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Nov 2020
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Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
用转位酶Oxa1L纯化HEK细胞线粒体核糖体   

Hanting YangHanting Yang* Nirupa DesaiNirupa Desai* (*共同第一作者)
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Abstract

Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells have been developed. However, the fast purification of mitoribosomes anchored to the mitochondrial inner membrane in complex with the Oxa1L translocase remains particularly challenging. Herein, we present a protocol recently developed and modified in our lab that provides details for the efficient isolation of intact mitoribosomes with its translocase Oxa1L. We combined the cell culture of PDE12-/- or wild-type HEK293 cell lines with the isolation of mitochondria and the purification steps used for the biochemical and structural studies of mitoribosomes and Oxa1L.


Graphic abstract:



Schematic procedure for the purification of mitoribosomes from HEK cells. The protocol described herein includes two main sections: 1) isolation of mitochondria from HEK cells; and 2) purification of mitoribosome-Oxa1L from mitochondria. RB: Resuspension Buffer (see Recipes) (Created with BioRender.com).


Keywords: Mitochondria (线粒体), Ribosome purification (核糖体净化), Mitoribosome (核糖体), Oxa1L (Oxa1L), Biochemistry (生物化学), Cryo-EM (Cryo-EM)

Background

Human mitochondria possess their own genome and house all the components necessary for transcription, RNA maturation, and translation of the encoded genetic information (Dennerlein et al., 2017). The mitoribosome, one of the most important machineries within mitochondria, is responsible for the translation of essential mitochondrial mRNAs encoding components of oxidative phosphorylation (OXPHOS) complexes (Ott et al., 2016). Since all proteins translated by the human mitoribosome are membrane proteins, knowledge of the mechanism of co-translational membrane protein insertion is central to understanding mitoribosome translation (Hildenbeutel et al., 2012; Stiller et al., 2016). In human mitochondria, the oxidase assembly 1-like (Oxa1L) translocase plays a central role in the membrane insertion of mitochondrially encoded products (Haque et al., 2010; Stiller et al., 2016); however, due to the unique features of mitochondria, little is known regarding the molecular and quality control mechanisms underlying mitochondrial translation.


Owing to the structures obtained by single-particle electron cryo-microscopy (cryo-EM), opportunities now arise to comprehensively study the molecular mechanisms underlying the translational action and quality control of the human mitoribosome (Rathore, 2020). Although the existence of quality control in mitochondria has been predicted based on analogy with bacterial and eukaryotic cytosols (Ayyub et al., 2020), biochemical or structural evidence of ribosome-associated quality control in mitochondria remain elusive. We reasoned that any attempt to induce translational stalling may trap mitoribosomes in various stages of the translation cycle during the act of nascent chain insertion into the inner mitochondrial membrane, and generate intermediates suitable for structural analysis by cryo-EM. To this end, we purified mitoribosomes from a genetically engineered human cell line lacking the 2’-5’ phosphodiesterase exonuclease 12 (PDE12). PDE12 facilitates the maturation of mt-tRNA; therefore, a PDE12 knockout can lead to aberrant polyadenylation of the 3’ ends of mt-tRNAs and, consequently, mitoribosome stalling (Rorbach et al., 2011; Pearce et al., 2017). As expected, we observed a substantial proportion of stalled mitoribosomes and discovered a mitoribosome-associated quality control pathway (mtRQC) to rescue the elongational stalling ribosomes (Desai et al., 2020). Our report of the first mitoribosome-associated quality control pathway provides novel insights into the regulation of mitochondrial translation. Since the cell needs to detect and respond rapidly even to subtle changes in translation rates, quality control is intimately coupled to elongation. During purification, we included GMPPCP, a non-hydrolysable GTP analog, to prevent dissociation of GTPases from mitoribosomes, and successfully captured five additional structures of virtually every elongating ribosomal state. Purifying the mitoribosome in complex with its translocase Oxa1L was challenging since membrane proteins are unstable and tend to dissociate during complicated isolation steps. Accordingly, we added n-dodecyl β-D-maltoside (β-DDM) along with cardiolipin to solubilize mitoribosomes anchored to the mitochondrial inner membrane via the Oxa1L translocase. We also trapped Oxa1L on all our active and stalled ribosomes, giving us a first glimpse at the co-translational insertion of mitochondrial inner membrane proteins.


This protocol was modified from prior methods developed in our lab (Amunts et al., 2015; Brown et al., 2017) and by others (Greber et al., 2014 and 2015; Aibara et al., 2018), as well as in our current work (Desai et al., 2020). The method described here can be used to obtain mitoribosomes in complex with Oxa1L, which has advantages such as the applicability for mitochondria isolation from large volumes of suspension cell culture, the use of common media and buffers, ease of handling, and absence of additional affinity purification steps. In our previous publication and experiments, we observed a good reproducibility between HEK293 cell batches using this methodology.

Materials and Reagents

  1. T75/T175 flasks (Thermo ScientificTM)

  2. 1 L/2 L Tissue Culture-treated roller bottles (CorningTM)

  3. 50 ml/250 ml/5 L polycarbonate Erlenmeyer flasks (CorningTM)

  4. Teflon/glass Dounce homogenizers (Cambridge Glassblowing Limited)

  5. Miracloth (Merck Millipore Calbiochem, catalog number: 475855)

  6. SW40 tubes for mitochondria gradient (Beckman, catalog number: 344060)

  7. Polycarbonate bottle with cap assembly for type 70Ti rotor, for cushion (Beckman, catalog number: 355618)

  8. TLS-55 tubes for mitoribosome gradient (Beckman, catalog number: 347356)

  9. Dulbecco’s Modified Eagle Medium (GibcoTM, Life technologies)

  10. Tetracycline-free fetal bovine serum (GibcoTM, Life technologies)

  11. Freestyle 293 Expression medium (GibcoTM, Life technologies)

  12. Dnase (RNA-free) (HT Biotechnology, N401a)

  13. n-Dodecyl β-D-maltoside (Anatrace, catalog number: D310)

  14. 18:1 Cardiolipin (TOCL; Avanti, catalog number: 710335C)

  15. β, γ-Methyleneguanosine 5′-triphosphate sodium salt (GMPPCP; Sigma, catalog number: M3509)

  16. Protease inhibitor (cOmpleteTM, Roche)

  17. Rabbit anti-mS27 (Abcam, catalog number: ab67157)

  18. Mouse anti-OXA1L (Abcam, catalog number: ab88975)

  19. Goat anti-mouse Alexa Fluor Plus 800 (ThermoFisher, catalog number: A32730)

  20. Goat anti-rabbit Alexa Fluor Plus 680 (ThermoFisher, catalog number: A32734)

  21. MIB Buffer (Mitochondria isolation buffer, see Recipes)

  22. SM4 Buffer (Sucrose/mannitol buffer, see Recipes)

  23. MIBSM Buffer (MIB+SM4: Experimental buffer, see Recipes)

  24. SEM Buffer (Sucrose EDTA MOPS buffer, see Recipes)

  25. 60%, 32%, 23%, and 15% Sucrose Gradient Buffer (see Recipes)

  26. Lysis Buffer (see Recipes)

  27. Sucrose Cushion Buffer (see Recipes)

  28. Resuspension Buffer (see Recipes)

  29. 15-30% Linear Sucrose Gradient Buffer (see Recipes)

Equipment

  1. Automated cell counter (Countess® II FL)

  2. Avanti-J26 XP centrifuge (Beckman Coulter)

  3. High speed centrifuge (Beckman Coulter, model: Avanti J-26XP)

  4. Ultracentrifuge (Beckman Coulter, model: Optima L-80 XP)

  5. Tabletop ultracentrifuge (Beckman Coulter, model: OptimaTM MAX)

  6. Centrifuge (Eppendorf, model: 5430 R)

  7. NanoDrop® 2000 (Thermo)

  8. Odyssey® CLx Infrared Imaging System (LI-COR Biosciences)

  9. Vitrobot Mark IV (Thermo Fisher Scientific)

  10. Titan Krios microscope (Thermo Fisher Scientific FEI)

Procedure

  1. Cell Culture

    1. Maintain PDE12-/- HEK293 cells in T75 flasks in DMEM supplemented with 10% FBS, at 37°C and 5% CO2. Cells should be ready for transfer to a T175 flask in ~2-3 days.

    2. Scale up by splitting every 2-3 days until the target cell mass is reached. At least 8 flasks with 30 ml cells per flask is recommended as a starting point for adapting to suspension culture.

    3. At 90% confluence, detach cells with 0.05% trypsin-EDTA for 10-15 min at 37°C, and monitor the cell rounding. Centrifuge at 200 × g to remove trypsin, then resuspend the pelleted cells 2-3 times in 10-20 ml Freestyle medium with 1% FBS to wash out old media.

    4. Resuspend the detached cells in 30-50 ml Freestyle media with 1% FBS, adjust cell density with media to around 0.5 × 106 cells/ml, and grow at 37°C, 8% CO2, 120 × g.

    5. Scale up by diluting and splitting with pre-warmed fresh Freestyle media with 1% FBS. Count cells using an automated cell counter and adjust cell concentration to 0.5 × 106 cells/ml.

    6. Observe and count the cells every other day, and proceed to split/dilute the cells if the cell density is above 1.5 × 106 cells/ml.

    7. The final cell density can range from 0.8 to 1.5 × 106 cells/ml, and the isolation of mitochondria requires at least 10 L as a starting point (Please see Notes for starting volume of wild-type).

    8. Harvest cells by centrifugation at 1,000 × g for 7 min at 4°C. Decant the supernatant carefully and resuspend the pelleted cells quickly in pre-cooled PBS to wash out the media.

    9. Centrifuge the resuspended cells at 1,200 × g for 10 min at 4°C. Decant the supernatant carefully and weigh the pellet. Continue to isolate the mitochondria from harvested cells, or snap-freeze the cells in liquid nitrogen and store at -80°C.


  2. Mitochondria isolation

    1. Pre-cool the Teflon/glass Dounce homogenizer O/N prior to use.

    2. Resuspend the pellet (e.g., 10 g) in 60 ml MIB buffer.

    3. Allow cells to swell by gently stirring in a 4°C cold room for 15 min.

    4. Add 20 ml SM4 buffer (1/3 of MIB buffer volume).

    5. Dounce homogenize the samples (> 60 up-and-down passes).

    6. Centrifuge the homogenized sample at 800 × g for 15 min at 4°C. Collect supernatant (Supernatant-1) through a Miracloth (or other cheese cloth with a typical pore size of ~22-25 µm). Keep the pellet.

    7. Resuspend the pellet in 20 ml MIBSM (3:1 MIB:SM) buffer, homogenize manually (~15 up-and-down passes), then centrifuge at 4°C, 800 × g for 15 min.

    8. Collect supernatant (Supernantant-2) through a Miracloth and combine with Supernatant-1 from B-6. Centrifuge at 1,000 × g for 15 min at 4°C. Collect supernatant.

    9. Centrifuge the supernatant from the previous step at 10,000 × g for 15 min at 4°C.

    10. Keep pellet, but carefully wash out loose pellet without disturbing the tight portion.

    11. Resuspend in 10 ml MIBSM buffer, add 100 U RNase-free DNase I per 10 g cells, rotate on a roller in a cold room for 20 min.

    12. Centrifuge at 10,000 × g for 15 min at 4°C.

    13. Resuspend the pellet in ~2 ml SEM buffer and homogenize gently with a small Dounce homogenizer. Perform no more than five up-and-down passes.

    14. Prepare a 15-23-32-60% sucrose gradient in SW40 tubes. Place 1.5 ml of the 60% sucrose stock buffer into the Beckman Ultra-Clear centrifuge tube. Carefully overlay 4.5 ml of the 32% sucrose stock buffer. Repeat with 1.5 ml of the 23% sucrose stock buffer and then with 1.5 ml of the 15% sucrose stock buffer.

    15. Load resuspended crude mitochondrial sample from Step B13 on top of the 15% sucrose in the density gradient prepared in Step B14. Centrifuge in an SW40 rotor at 28, 000 × g for 1 h at 4°C.

    16. The intact mitochondria form a brown band at the 60%-32% sucrose interface. Carefully remove the sucrose from the top until reaching the mitochondrial band, then take the brown band with a pipette at the interface of 32% and 60% sucrose.

    17. Snap-freeze the purified mitochondria in liquid nitrogen and store at -80°C.


  3. Mitoribosome-Oxa1L purification

    1. Defrost the frozen mitochondria on ice.

    2. Add 2 volumes of lysis buffer to mitochondria (e.g., Add 8 ml lysis buffer to 4 ml mitochondria). Mix immediately by inverting the tube several times.

    3. Homogenize with a small Teflon/glass Dounce homogenizer to assist lysis.

    4. Rotate on a roller in a cold room for 20 min to complete the lysis.

    5. Centrifuge the lysed material (approximately 9 ml) at 30,000 × g for 20 min at 4°C to remove the insoluble material. Decant the supernatant carefully from the pellet and discard the pellet.

    6. Prepare the sucrose cushion in Ti70 (tube vol approx. 24 ml): underlay 7 ml 1 M (34%, w/v) Sucrose Cushion Buffer at the bottom of the tube.

    7. Layer approximately 17 ml lysed mitochondrial sample on the sucrose cushions carefully, resulting in a lysate:cushion ratio of 2.5:1.

    8. Centrifuge the sample at ~231,550 × g for 60 min at 4°C.

    9. Discard the supernatant and rinse the tubes sequentially with resuspension buffer to remove residual sucrose (without disturbing the pellet).

    10. Resuspend pellets in a total of 100 µl resuspension buffer.

    11. Measure mitoribosome absorption at A260.

    12. Load the entire sample onto a linear 15-30% sucrose gradient tube. Centrifuge in a TLS-55 rotor at 213, 626 × g for 60-90 min at 4°C.

    13. Fractionate the gradient, determine the optical density at A260, and pool the fraction corresponding to the nucleic acid peak together. The typical A260:A280 ratio of the peak is >1.6.

    14. Exchange the buffer if necessary and dilute the sample with at least 5-10 volumes of final buffer; concentration or dialysis are recommended. Calculate the final concentration using a NanoDrop and the conversion: 1 A260 = 0.1 mg/ml.

    15. Use the purified mitoribosome sample for subsequent experiments or snap-freeze in resuspension buffer and store at -80°C.

Data analysis

The mitoribosome-Oxa1L purification protocol described here employs PDE12-/- HEK293 cells, but is applicable to wild-type or other HEK293-derived cells. The high-quality purified sample enables biochemical and structural analysis, which can be applied to study human mitoribosomal translation. Representative results for structural studies are presented as figures below; these analyses are just briefly described to work as quality controls for the sample preparation in this protocol.

 Following subsequent centrifugation steps, the intact mitochondria are separated on the sucrose gradient (Figure 1); special care is needed to collect the brown band at the 60%-32% interface with minimal contamination from the surrounding buffer. Preparation of high-quality intact mitochondria can be extrapolated to purify or study other mitochondrial macromolecules.

 Mitochondria that have been successfully isolated are lysed, and mitoribosome-Oxa1L are finally purified with a sucrose cushion followed by a continuous linear 15-30% sucrose gradient. The sucrose gradient can then be fractionated, either automatically or manually, by carefully taking fractions with a pipette or punching the tube bottom with a 21 G needle and collecting the drops. Two major mitoribosomal populations are identified in the gradient: the 55S monosome and the large 39S subunit, as illustrated in Figure 2. An additional peak located closer to the bottom may appear in preparations contaminated with 80S cytoplasmic ribosomes. Oxa1L was present in our samples, as confirmed by western blotting (Figure 2B) and cryo-EM maps (Figure 3). To investigate nascent chain translocation, we performed focused classification with signal subtraction (FCwSS) on the mitoribosomal exit tunnel. For further information or other in silico processing, please see Figure S3, S12A in our published paper (Desai et al., 2020).



Figure 1. Isolation of mitochondria on a sucrose gradient. Mitochondria were separated on a discontinuous sucrose gradient. The purified mitochondria are found at the 32%/60% interface.



Figure 2. Purification of mitoribosomes with Oxa1L on a sucrose gradient. A. Absorbance at 260 nm of the sucrose gradient fractions. B. Western blotting of mitoribosomal protein mS27 and OXA1L in fractions, showing that Oxa1L co-pellets with mitoribosomes in our preparation.



Figure 3. cryo-EM micrograph, 2D classes, and 3D initial reconstruction. A. A micrograph of the mitoribosome-Oxa1L sample. B. Representative processing data (2D classes). C. 3D reconstruction of the mitoribosome-Oxa1L complex.

Notes

  1. Notes for cell culture:

    The cell viability should be maintained at >90%, and for the final culture that is harvested >95%. The cell culture procedure is specifically for PDE12-/- cells used in this manuscript.

    For wild-type cells, all steps are the same except two:

    1. Grow cells in FreeStyle media + 5% FBS in suspension culture. Split when the cell density reaches 2 × 106 cells/ml (compared with A3-4).

    2. Harvest cells at a concentration of ~3-4 × 106 cells/ml.

      Once you have 10-20 g cell pellets (from 10-15 L PDE12-/- HEK cells or 2 L wild-type HEK293 cells) as a starting point, there are no differences in the following steps for mitochondria isolation and mitoribosome purification.

  2. Notes for mitochondria isolation:

    1. It is important to work quickly and keep everything on ice throughout the procedure.

    2. Besides a Dounce homogenizer in Step B5; alternatively, you could use nitrogen cavitation at a pressure of 500 psi for 20 min (Aibara et al., 2018). Pre-cool the nitrogen cavitation chamber O/N at 4°C.

  3. Notes for mitoribosome-Oxa purification:

    1. You can always start with a smaller volume of cells and subsequently prepare a smaller volume of buffers to keep the ratio the same.

    2. The small sucrose gradient with the TLS-55 rotor in this protocol allows for the rapid purification of mitoribosome-Oxa1L; however, there is normally an overlap between 55S monosome and 39S large subunit peaks. Separation of the 55S and 39S populations is not necessary for cryo-EM studies, given that further processing will be performed. If a better separation is needed in other situations, larger tubes (e.g., SW40) and a longer running time (e.g., 16 h) are recommended.

    3. In our dataset, among all the mitoribosome samples, the proportion of complexes of mitoribosome with Oxa1L is around 12%.

Recipes

Buffers for mitochondria isolation

  1. Mitochondrial isolation buffer (MIB)

    50 mM HEPES-KOH, pH 7.4

    10 mM KCl

    1.5 mM MgCl2

    1 mM EDTA

    1 mM EGTA

    1 mM DTT

    Protease inhibitors (1 tablet/50 ml)

  2. Sucrose/mannitol buffer (SM4)

    280 mM sucrose

    840 mM mannitol

    50 mM HEPES-KOH, pH 7.5

    10 mM KCl

    1.5 mM MgCl2

    1 mM EDTA

    1 mM EGTA

    1m M DTT

    Protease inhibitors (1 tablet/50 ml)

  3. Experimental buffer (MIBSM)

    3 volumes MIB mix with 1 volume SM4 (e.g., 120 ml MIB buffer + 40 ml SM4 buffer)

  4. Sucrose-EDTA MOPS (SEM) buffer

    250 mM sucrose

    1 mM EDTA

    20 mM MOPS-KOH (alternative: HEPES-KOH, pH 7.4)

  5. Sucrose gradient (SG) buffer

    20 mM HEPES-KOH, pH 7.4

    1 mM EDTA and 60%/32%/23% and 15% sucrose, respectively (make separate stock solutions of 4 different sucrose concentrations for the preparation of the stepwise sucrose gradient).


Buffers for mitoribosome purification

  1. Lysis buffer

    25 mM HEPES-KOH, pH 7.4

    150 mM KCl

    50 mM MgOAc

    1.5% β-DDM

    0.15 mg/ml Cardiolipin

    500 mM GMPPCP

    2 M DTT

    Protease inhibitors (1 tablet/50 ml)

  2. Sucrose cushion (SC)

    1 M sucrose (34% w/v)

    20 mM HEPES-KOH, pH 7.4

    100 mM KCl

    20 mM MgOAc

    0.6% β-DDM

    0.06 mg/ml Cardiolipin

    250 mM GMPPCP

    2 mM DTT

  3. Resuspension buffer (RB)

    20 mM HEPES-KOH, pH 7.4

    100 mM KCl

    5 mM MgOAc

    0.15% β-DDM

    0.015 mg/ml Cardiolipin

    250 mM GMPPCP

    2 mM DTT

  4. 15-30% linear sucrose gradients

    15-30% linear sucrose in 20 mM HEPES-KOH, pH 7.4

    100 mM KCl

    5 mM MgOAc

    0.05% β-DDM

    0.005 mg/ml Cardiolipin

    250 mM GMPPCP

    2 mM DTT

Acknowledgments

This work received funding from the following sources: UK Medical Research Council (MC_U105184332); Wellcome Trust Senior Investigator award (WT096570); Agouron Institute; and the Louis-Jeantet Foundation. H.Y. is funded by an EMBO Long-Term Fellowship (EMBO ALTF 806-2018), and N.D. is funded by a Wellcome Trust Clinical PhD Fellowship (110301/Z/15/Z). This protocol was taken from Desai et al. (2020) with minor modifications.

Competing interests

The authors declare no competing financial or non-financial interests.

References

  1. Aibara, S., Andrell, J., Singh, V. and Amunts, A. (2018). Rapid Isolation of the Mitoribosome from HEK Cells. J Vis Exp(140).
  2. Amunts, A., Brown, A., Toots, J., Scheres, S. H. W. and Ramakrishnan, V. (2015). Ribosome. The structure of the human mitochondrial ribosome. Science 348(6230): 95-98.
  3. Ayyub, S. A., Gao, F., Lightowlers, R. N. and Chrzanowska-Lightowlers, Z. M. (2020). Rescuing stalled mammalian mitoribosomes - what can we learn from bacteria? J Cell Sci 133(1).
  4. Brown, A., Rathore, S., Kimanius, D., Aibara, S., Bai, X. C., Rorbach, J., Amunts, A. and Ramakrishnan, V. (2017). Structures of the human mitochondrial ribosome in native states of assembly. Nat Struct Mol Biol 24(10): 866-869.
  5. Dennerlein, S., Wang, C. and Rehling, P. (2017). Plasticity of Mitochondrial Translation. Trends Cell Biol 27(10): 712-721.
  6. Desai, N., Yang, H., Chandrasekaran, V., Kazi, R., Minczuk, M. and Ramakrishnan, V. (2020). Elongational stalling activates mitoribosome-associated quality control. Science 370(6520): 1105-1110.
  7. Greber, B. J., Boehringer, D., Leitner, A., Bieri, P., Voigts-Hoffmann, F., Erzberger, J. P., Leibundgut, M., Aebersold, R. and Ban, N. (2014). Architecture of the large subunit of the mammalian mitochondrial ribosome. Nature 505(7484): 515-519.
  8. Greber, B. J., Bieri, P., Leibundgut, M., Leitner, A., Aebersold, R., Boehringer, D. and Ban, N. (2015). Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome. Science 348(6232): 303-308.
  9. Haque, M. E., Elmore, K. B., Tripathy, A., Koc, H., Koc, E. C. and Spremulli, L. L. (2010). Properties of the C-terminal tail of human mitochondrial inner membrane protein Oxa1L and its interactions with mammalian mitochondrial ribosomes. J Biol Chem 285(36): 28353-28362.
  10. Hildenbeutel, M., Theis, M., Geier, M., Haferkamp, I., Neuhaus, H. E., Herrmann, J. M. and Ott, M. (2012). The membrane insertase Oxa1 is required for efficient import of carrier proteins into mitochondria. J Mol Biol 423(4): 590-599.
  11. Ott, M., Amunts, A. and Brown, A. (2016). Organization and Regulation of Mitochondrial Protein Synthesis. Annu Rev Biochem 85: 77-101.
  12. Pearce, S. F., Rorbach, J., Van Haute, L., D'Souza, A. R., Rebelo-Guiomar, P., Powell, C. A., Brierley, I., Firth, A. E. and Minczuk, M. (2017). Maturation of selected human mitochondrial tRNAs requires deadenylation. Elife 6: e27596.
  13. Rathore, S. (2020). Structural characterisation of mitochondrial macromolecular complexes using cryo-EM: Mitoribosome biogenesis and respiratory chain supercomplex Doctoral dissertation. Stockholm University.
  14. Rorbach, J., Nicholls, T. J. and Minczuk, M. (2011). PDE12 removes mitochondrial RNA poly(A) tails and controls translation in human mitochondria. Nucleic Acids Res 39(17): 7750-7763.
  15. Stiller, S. B., Hopker, J., Oeljeklaus, S., Schutze, C., Schrempp, S. G., Vent-Schmidt, J., Horvath, S. E., Frazier, A. E., Gebert, N., van der Laan, M., Bohnert, M., Warscheid, B., Pfanner, N. and Wiedemann, N. (2016). Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins. Cell Metab 23(5): 901-908.

简介

[摘要]线粒体核糖体(mitoribosomes)在线粒体内进行蛋白质合成,线粒体是真核细胞中负责能量转换和三磷酸腺苷(ATP)产生的细胞器。为了研究它们的功能和结构,已经开发出从富含线粒体的动物组织或 HEK 细胞中大规模纯化完整的线粒体。然而,锚定在线粒体内膜上与 Oxa1L 转位酶复合的线粒体糖体的快速纯化仍然特别具有挑战性。在此,我们提出最近开发并在我们的实验室修改的协议,这provid ES 使用其转位酶 Oxa1L 有效分离完整线粒体糖体的详细信息。我们将PDE12 -/-或野生型 HEK293 细胞系的细胞培养与线粒体的分离以及用于线粒体和 Oxa1L 的生化和结构研究的纯化步骤相结合。


图文摘要:


示意性过程的mitoribosome的纯化š从HEK细胞。该协议在此描述includ ES 2个主要部分:1)mitocho的隔离从HEK细胞ndria; 和2) 从线粒体中纯化mitoribosome-Oxa1L。RB:重悬缓冲液(参见配方)(由BioRender.com创建)。


[背景]人类线粒体拥有自己的基因组中并且房子所有的必需的转录,RNA成熟部件,和的编码遗传信息翻译(DENNERLEIN等人,2017)。第m itoribosome,线粒体内的最重要的机械之一,是负责必不可少线粒体mRNAs的翻译连接的编码部件氧化磷酸化(OXPHOS)配合物(奥特等人,2016)。由于人类线粒体翻译的所有蛋白质都是膜蛋白,因此共翻译膜蛋白插入机制的知识对于理解线粒体翻译至关重要(Hildenbeutel等人,2012 年;Stiller等人,2016 年)。在人类线粒体中,氧化酶组装体 1 - like (Oxa1L) 转位酶在线粒体编码产物的膜插入中起核心作用(Haque等人,2010 年;Stiller等人,2016 年);ħ H但是,由于线粒体的独特功能,小我已探明的关于分子和质量控制机制小号底层线粒体翻译。

由于由单一的得到的结构-粒子电子低温显微术(冷冻电镜),机会现在出现全面研究的基础的翻译的分子机制人人类mitoribosome的动作和质量控制(拉索,2020)。虽然质量控制的线粒体中的存在ħ如已经基于与细菌类比预测升和真核胞液(阿尤布等人,2020),在线粒体的核糖体相关联的质量控制的生化或结构证据仍然是难以捉摸的。我们推断,任何企图诱导平移失速米AY在翻译周期的各个阶段陷阱mitoribosomes期间新生链插入行为变成线粒体内膜,并产生适合于结构analys中间体我通过Cryo-EM秒。为此,瓦特从基因工程人细胞线e纯化mitoribosomes缺乏荷兰国际集团的2'-5'核酸外切酶磷酸二酯酶12(PDE12)。PDE12 促进 mt-tRNA 的成熟;因此,一个PDE12敲除可导致MT-tRNA的3'末端的多腺苷酸化异常和,因此,mitoribosome失速(Rorbach等人,2011 ;皮尔斯。等人,2017)。正如预期的那样,我们观察到大量停滞的线粒体糖体,并发现了线粒体相关的质量控制途径 (mtRQC) 来挽救伸长的停滞核糖体(Desai等,2020)。我们关于第一个线粒体相关质量控制途径的报告为线粒体翻译的调节提供了新的见解。由于细胞需要快速检测并快速响应翻译速率的细微变化,因此质量控制与延伸率密切相关。在纯化过程中,我们加入了 GMPPCP,一种不可水解的 GTP 类似物,以防止 GTPases 从 mitoribosomes 解离,并成功捕获了几乎每个伸长核糖体状态的五个额外结构。净化的mitoribosome在复杂的转位其OXA1L是具有挑战性的,因为膜蛋白是不稳定的,容易分解过程中复杂的隔离措施。因此,我们添加了 正d odecylβ-d麦芽糖苷(β-DDM)心磷脂溶解经由OXA1L移位酶锚定到线粒体内膜mitoribosomes沿。我们还被困OXA1L我们所有的活跃和停滞的核糖体,给我们的第一一瞥在在线粒体内膜蛋白共翻译插入。

该协议是由我们实验室开发的现有方法修改(Amunts等人,2015;布朗等人,2017)和由他人(Greber等人,2014和2015; Aibara 。等人,2018),以及在我们目前的工作中(Desai等人,2020 年)。该方法这里描述的可用于获得在复杂mitoribosomes与OXA1L,其具有的优点,如可应用用于从大体积的细胞悬浮培养,使用普通的媒体和缓冲液,便于线粒体隔离的HANDL ING ,和不存在的附加亲和纯化步骤。在我们之前的出版物和实验中,我们使用这种方法观察到 HEK293 细胞批次之间的良好重现性。

关键字:线粒体, 核糖体净化, 核糖体, Oxa1L, 生物化学, Cryo-EM

材料和试剂
 
T75 / T175 ˚F lasks物(Thermo Scientific TM )
1 L / 2升组织培养-t reated ř奥勒b ottles(康宁TM )
50米升/250米升/5μL p olycarbonate锥形˚F lasks(康宁TM )
Teflon/玻璃 Dounce 均质器(Cambridge Glassblowing Limited)
Miracloth(Merck Millipore Calbiochem ,目录号:475855)
用于线粒体梯度的 SW40 管(Beckman,目录号:344060)
聚碳酸酯b ottle与Ç AP一个ssembly用于吨YPE 70Ti转子,用于缓冲(贝克曼,目录号:355618)
用于线粒体梯度的 TLS-55 管(Beckman,目录号:347356)
Dulbecco 改良 Eagle 培养基(Gibco TM ,Life Technologies)
不含四环素的胎牛血清(Gibco TM ,Life Technologies)
Freestyle 293 表达培养基(Gibco TM ,Life Technologies)
DNase(无 RNA)(HT Biotechnology,N401a)
正十二烷基β-D-麦芽糖苷(Anatrace ,目录号:D310)
18:1 心磷脂(TOCL ; Avanti ,目录号:710335C)
β ,γ-亚甲基鸟苷 5'-三磷酸钠盐(GMPPCP ;Sigma ,目录号:M3509)
P rotease抑制剂(完全TM ,Roche)的
R abbit anti-mS27(Abcam ,目录号:ab67157 )
中号乌斯抗OXA1L(Abcam公司,目录号:ab88975 )
山羊抗小鼠Alexa Fluor Plus 800(ThermoFisher ,目录号:A32730 )
山羊抗兔Alexa Fluor Plus 680(ThermoFisher ,目录号:A32734 )
MIB 缓冲液(线粒体分离缓冲液,参见配方)
SM4 缓冲液(蔗糖/甘露醇缓冲液,参见配方)
MIBSM 缓冲液(MIB+SM4:实验缓冲液,见配方)
SEM 缓冲液(蔗糖 EDTA MOPS缓冲液,参见配方)
60%、32%、23%和15% 蔗糖梯度缓冲液(见配方)
裂解缓冲液(见配方)
蔗糖Ç ushion缓冲区(见食谱)
重悬缓冲液(见配方)
15-30%线性小号ucrose ģ radient缓冲液(见配方)
 
设备
 
自动细胞计数仪 (Countess ® II FL)
阿凡提-J26 XP离心机(贝克曼Ç oulter)
高速离心机(贝克曼库尔特,型号:Avanti J-26XP)
超速离心机(Beckman Coulter,型号:Optima L-80 XP)
桌面ù ltracentrifuge(贝克曼Ç oulter,型号:的Optima TM MAX)
离心机(Eppendorf,型号:5430 R)
纳米d ROP ® 2000(温)
Odyssey ® CLx 红外成像系统 (LI-COR Biosciences)
Vitrobot Mark IV (Thermo Fisher Scientific)
Titan Krios 显微镜(Thermo Fisher Scientific FEI)
 
程序
 
细胞培养
将 PDE12 -/- HEK293 细胞保持在T75 烧瓶中的DMEM 中,并在 37°C 和 5% CO 2下补充 10% FBS 。细胞应准备好在约 2-3 天内转移到 T175 烧瓶中。
通过拆分规模达每2-3天,直到在到达靶细胞质量。推荐至少 8 个培养瓶,每个培养瓶含有 30 ml细胞,作为适应悬浮培养的起点。
在90%confluenc ë ,分离的细胞用0.05%胰蛋白酶-EDTA 10-15分钟,在37℃ ,和监测细胞四舍五入。Ç在200 entrifuge ×克以除去胰蛋白酶,然后重新悬浮沉淀的细胞2 -3倍10〜20毫升自由式MEDI微米有1个%FBS洗出旧的介质。
重悬分离的细胞在30 - 50米升Freestyle培养基含有1%FBS,调整与媒体细胞密度大约为0.5× 10 6 个细胞/ml,并在37°C、8% CO 2 、120 × g下生长。
通过使用含有 1% FBS 的预热新鲜 Freestyle 培养基进行稀释和拆分来扩大规模。使用自动细胞计数器对细胞进行计数并将细胞浓度调整为 0.5 × 10 6细胞/ml。
每隔一天观察和计数细胞,如果细胞密度高于1.5 × 10 6 个细胞/ml ,则继续分裂/稀释细胞。
最终的细胞密度的范围可以从0.8到1.5 × 10 6个细胞/米升,和线粒体的分离需要至少10升作为起始点(请参见注释用于开始野生型的体积)。
在4°C 下以 1,000 × g离心7 分钟收获细胞。小心倒出上清液,并在预冷的 PBS 中快速重悬沉淀的细胞以洗掉培养基。
将重悬的细胞在4°C 下以 1,200 × g离心10 分钟。小心倒出上清液,并权衡PELLE牛逼。继续从收获的细胞中分离线粒体,或在液氮中快速冷冻细胞并储存在 -80°C。
 
线粒体分离
预-冷却聚四氟乙烯/玻璃Dounce匀浆O / N在使用之前。
重悬沉淀(例如,10克)在60毫升缓冲液的MIB。
在 4°C 的冷室中轻轻搅拌 15 分钟,让细胞膨胀。
添加 20 ml SM4 缓冲液(MIB 缓冲液体积的1/3 )。
Dounce 均质化样品(> 60 次上下通道)。
离心机中以800匀化样品×克为15分钟,在4℃下。收集上清液(上清液1)通过的Miracloth(或与其它干酪布一个典型孔尺寸的〜22-25微米)。保留颗粒。
重悬沉淀在20毫升MIBSM(3:1 MIB:SM)缓冲液,搅匀手动(〜15向上和向下次),然后离心在4℃下,800 ×克为15分钟。
通过 Miracloth收集上清液 ( S upernant-2) 并与来自 B-6 的 Supernatant-1 结合。离心机在1000 ×克为15分钟,在4℃下。收集上清液。
离心从上清先前以10,000步骤×克为15分钟,在4℃下。
保留颗粒,但小心地洗掉松散的颗粒,不要打扰紧的部分。
在 10 ml MIBSM 缓冲液中重悬,每 10 g 添加 100 U RNase-free DNase I 细胞,旋转对辊一冷室中静置20分钟。
离心机以10,000 ×克为15分钟,在4℃下。
将沉淀重悬在 ~2 ml SEM 缓冲液中,并用小型 Dounce 匀浆器轻轻匀浆。执行不超过五次上下传球。
在 SW40 管中准备1 5-23-32-60% 蔗糖梯度。地方加入1.5ml的60%蔗糖储备缓冲到贝克曼超清晰离心管中。小心地覆盖 4.5 ml 的 32% 蔗糖储备缓冲液。用 1.5 ml的23% 蔗糖储备缓冲液重复,然后用 1.5 ml的15% 蔗糖储备缓冲液重复。
负载从再悬浮粗线粒体试样小号TEP B13上的顶部的15%蔗糖中制备的密度梯度小号TEP B14。离心机在一个SW40转子以28,000 ×克为1个小时,在4℃下。
完整的线粒体在 60%-32% 蔗糖界面形成棕色带。小心地取下了从蔗糖的顶部,直到到达线粒体带,然后用移液管在32%和60%蔗糖的界面上取棕色带。
在液氮中快速冷冻纯化的线粒体并储存在 -80°C。
 
Mitoribosome-Oxa1L 纯化
在冰上解冻冷冻的线粒体。
向线粒体中加入2倍体积的裂解缓冲液(例如,向 4 ml 线粒体中加入 8 ml 裂解缓冲液)。混合立即被颠倒的管几倍。
均化用一个小聚四氟乙烯/玻璃的Dounce匀浆器以协助裂解。
[R上为辊otate一冷室中静置20分钟,以完成的裂解。
离心机的裂解材料(约9毫升)在30000 ×克为20分钟,在4℃下,以除去所述不溶性材料。小心地从沉淀中倒出上清液并丢弃沉淀。
制备的蔗糖垫在Ti70(管体积约24毫升):底图7毫升1 M(34%,W / V)在管的底部蔗糖垫缓冲液。
层大约17 ml裂解线粒体样品上的蔗糖垫小心,从而导致在一个裂解物:缓冲比率的2.5:1。
在 4°C 下以 ~231,550 × g离心样品60 分钟。
丢弃的上清液和冲洗的管依次用重悬缓冲液以除去残留的蔗糖(在不干扰颗粒)。
重悬粒料一个总的100μl的重悬缓冲液中。
在A 260测量 mitoribosome 吸收。
加载的整个样品到一个线性15-30%的蔗糖梯度管。离心机在一个TLS-55转子在213,626 ×克为60-90分钟,在4℃下。
对梯度进行分级,确定A 260处的光密度,并将与核酸峰对应的分数汇集在一起。峰的典型A 260 : A 280比> 1.6。
如有必要,更换缓冲液并用至少 5-10 体积的最终缓冲液稀释样品;建议浓缩或透析。计算最终的浓度使用Ñ ANO d ROP和转换:1个甲260 = 0.1毫克/毫升。
用于该纯化的mitoribosome样品后续实验ö ř管理单元-在再悬浮缓冲液和储存冷冻在-80℃。
 
数据分析
 
所述mitoribosome-OXA1L纯化方案describ编这里采用PDE12 - / - HEK293细胞,但是APPLI电缆到野生型或其它HEK293衍生的细胞。的高-质量纯化样品使生物化学和结构分析,其可以被应用到研究人类mitoribosomal翻译。结构研究的代表性结果如下图所示;这些分析都只是简要描述工作作为质量控制用于样品制备在此协议。
  在随后的离心步骤之后,完整的线粒体在蔗糖梯度上分离(图1 );需要特别注意收集 60%-32% 界面处的棕色条带,同时尽量减少周围缓冲液的污染。高质量完整线粒体的制备可用于纯化或研究其他线粒体大分子。
  线粒体已成功地分离被溶解,并mitoribosome-OXA1L最终纯化,使用一个蔗糖垫随后是连续的线性15-30%蔗糖梯度。然后可以自动或手动分离蔗糖梯度,方法是用移液管小心地取分数或用 21 G 针头冲管底部并收集液滴。两个主要mitoribosomal群体在梯度确定:在55S monosome和所述大39S亚基,如图中图2 。位于更靠近底部的额外峰可能出现在制剂铅污染ED与80S核糖体细胞质。OXA1L存在我们的样品中,通过蛋白质印迹(如确认图2乙)和冷冻电镜图(图3 )。为了研究新生的链易位,我们在核糖体出口隧道上使用信号减法 (FCwSS) 进行了重点分类。有关更多信息或其他计算机处理,请参见我们已发表论文(Desai等人,2020 年)中的图 S3、S12A 。
 
 
图1 。在蔗糖梯度上分离线粒体。在不连续的蔗糖梯度上分离线粒体。纯化的线粒体位于 32%/60% 界面。
 
 
图2 。P与OXA1L mitoribosomes对蔗糖梯度urification。A. 蔗糖梯度级分在 260 nm 处的吸光度。B.对馏分中的核糖体蛋白 mS27 和 OXA1L 进行蛋白质印迹,表明 Oxa1L在我们的制备中与核糖体s共沉淀。
 
 
图3 。冷冻电镜显微照片、2D 类别和 3D 初始重建。A.显微照片的所述mitoribosome-OXA1L样品。B. [R具有代表性处理数据(2D类)。C.三维重建的mitoribosome-OXA1L复杂。
 
笔记
 
细胞培养注意事项:
细胞活力应保持在>90%,对于收获的最终培养物 >95%。细胞培养过程是专门为PDE12 - / -在第使用的细胞是手稿。
对于野生型细胞,所有步骤都在相同的但有两个:
在 FreeStyle 培养基 + 5% FBS 悬浮培养中培养细胞。小号PLIT时的细胞密度达到2 × 10 6个细胞/ ml(用A3-4相比)。
以~3-4 × 10 6 个细胞/毫升的浓度收获细胞。
一旦你甲肝Ë 10-20克细胞沉淀(从10-15大号PDE12 - / - HEK细胞或2L的野生-类型的HEK293细胞)作为起始点,存在有在下面的步骤没有差异为线粒体分离和纯化mitoribosome .
线粒体分离注意事项:
在整个过程中快速工作并保持一切都在冰上很重要。
除了一个d盎司搅匀ř在小号TEP B5 ; 或者,您可以在500 psi的压力下使用氮气空化20 分钟(Aibara等人,2018 年)。P重新冷却氮气穴室O / N在4 ℃下。
mitoribosome- O xa 纯化注意事项:
你Ç一个总是开始一个细胞的更小的体积和随后的LY准备一个缓冲器的更小的体积,以柯Ë P上的比率是相同的。
该协议中TLS-55转子的小蔗糖梯度允许快速纯化 mitoribosome-Oxa1L ;然而,55S 单体和 39S 大亚基峰之间通常存在重叠。在55S和39S群体的分离是没有必要的冷冻电镜研究,给出个在进一步的处理将被执行。如果需要在其它情况更好地分离小号,较大的管(例如,SW40)和一个较长的运行时间(例如,16小时)的建议。
在我们的数据集中,在所有 mitoribosome 样本s 中,mitoribosome 与 Oxa1L的复合物es的比例约为 12%。
 
食谱
 
线粒体分离缓冲液
1.线粒体分离缓冲液 (MIB)      
50 mM HEPES-KOH,pH 7.4
10 毫米氯化钾
1.5 毫米氯化镁2
1 mM EDTA
1 mM EGTA
1 毫米 DTT
P rotease抑制剂(1片/ 50ml)中
2.蔗糖/甘露醇缓冲液 (SM4)      
280 mM 蔗糖
840 mM 甘露醇
50 mM HEPES-KOH,pH 7.5
10 毫米氯化钾
1.5 毫米氯化镁2
1 mM EDTA
1 mM EGTA
1 米 M DTT
P rotease抑制剂(1片/ 50ml)中
3.实验缓冲液(MIBSM)      
3体积小号MIB混合物用1倍体积SM4(例如,120毫升MIB缓冲液+40毫升SM4缓冲液)
4.蔗糖-EDTA MOPS (SEM) 缓冲液      
250 mM 蔗糖
1 mM EDTA
20 mM MOPS-KOH(替代:HEPES-KOH,pH 7.4)
5.蔗糖梯度 (SG) 缓冲液      
20 mM HEPES-KOH,pH 7.4
1mM EDTA和60%/ 32%/ 23%和15%的蔗糖,分别(使分离的储备溶液的4个不同的蔗糖浓度小号用于该逐步蔗糖梯度的制备)。
 
用于线粒体纯化的缓冲液
裂解缓冲液
25 mM HEPES-KOH ,pH 7.4
150 毫米氯化钾
50 mM MgOAc
1.5% β -DDM
0.15毫克/毫升心磷脂
500 mM GMPPCP
2 M 数字地面电视
P rotease抑制剂(1片/ 50ml)中
蔗糖垫 (SC)
1 M 蔗糖(34% w/v)
20 mM HEPES-KOH ,pH 7.4
100 毫米氯化钾
20 mM MgOAc
0.6% β -DDM
0.06 毫克/毫升心磷脂
250 mM GMPPCP
2 毫米 DTT
重悬缓冲液 (RB)
20 mM HEPES-KOH,pH 7.4
100 毫米氯化钾
5 mM MgOAc
0.15% β -DDM
0.015 毫克/毫升心磷脂
250 mM GMPPCP
2 毫米 DTT
15-30% 线性蔗糖梯度
15-30%线性小号在20mM HEPES-KOH ucrose,pH 7.4中
100 毫米氯化钾
5 mM MgOAc
0.05% β -DDM
0.005 毫克/毫升心磷脂
250 mM GMPPCP
2 毫米 DTT
 
致谢
 
这项工作获得了以下来源的资助:英国医学研究委员会 (MC_U105184332);威康信托高级研究员奖 (WT096570);阿古隆研究所;和路易斯-让泰基金会。HY由EMBO资助长期研究金(EMBO ALTF 806-2018) ,和N.D.由Wellcome基金会临床研究金博士(110301 / Z / 15 / Z)的资助。该协议取自Desai等人。( 2020 )稍作修改。
 
利益争夺
 
作者声明没有相互竞争的财务或非财务利益。
 
参考
 
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yang, H. and Desai, N. (2021). Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells. Bio-protocol 11(15): e4110. DOI: 10.21769/BioProtoc.4110.
  2. Desai, N., Yang, H., Chandrasekaran, V., Kazi, R., Minczuk, M. and Ramakrishnan, V. (2020). Elongational stalling activates mitoribosome-associated quality control. Science 370(6520): 1105-1110.
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