store at 4°C and prewarm to 37°C before use

    3. Aliquots can be kept frozen at -20°C

  4. 1× buffer M

    220 mM mannitol

    70 mM Sucrose

    5 mM HEPES-KOH, pH 7.4

    1 mM EGTA-KOH, pH 7.4

    Prepare freshly and store at 4°C before use

  5. 1× sample buffer

    2% SDS

    60 mM Tris(hydroxymethyl)aminomethane (Tris-HCl) pH 6.8

    10% glycerol

    0.005% bromophenol blue

    50 mM dithiothreitol (DTT)

  6. 20 µg ml-1 peqGOLD proteinase K

    PeqGOLD proteinase K in SEH buffer. See Note 5.

  7. SEH buffer

    250 mM Sucrose

    1 mM EDTA

    20 mM HEPES/KOH pH 7.4

  8. 20 µg/import mitochondria

    Isolated mitochondria must be used immediately for import experiments. After isolation, mitochondria concentration is assessed with the Bradford assay. The concentration of mitochondria for each import reaction is calculated from this and directly pipetted (20 µg/import). Mitochondria can be diluted in buffer M if required.

Acknowledgments

The Deutsche Forschungsgemeinschaft (DFG) funds the research in the Laboratory of JR (RI2150/2-2 – project number 251546152, RI2150/5-1 – project number 435235019, CRC1218 / TP B02 – project number 269925409, and RTG2550/1 – project number 411422114). The protocol was used in the following original research papers: Saita et al. (2018), MacVicar et al. (2019), and Murschall et al. (2020).

Competing interests

The authors declare that they have no competing interests.

References

  1. Banci, L., Barbieri, L., Luchinat, E. and Secci, E. (2013). Visualization of redox-controlled protein fold in living cells. Chem Biol 20(6): 747-752.
  2. Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. and Pfanner, N. (2009). Importing mitochondrial proteins: machineries and mechanisms. Cell 138(4): 628-644.
  3. Chiusolo, V., Jacquemin, G., Yonca Bassoy, E., Vinet, L., Liguori, L., Walch, M., Kozjak-Pavlovic, V. and Martinvalet, D. (2017). Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis.Cell Death Differ 24(4): 747-758.
  4. Durigon, R., Wang, Q., Ceh Pavia, E., Grant, C. M. and Lu, H. (2012). Cytosolic thioredoxin system facilitates the import of mitochondrial small Tim proteins.EMBO Rep 13(10): 916-922.
  5. Endo, T. and Tamura, Y. (2018). Shuttle mission in the mitochondrial intermembrane space. EMBO J 37(4).
  6. Habich, M., Salscheider, S. L., Murschall, L. M., Hoehne, M. N., Fischer, M., Schorn, F., Petrungaro, C., Ali, M., Erdogan, A. J., Abou-Eid, S., Kashkar, H., Dengjel, J. and Riemer, J. (2019a). Vectorial Import via a Metastable Disulfide-Linked Complex Allows for a Quality Control Step and Import by the Mitochondrial Disulfide Relay. Cell Rep 26(3): 759-774 e755.
  7. Habich, M., Salscheider, S. L. and Riemer, J. (2019b). Cysteine residues in mitochondrial intermembrane space proteins: more than just import. Br J Pharmacol 176(4): 514-531.
  8. Hansen, K. G. and Herrmann, J. M. J. T. p. j. (2019). Transport of proteins into mitochondria. 38(3): 330-342.
  9. Hartl, F. U., Pfanner, N., Nicholson, D. W. and Neupert, W. (1989). Mitochondrial protein import. Biochim Biophys Acta 988(1): 1-45.
  10. MacPherson, L. and Tokatlidis, K. (2017). Protein trafficking in the mitochondrial intermembrane space: mechanisms and links to human disease.Biochem J 474(15): 2533-2545.
  11. MacVicar, T., Ohba, Y., Nolte, H., Mayer, F. C., Tatsuta, T., Sprenger, H. G., Lindner, B., Zhao, Y., Li, J., Bruns, C., Kruger, M., Habich, M., Riemer, J., Schwarzer, R., Pasparakis, M., Henschke, S., Bruning, J. C., Zamboni, N. and Langer, T. (2019). Lipid signalling drives proteolytic rewiring of mitochondria by YME1L. Nature 575(7782): 361-365.
  12. Mokranjac, D. and Neupert, W. (2007). Protein import into isolated mitochondria. Mitochondria, Springer: 277-286.
  13. Murschall, L. M., Gerhards, A., MacVicar, T., Peker, E., Hasberg, L., Wawra, S., Langer, T. and Riemer, J. (2020). The C-terminal region of the oxidoreductase MIA40 stabilizes its cytosolic precursor during mitochondrial import. BMC Biol 18(1): 96.
  14. Pfanner, N., Warscheid, B. and Wiedemann, N. J. (2019). Mitochondrial protein organization: from biogenesis to networks and function. Nat Rev Mol Cell Biol 20(5): 267.
  15. Richter, F., Dennerlein, S., Nikolov, M., Jans, D. C., Naumenko, N., Aich, A., MacVicar, T., Linden, A., Jakobs, S., Urlaub, H., Langer, T. and Rehling, P. (2019). ROMO1 is a constituent of the human presequence translocase required for YME1L protease import. J Cell Biol 218(2): 598-614.
  16. Saita, S., Tatsuta, T., Lampe, P. A., Konig, T., Ohba, Y. and Langer, T. (2018). PARL partitions the lipid transfer protein STARD7 between the cytosol and mitochondria. EMBO J 37(4).
  17. Schmidt, O., Pfanner, N. and Meisinger, C. (2010). Mitochondrial protein import: from proteomics to functional mechanisms. Nat Rev Mol Cell Biol 11(9): 655-667.
  18. Spinelli, J. B. and Haigis, M. C. (2018). The multifaceted contributions of mitochondria to cellular metabolism. Nat Cell Biol 20(7): 745-754.
  19. Tang, B. L. and Tang (2015). Membrane Trafficking: Second Edition. Springer.
  20. Vafai, S. B. and Mootha, V. K. (2012). Mitochondrial disorders as windows into an ancient organelle. Nature 491(7424): 374-383.
  21. Weckbecker, D. and Herrmann, J. M. (2013). Methods to study the biogenesis of membrane proteins in yeast mitochondria. Methods Mol Biol 1033: 307-322.

简介

[摘要]线粒体是必不可少的细胞器,含有大约 1 , 500 种蛋白质。这些蛋白质中只有大约1% 是在线粒体内合成的,而其余 99% 作为前体在细胞质核糖体上合成并输入细胞器。V arious工具和技术来分析导入过程中得到了发展。其中,体外重组进口系统对于详细研究这些过程具有重要意义。这些实验监测先前在无细胞环境中放射性标记的线粒体前体的输入反应。然而,所描述的方法已经主要在从分离线粒体进行酿酒酵母。在这里,我们描述了这种强大的检测方法的适应性,以将蛋白质导入从人类组织培养细胞中分离出来的粗线粒体。



图文摘要:







监测蛋白质输入到从人类细胞中分离的线粒体的检测概述

[背景]线粒体蛋白质在能量守恒,产热,脂质和铁代谢,信令执行关键功能,和细胞死亡途径(Vafai和Mootha ,斯皮内利和Haigis; 2012 ,2018; Habich 。等人,2019b ;普凡纳等。, 2019) 。人类线粒体包含大约1500 种不同的蛋白质,但只有 13 种在线粒体核糖体的基质中被翻译。因此,绝大多数蛋白质在胞质核糖体上作为前体合成,然后输入到线粒体中。d ifferent进口途径已经确定,直接蛋白质的正确submitochondrial本地化(哈特尔等,1989; Chacinska等,2009;施密特等人。,2010;麦弗逊和Tokatlidis ,2017年;远藤和田村,2018;汉森和赫尔曼,2019 年)。大多数这些途径已使用少量模型底物进行表征。然而,越来越清楚的是,许多蛋白质对进口机械组件有不同的依赖性,并且驱动进口的内在蛋白质特性可能会有所不同。使用分离的线粒体的体外重组系统是强大的工具,可以快速分析线粒体蛋白的导入。这些测定允许对蛋白质输入及其决定因素进行快速和平行分析。与完整细胞中的分析相比,胞质过程 –包括蛋白酶体降解、不同的蛋白质转录/翻译率、与其他前体的竞争以及特定伴侣系统的贡献–可以在使用分离线粒体的导入分析中排除。

大多数描述进口的实验已经从分离的线粒体进行酿酒酵母(Mokranjac和纽珀特,; Weckbecker和赫尔曼2007 ,2013;唐和唐,2015年),主要是因为酵母遗传学允许详细的机械分析和洞察到进口途径。我们调整了监测蛋白质输入到从人体组织培养细胞中富集的线粒体的协议。与CRISPR-Cas9介导的基因编辑(敲除和敲插件),siRNA介导的蛋白消耗的组合,并稳定细胞系过表达人类的进口机器的部件让人类线粒体蛋白进口的详细调查。这使得能够调查人类和酵母线粒体输入之间的差异以及人类患者突变的分析。

关键字:细胞器中导入, 分离线粒体, 放射性同位素标记蛋白, 无细胞蛋白质合成, 人体组织培养细胞



材料和试剂

HEK293细胞的培养
1.细胞培养皿100 × 20 mm(Sarstedt ,目录号:83.3902)      


2.细胞培养皿150 × 20 mm(Sarstedt ,目录号:83.3903)      


3.高压灭菌一次性玻璃巴斯德移液器,无棉垫(VWR,目录号:HECH40567001)      


4.血清移液管:无菌独立包装,      


2 ml移液器(Sarstedt ,目录号:83.3903)


5 ml移液器(Sarstedt ,目录号:86.1252.001)


10 ml移液器(Sarstedt ,目录号:86.1254.001)


25 ml移液器(Sarstedt ,目录号:86.1685.001)


5. Flp -In T- REx HEK293 细胞(人胚胎肾 293,Invitrogen,目录号:R78007)      


6. Dulbecco 改良的 Eagle 培养基 (DMEM) 培养基完全(见食谱)      


10%胎牛血清(FCS,Sigma-Aldrich,目录号:F0804)和


1% 青霉素/链霉素(P/S,Sigma-Aldrich,目录号:P0781-100ML)


DMEM 高葡萄糖(Thermo Fisher,目录号:41965-062)


7. Dulbecco 磷酸盐缓冲盐水 (PBS) (见食谱)      


PBS 粉末(Sigma-Aldrich,目录号:D5652)


8.胰蛋白酶- EDTA溶液(见配方)      


10 ×胰蛋白酶- EDTA溶液(Sigma-Aldrich,目录号:T4174)

从 HEK293 细胞中分离线粒体
细胞刮刀 16 mm(Sarstedt ,目录号:831832)
15 ml F alcon 管(VWR,目录号:734-0451)
3 × 150 mm 铺有 HEK293 细胞的汇合培养皿
注意:细胞在线粒体分离前 3-4 天播种。细胞生长至 90% 汇合。见注 2 。


完整的TM Protease Inhibitor Cocktail 不含 EDTA 片剂(Roche,目录号:11873580001)。使用前新鲜添加。等分试样可以储存在 -20°C
PBS,使用前4°C保存
Bradford Reagent ROTI ® Quant(Roth,目录号:K015.1)
1 ×缓冲液M (见配方)
甘露醇(默克,目录号:1.05983)


蔗糖(Sigma-Aldrich,目录号:S0389)


HEPES-KOH,pH 7.4(VWR,目录号:441487)


EGTA-KOH,pH 7.4(Roth,目录号:3054.1)

[ 35 S]-标记前体的合成
TNT ®快速耦合转录/翻译系统 SP6 启动子(Promega,目录号:L2080)。见注 3 。
EasyTag TM EXPRESS 35 S 蛋白质标记混合物,[ 35 S]-,7 mCi ,1175 Ci/ mmol (PerkinElmer,目录号:NEG772007MC)。见注 4 。
DNA 模板 (1 µg ml -1 ) 包含插入到 SP6 启动子下游的线粒体前体基因。质粒载体:pGEM-3/4 (Promega)。储存在-20°C。
无核酸酶双蒸水
200 mM L-甲硫氨酸(Merck,目录号:1057070100)在无核酸酶的双蒸水中。4°C 保存
50 mM 乙二胺四酸(EDTA)二钠盐 2-水合物,pH 8(AppliChem,目录号:A2937)在无核酸酶的双蒸水中
1 ×样品缓冲液(见配方)
三(羟甲基)氨基甲烷(Tris,VW R ,目录号:0497)


G甘油(Sigma-Aldrich,目录号:G7757)


B溴酚蓝(Roth,目录号:A512.1)


d ithiothreitol(DTT,APPLICHEM,目录号:A1101)

将 [ 35 S] 标记的前体导入分离的线粒体
印迹膜,硝酸纤维素,Amersham TM Protran ® (VW R ,目录号:10600001),0.2 µm 孔径
20 mM羰基氰化物3-氯苯腙(CCCP,Sigma-Aldrich,目录号:C2759)
200 mM苯甲基磺酰氟(PMSF,AppliChem,目录号:A0999.0100)
碎冰
Triton X-100(AppliChem,目录号:A1388)
1 ×缓冲液M (见配方)
20 µg/进口反应线粒体(参见:食谱)
20 µg ml -1 peqGOLD蛋白酶 K(见食谱)
1 ×样品缓冲液(见配方)
SEH缓冲液(见配方)
 


设备

HEK293细胞的培养
层流罩 II 级(ENVAIR 生态)
CO 2培养箱
真空泵
显微镜
离心机(例如,微量离心,型号:5702R)
配备的细胞培养实验室,包括,例如,II 级层流罩 (ENVAIR eco)、设置在 37°C 和 5% CO 2 的用于细胞培养的 CO 2培养箱、用于使用无菌玻璃巴斯德吸管去除培养基的真空泵,显微镜,和细胞计数器和一个冷却离心机。

从 HEK293 细胞中分离线粒体
电动搅拌器 ( Heidolph RZR 2020)
Potter S 均质器(15 ml),带有紧密配合的 PTFE(聚四氟乙烯)柱塞(Sartorius,目录号:8542406)
间隙的范围(即,在杵和汽缸壁之间的平均距离)的最佳线粒体隔离由此从0.045 - 0.065毫米。


离心机(Eppendorf ,型号:5702R )
离心机(Eppendorf ,型号:5417R )
真空泵(KNF laboport微型泵),用于使用无菌玻璃巴斯德移液管除去介质。
 


[ 35 S]-标记的前体合成
缺氧手套箱和柜子(Coy Laboratory Products,“Coy 1 Person O 2 Control Glove Box”,目录号:031615);如果前体蛋白不容易被空气氧化(例如,不包含或仅包含很少的半胱氨酸残基),则不需要。

将 [ 35 S] 标记的前体导入分离的线粒体
ThermoMixer Eppendorf (F1.5, #EP5384000012)
离心机(Eppendorf ,型号:5417R )
用于 SDS-PAGE 电泳和(半干)印迹的系统(Bio - Rad,MiniPROTEAN Tetra Cell,目录号:1658000)
成像系统台风 FLA 9500(GE Healthcare,目录号:9351515)
Image Eraser FLA(GE Healthcare,目录号:12999750)
 


软件

ImageQuant TL 8.1(GE 医疗生命科学)。
 


程序

图 1描述了该过程的概述。

 


图 1. “ inorganello import assay”协议中的步骤示意图

HEK293细胞的培养
HEK293 细胞在完全含有高葡萄糖、FCS 和青霉素/链霉素抗生素混合物的 DMEM 培养基中培养。对于传代,将 HEK293 细胞培养在 100 毫米培养皿中的 10 毫升 DMEM 培养基中,直至达到 90% 汇合。对于所描述的实验,将 HEK293 细胞铺在 30 毫升 DMEM 培养基中的 150 毫米培养皿上,并培养至汇合。特别小心,必须采取一些细胞,在其中线粒体蛋白质(例如,线粒体进口机厂的Ý )已经被耗尽或者或过表达; 可能需要不同的培养条件。


在 37°C 和 5% CO 2的培养箱中,在 10 ml DMEM 完全培养基中的 100 mm 培养皿上培养 HEK293 细胞,直到它们达到 90% 汇合。
将 HEK293 菜肴转移到经过表面消毒的层流罩 II 类中。
转移的无菌DMEM培养基完整,无菌PBS,并在无菌胰蛋白酶- EDTA进层流罩。所有溶液应预热至 37°C。
使用高压灭菌的一次性玻璃巴斯德吸管和真空泵取出介质。
小心地加入10ml冲洗细胞的在使用血清移液管和移液管男孩培养皿的边缘无菌PBS。
使用高压灭菌的一次性玻璃巴斯德吸管和真空泵取出 PBS。
加入1ml的无菌胰蛋白酶- EDTA滴加到使用血清移液管和移液管男孩细胞。
在 37°C 和 5% CO 2下孵育细胞 5 分钟,直到细胞从培养皿中分离。用手指轻弹盘子。
将培养皿放回经过表面消毒的层流罩中,并加入 9 ml的DMEM 培养基。
使用 10 ml 血清移液器和移液器男孩将细胞单一化。将移液器尖端轻轻按在盘子底部。
将 2 ml 细胞悬浮液 3 次转移到含有 28 ml DMEM 培养基的 150 mm 培养皿中。在这一步之后,你应该有三个细胞数相同的培养皿。
9毫升添加的DMEM培养基完整的“旧” 100mm培养皿中用于进一步培养。
将所有板转移到 37°C 和 5% CO 2的培养箱中,培养铺在三个 150 毫米培养皿上的 HEK293 细胞,直到它们达到汇合。
 


从 HEK293 细胞中分离线粒体
该协议不会产生高纯度的线粒体,而是富含线粒体的一小部分。这足以监测蛋白质进入线粒体。为实现高效的隔离程序,请确保所有缓冲液、材料和试剂均预冷至 4°C。始终在碎冰上工作。分离的线粒体很脆弱(线粒体膜很容易被破坏);b吹打含有线粒体和使用切割的枪头解决方案,当E小心。


冲洗的镀有HEK293细胞(约30层3种汇合150毫米菜肴× 10 6每150毫米培养皿的细胞)用10毫升的冰冷PBS中。
加入10 mL的冰冷PBS中,轻轻刮去关闭单元的使用细胞刮。转移的细胞溶液小号成3 × 15毫升˚F爱尔康管小号。
使用离心机 (Eppendorf 5702R)将细胞在4°C 下以 500 × g沉淀在 15 ml F alcon 管中5 分钟。使用真空泵取出 PBS。
使用 10 ml 血清移液管和移液器将所有三个细胞沉淀重悬在总共 5 ml 1 × M 缓冲液中,其中含有 1 × Complete TM Protease Inhibitor Cocktail。见注 6 。
将细胞转移到预冷的 Potter S 均质器圆筒。在碎冰 (1 , 000 rpm)上使用预冷的 potter 匀浆器匀浆细胞。向上和缓慢向下移动气缸(研杵/柱塞固定在搅拌器中)(向上和向下 1 次计为一次冲程)。执行 15 次冲程。在这一步中,质膜被打开,细胞器和细胞质被释放。见注 7 。
细胞裂解可以通过吸取20μ被监控升悬浮液到一个载玻片,并把一个在顶部盖玻片用台盼蓝染色的光学显微镜下观察细胞的完整性之前。
转移的细胞匀浆至15ml ˚F爱尔康管。使用离心机 (Eppendorf 5702R) 在 4°C 下以 600 × g将匀浆的细胞沉淀 5 分钟。
收集上清液并储存于 4°C。
如果你怀疑细胞裂解可能是效率不高,执行小号TEPS乙10乙12 ; 否则,继续进行小号TEP乙13 。
重悬从沉淀小号TEP乙7,其可能仍包含完整的细胞,在5毫升1 ×米缓冲区,并重复步骤波特(15个冲程,1 ,000rpm下)。
使用离心机 (Eppendorf 5702R)在 15 ml F alcon 管中于 4°C 下以 600 × g离心5 分钟沉淀匀浆细胞。
收集上清液并用池从上清液小号TEP乙7。
使用切割的 1 ml 移液器吸头将 2 ml 上清液分装到 2 ml 试管中。在这一步您应该有三个完全装满的 2 毫升管。使用离心机 (Eppendorf 5417R) 在 4°C 下以 600 × g离心 5 分钟以沉淀细胞核。
使用切开的 1 ml 移液器吸头(避免用移液器吸头接触沉淀物)小心收集步骤 13 中的上清液,然后转移到新的 2 ml 管中。使用离心机 (Eppendorf 5417R) 在 4°C 下以 8,000 × g离心上清液10 分钟。
注:牛逼,他从沉淀这个步骤包含原油线粒体。


仔细收集上清并丢弃它。避免与移液管尖端接触粒料,但仍除去的完全上清液。
遵循此协议将产生 3 × 2 ml 含有颗粒的管。用切割的 200 µl 移液器吸头轻轻重悬这些颗粒,这些颗粒在总共 1 ml 冰冷的 1 × M 缓冲液(不含完整的蛋白酶抑制剂混合物)中含有粗线粒体。将颗粒合并到一个 2 毫升管中。添加另一个1毫升冰冷的1 ×米缓冲区(没有的完全蛋白酶抑制剂混合物)。见注 8 。
使用离心机 (Eppendorf 5417R) 在 4°C 下以 6,000 × g 的速度旋转 2 ml 管10 分钟,并小心去除上清液。
注:牛逼,他从沉淀这个步骤包含线粒体。


于400μl使用切200μl的移液管尖端重悬线粒体级分的冰冷1 ×米缓冲区。
储存在冰上,直到确定蛋白质含量。随后,与立即进行了进口实验。
根据制造商的说明,使用Bradford Reagent ROTI ® Quant Assay量化分离的线粒体的浓度。使用一系列不同稀释度的线粒体溶液进行蛋白质测定,以确保至少有一个样品在标准范围内。对于野生-类型的HEK293细胞,一个良好的开端是在1:10稀释线粒体溶液的蛋白质测定。             
 


[ 35 S]-标记的前体合成
胞质核糖体合成处于还原状态的前体(即,半胱氨酸残基以硫醇形式存在)。Ç ytosolic酶保持这些前体在还原态吨ö实现有效易位至线粒体(Durigon等人,2012;颁赐。等人,2013; Habich等人。,2019一)。要生成减少的放射性标记前体,请在含 0.5% 残留 O 2的缺氧手套箱和机柜中执行以下步骤。Sulfur-35 [ 35 S] 是一种低能量的β发射体。使用放射性时请参阅一般安全预防措施。


计划好放射性标记前体的合成。一般来说,合成应至少在线粒体分离和蛋白质导入前一天进行。
为了合成将50μl[的35 S] -标记的前体裂解物,米IX 40微升的TNT ®快速偶联的转录/翻译混合物和2微升的的EasyTag TM EXPRESS 35 S蛋白标记混合物用1μg的DNA模板。用无核酸酶的双蒸水将混合物填充至 50 µl。
在低氧2浓度下在缺氧手套箱柜中在 30°C 下孵育 90 分钟。
为了终止反应,加入1微升的200mM的L-蛋氨酸。             
在 37°C 下孵育 5 分钟。
添加 1 µl 50 mM的EDTA,它会启动核糖体的解离。             
将裂解物储存在 -80°C。见注 9 。
为了在线粒体导入过程之前测试快速转录/翻译反应的效率,将 0.5 µl 放射性标记的裂解物重悬在 20 µl含有 50 mM DTT的1 ×样品缓冲液中。
将所有样品在 95°C 下煮沸 5 分钟。
通过 SDS-PAGE 分析样品。
将蛋白质转移到硝酸纤维素印迹膜上并干燥。
将硝酸纤维素印迹膜暴露在放射自显影胶片/屏幕上。
16 小时后,使用成像系统(例如,台风)开发放射自显影胶片。蛋白质信号应该在正确的分子量下清晰可见。如果 16 小时后信号非常强,则相应地稀释裂解液。如果信号较弱,则合成可能无法正常进行,或者目标蛋白质中的甲硫氨酸残基太少。见注 10 。
 


[ 35 S] 标记的前体体外导入分离的线粒体
在导入实验中,监测线粒体前体跨线粒体外膜的易位效率。非进口的前蛋白可以通过丝氨酸蛋白酶蛋白酶 K 处理降解,而易位前体在细胞器内受到保护。该协议测试的不同变化的膜电位或特定成分的影响的进口机厂ÿ上的前体进口(见注11 )。例如,可以使用质子梯度解偶联器 CCCP 来测试膜电位的影响。在我蛋白质的正确定位线粒体内可以M端口通过线粒体分馏在导入后进行测试。


从 HEK293 细胞中分离出的线粒体非常脆弱。移液线粒体时要小心,并在线粒体分离的同一天进行导入反应。


计划实验FO的布局[R确定进口动力学,例如,需要多少时间点来评估线粒体进口。用于初始实验良好的时间点是,例如,1,10 ,和30分钟。此外,还需要控制。“输入”控制允许CALCULAT荷兰国际集团的进口效率和COMPAR ING生物学重复。了“的Triton X-100”控制允许评估荷兰国际集团展开/折叠的前体是否易受蛋白酶处理。这很重要,因为对降解的抗性将阻止区分进口和非进口蛋白质(= 被蛋白酶降解)。“无膜电位”控制对于测试感兴趣的特定前体的输入是否取决于膜电位很重要。所有这些样本和控制加起来就是必需的导入反应总数。
分发20微克的在反应管进口每线粒体(可在每个时间点独立地进行导入,即,每个时间点,或者使用从该等分试样在每个时间点画出的主混合物一个反应管)。使用离心机(Eppendorf 5417R)在 4°C 下以 8,000 × g离心5 分钟。去除上清液。沉淀含有线粒体。为“正常”的进口动力学评估,小心混合20微克沉淀粗线粒体与4μl的[ 35 S] -标记的前体裂解物(溶胞产物试验后潜在稀释,见S TEP乙使用切口10微升移液管尖端13)。避免涡旋,因为这会破坏线粒体膜。对于每个时间点,在冰上准备一个单独的反应管。见注 12 。
 


对于“无膜电位”控制:混合仔细20微克的沉淀粗线粒体与1微升的20毫CCCP。允许 5 分钟的孵育时间。加入4微升的[ 35 S] -标记的前体裂解物。
一旦线粒体和裂解物混合,导入反应就开始了。在导入反应过程中,将样品置于 30°C,同时在ThermoMixer 中以 600 rpm 的速度摇动。
通过将样品放在冰上,在所需的时间点停止反应。直接以 8 , 000 × g离心 5 分钟。去除上清液。颗粒含有带有进口蛋白质的线粒体。
对于蛋白酶K处理,于400μl重悬线粒体的1 ×米缓冲区含有20μg的毫升-1蛋白酶K.
在冰上孵育 20 分钟。
对于“的Triton X-100”的控制,在步骤6中于400μl重悬线粒体的1 ×米缓冲区含有20μg毫升-1的蛋白酶K和0.5%的Triton X-100。
加入2μl的停止消化的200mM的PMSF(1mM的终浓度)。
使用离心机(Eppendorf 5417R)在 4°C 下以 10,000 × g离心5 分钟。去除上清液。颗粒含有带有进口蛋白质的线粒体。
加入 400 µl含有 1 mM PMSF的缓冲液 M,并使用离心机(Eppendorf 5417R)在 4°C 下以 10,000 × g离心 5 分钟。去除上清液。颗粒含有带有进口蛋白质的线粒体。使用离心机(Eppendorf 5417R)在 4°C 下以 10,000 × g离心1 分钟。去除残留的上清液。颗粒含有带有进口蛋白质的线粒体。
将沉淀重悬在 40 µl含有 50 mM DTT的1 ×样品缓冲液中。见注 13 。
将所有样品在 95°C 下煮沸 5 分钟。
通过 SDS-PAGE 分析样品。
将蛋白质转移到硝酸纤维素印迹膜上并干燥。
将硝酸纤维素印迹膜暴露在放射自显影胶片/屏幕上。
一天到几天后,使用成像系统(例如,台风)显影放射自显影胶片/屏幕。这种实验的一个例子如图 2所示。
 

图2.在organello线粒体基质蛋白SOD2(含有线粒体靶向信号,MTS)和IMS蛋白的进口测定,Cox19(无MTS) 。A. SOD2 和 COX19 分别位于基质和 IMS 中。线粒体膜电位对于基质蛋白的输入以及人类细胞中不含 MTS 的 IMS 蛋白都很重要。B.放射性标记的 SOD2 导入分离的 HEK293 线粒体的动力学。SOD2 前体在到达基质时进行处理(成熟形式)。膜电位 (CCCP) 的消耗会阻止输入。线粒体定位的 SOD2 对蛋白酶 K (PK) 处理没有抵抗力,因为它的信号在 Triton X-100 处理后丢失。通过放射自显影(18 小时暴露)可视化导入的 SOD2。C.将 Cox19 导入 HEK293 线粒体。COX19 不包含 MTS。因此,进口和非-进口COX19迁移在同一高度。COX19 的导入取决于膜电位(CCCP 控制中的较低信号)。

数据分析

使用ImageQuant TL 8.1 (GE Healthcare Life Science)量化蛋白质信号并使用 Microsoft Excel 绘图。实验通常重复 3 次,并以具有标准偏差的平均值表示。

笔记

与受损的进口机厂细胞Ý (例如,通过CRISPR-Cas9-居间获得d缺失进口机械部件的)可能具有在正常培养基中生长困难。在这种情况下,尿苷(50微克/毫升的最终,Sigma-Aldrich公司,#U3003),丙酮酸钠(Sigma-Aldrich公司,#S8636) ,和非必需氨基酸(Sigma-Aldrich公司,#M7145)可以加入。
从3 ×汇合HEK293细胞为150mm板,共0.5 -可以得到1.0毫克粗线粒体。不同的组织培养细胞可以产生完全不同的产量。例如,对于HeLa细胞,共0.2 -可以得到0.3毫克粗线粒体。
根据您的起始质粒,还可以订购T NT ®快速偶联转录/翻译系统以容纳 T7 启动子。
所述的EasyTag TM EXPRESS 35 S蛋白标记混合物,[ 35 S] - ,7mCi,1175次/来自Perkin Elmer的毫摩尔的效果最好为在organello进口测定。
将蛋白酶 K 保持在一次性等分试样中,并避免多次冻融循环,因为这会消除酶活性。等分试样可在 -20°C 下储存数月。
在 potter 匀浆之前用于悬浮细胞的 1 × M 缓冲液的体积取决于所用的细胞量。对于3 × 150毫米板,使用5毫升的1 ×米缓冲区。对于更多的单元格,相应地乘以这个数量。为更少的细胞,仍使用5毫升的1 ×米缓冲区。
在这一步孵育会导致细胞肿胀,这有利于陶器匀浆过程中的细胞裂解。在膨胀时,可以通过将 PTFE 柱塞拧到电动搅拌器上来组装陶器均质器。将电动搅拌器设置为 1 , 000 rpm。乙EFORE你开始搅匀,测试如果活塞紧紧地组装并且没有显示出任何水平运动。只有在柱塞旋转时才移动装有细胞悬浮液的圆柱体,以避免真空和气泡。此外,避免在上冲程中产生真空,以在均质过程中保持线粒体完整。这是非常缓慢的进行了行程移动完成光年。
从这一点开始,1 × M 缓冲液不含蛋白酶抑制剂混合物,以避免在导入检测中抑制蛋白酶 K。
请注意,35 S的放射性衰变半衰期为 87 天。放射性裂解物应尽可能新鲜使用,以获得最大的信号强度。此外,一个较长的裂解液存放时间会导致的半胱氨酸残基的氧化,这可能会导致进口。
如果(成熟)蛋白质不包含足够的甲硫氨酸残基(以及不太重要的足够半胱氨酸),可以添加额外的甲硫氨酸残基以增加信号强度(例如,在 C 端添加 4 个甲硫氨酸残基)。或者,前体也可以非放射性翻译(或从大肠杆菌中表达和纯化并展开),然后通过其他方法检测,包括通过针对附加标签的免疫印迹。
V所描述的进口反应的ariation可能被实现吨ö分析机理细节进口通路。
分析导入蛋白的亚软骨定位:导入后进行亚软骨分离,通过毛地黄皂苷滴定或低渗溶胀结合 PK 处理,去除外膜和 IMS 蛋白。
与进口机械部件前体互动:进口后进行免疫沉淀实验,可能使用交联方法来稳定的相互作用。
导入过程中的氧化还原过程分析:在存在不同量的膜渗透 (DTT) 或不渗透(TCEP、谷胱甘肽)还原剂的情况下执行导入。对这些试剂的依赖表明在进口过程中发生了氧化还原过程(例如,由于前体的氧化折叠)。
进口通路的分析:Ñ已经产生umerous细胞系缺乏导入机器的组分(Chiusolo 。等人,2017; Habich 。等人,2019一个;里氏等人,2019); p erforming导入实验成从这些细胞中分离出的线粒体允许小号映射在机械细节的特定进口通路。
与新鲜分离的线粒体导入反应,该加成再生能量成分的(如ADP,苹果酸盐,NADH,等等。)出现未必要的。尽管如此,对于某些前体蛋白质,这可能被认为是进一步提高进口效率。
对于 SDS-PAGE,20 µl 样品足以在较短的曝光时间内进行良好的检测。
 


食谱

1. Dulbecco 改良 Eagle 培养基 (DMEM) 培养基-完全      


将 10% 的胎牛血清和 1% 的青霉素/链霉素添加到一瓶新鲜的 DMEM 高葡萄糖中。储存于 4°C 并在使用前预热至 37°C。见注 1 。


2. Dulbecco 的磷酸盐缓冲盐水 (PBS)      


溶解一种瓶PBS粉末在双蒸瓦特一个根据制造商的说明之三
高压灭菌
储存于 4°C 并在使用前预热至 37°C
3.胰蛋白酶- EDTA溶液      


用 PBS 1:10稀释 10 × Trypsin-EDTA溶液
储存于 4°C 并在使用前预热至 37°C
等分试样可在 -20°C 下冷冻保存
4. 1 ×缓冲M      


220 mM 甘露醇


70 mM 蔗糖


5 mM HEPES-KOH,pH 7.4


1 mM EGTA-KOH,pH 7.4


使用前新鲜制备并储存于 4°C


5. 1 ×样品缓冲液      


2% 安全数据表


60 mM 三(羟甲基)氨基甲烷 (Tris-HCl ) pH 6.8


10% 甘油


0.005% 溴酚蓝


50 mM 二硫苏糖醇 (DTT )


6. 20 µg ml -1 peqGOLD蛋白酶 K      


PeqGOLD蛋白酶K的SEH缓冲。见注 5 。


7. SEH缓冲液      


250 mM 蔗糖


1 mM EDTA


20 mM HEPES/KOH pH 7.4


8. 20 µg/进口线粒体      


我分离的线粒体必须立即用于导入实验。分离后,用布拉德福德试验评估线粒体浓度。每个导入反应的线粒体浓度由此计算并直接移液(20 µg/导入) 。如果需要,线粒体可以在缓冲液 M 中稀释。

致谢

Deutsche Forschungsgemeinschaft (DFG) 资助了JR 实验室的研究(RI2150/2-2 – 项目编号 251546152、RI2150/5-1 – 项目编号 435235019、CRC1218 / TP B02 – 项目编号 26992054 – 项目编号 26992054 –编号 411422114)。该协议用于以下原始研究论文:Saita等人。(2018 年),MacVicar等人。( 2019 )和Murschall等人。( 2020) 。

利益争夺

作者声明他们没有竞争利益。

参考


1. Banci, L.、Barbieri, L.、Luchinat, E. 和 Secci, E. (2013)。活细胞中氧化还原控制的蛋白质折叠的可视化。化学生物学20(6):747-752。                       


2. Chacinska, A.、Koehler, CM、Milenkovic, D.、Lithgow, T. 和 Pfanner, N. (2009)。导入线粒体蛋白:机器和机制。单元格138(4):628-644。         


3. Chiusolo, V.、Jacquemin, G.、Yonca Bassoy, E.、Vinet, L.、Liguori, L.、Walch, M.、Kozjak-Pavlovic, V. 和 Martinvalet, D.(2017 年)。颗粒酶 B 以 Sam50、Tim22 和 mtHsp70 依赖性方式进入线粒体以诱导细胞凋亡。细胞死亡差异24(4):747-758。         


4. Durigon, R.、Wang, Q.、Ceh Pavia, E.、Grant, CM 和 Lu, H. (2012)。胞质硫氧还蛋白系统促进线粒体小 Tim 蛋白的导入。EMBO 代表13(10):916-922。         


5. Endo, T. 和 Tamura, Y. (2018)。线粒体膜间空间中的航天飞机任务。EMBO J 37(4)。         


6. Habich, M., Salscheider, SL, Murschall, LM, Hoehne, MN, Fischer, M., Schorn, F., Petrungaro, C., Ali, M., Erdogan, AJ, Abou-Eid, S., Kashkar, H.、Dengjel, J. 和 Riemer, J. (2019 a )。通过亚稳态二硫键连接复合物进行载体导入允许进行质量控制步骤并通过线粒体二硫键中继进行导入。细胞代表26(3):759-774 e755。                       


7. Habich, M.、Salscheider, SL 和 Riemer, J. (2019 b )。线粒体膜间隙蛋白中的半胱氨酸残基:不仅仅是进口。Br J Pharmacol 176(4): 514-531。         


8. Hansen, KG 和 Herrmann, JMJT pj (2019)。将蛋白质转运到线粒体中。  38(3):330-342。                       


9. Hartl, FU, Pfanner, N., Nicholson, DW 和 Neupert, W. (1989)。线粒体蛋白导入。Biochim Biophys Acta 988(1): 1-45。                       


10. MacPherson, L. 和 Tokatlidis, K.(2017 年)。线粒体膜间隙中的蛋白质运输:机制和与人类疾病的联系。生物化学杂志474(15):2533-2545。     


11. MacVicar, T., Ohba, Y., Nolte, H., Mayer, FC, Tatsuta, T., Sprenger, HG, Lindner, B., Zhao, Y., Li, J., Bruns, C., Kruger, M., Habich, M., Riemer, J., Schwarzer, R., Pasparakis, M., Henschke, S., Bruning, JC, Zamboni, N. 和 Langer, T. (2019)。脂质信号通过 YME1L 驱动线粒体的蛋白水解重新布线。自然575(7782):361-365。                   


12. Mokranjac, D. 和 Neupert, W. (2007)。蛋白质导入分离的线粒体。线粒体,施普林格:277-286。     


13. Murschall, LM, Gerhards, A., MacVicar, T., Peker, E., Hasberg, L., Wawra, S., Langer, T. 和 Riemer, J. (2020)。氧化还原酶 MIA40 的 C 端区域在线粒体输入期间稳定其胞质前体。BMC 生物学18(1):96。                   


14. Pfanner, N.、Warscheid, B. 和 Wiedemann, NJ(2019 年)。线粒体蛋白质组织:从生物发生到网络和功能。Nat Rev Mol Cell Bio l 20(5): 267。                   


15. Richter, F., Dennerlein, S., Nikolov, M., Jans, DC, Naumenko, N., Aich, A., MacVicar, T., Linden, A., Jakobs, S., Urlaub, H. , Langer, T. 和 Rehling, P. (2019)。ROMO1 是 YME1L 蛋白酶导入所需的人类前序列转位酶的组成部分。J Cell Biol 218(2):598-614。     


16. Saita, S., Tatsuta, T., Lampe, PA, Konig, T., Ohba, Y. 和 Langer, T. (2018)。PARL 在细胞质和线粒体之间分配脂质转移蛋白 STARD7。EMBO J 37(4)。     


17. Schmidt, O.、Pfanner, N. 和 Meisinger, C. (2010)。线粒体蛋白质导入:从蛋白质组学到功能机制。Nat Rev Mol Cell Biol 11(9): 655-667。     


18. Spinelli, JB 和 Haigis, MC (2018)。线粒体对细胞代谢的多方面贡献。Nat Cell Biol 20(7): 745-754。     


19. Tang, BL 和 Tang (2015)。膜运输:第二版。斯普林格。     


20. Vafai, SB 和 Mootha, VK (2012)。线粒体疾病作为进入古老细胞器的窗口。自然491(7424):374-383     


21. Weckbecker, D. 和 Herrmann, JM (2013)。研究酵母线粒体中膜蛋白生物发生的方法。方法 Mol Biol 1033:307-322。     
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引用:Murschall, L. M., Peker, E., MacVicar, T., Langer, T. and Riemer, J. (2021). Protein Import Assay into Mitochondria Isolated from Human Cells. Bio-protocol 11(12): e4057. DOI: 10.21769/BioProtoc.4057.
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