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Biochimica

用完全纯化的组分重构Msp1提取活性

Published: August 10, 2021
doi:
10.3791/62928
,
1Department of Chemistry & Biochemistry,University of Toledo

Summary

在这里，我们提出了一个详细的方案，用于在定义的蛋白脂质体中用完全纯化的组分重建Msp1提取活性。

Abstract

作为氧化磷酸化和凋亡调节的中心，线粒体在人体健康中起着至关重要的作用。适当的线粒体功能取决于强大的质量控制系统来维持蛋白质稳态（蛋白质稳态）。线粒体蛋白质平衡的下降与癌症，衰老，神经变性和许多其他疾病有关。Msp1是一种锚定在外线粒体膜中的AAA + ATP酶，通过去除错位的尾锚定蛋白来维持蛋白质平衡。使用重组成蛋白脂质体的纯化组分，我们已经证明Msp1是必要且足以从脂质双层中提取模型尾锚定蛋白的。我们简化的重组系统克服了阻碍膜蛋白提取详细研究的几个技术障碍。在这里，我们提供了脂质体生成，膜蛋白重建和Msp1提取测定的详细方法。

Introduction

适当的细胞功能取决于称为蛋白质稳态的过程，该过程确保功能蛋白处于正确的浓度和细胞位置1。蛋白质稳态的失败导致细胞器功能受损，并与许多神经退行性疾病2，3，4有关。膜蛋白对蛋白质稳态网络提出了独特的挑战，因为它们必须靶向正确的膜，同时避免疏水性跨膜结构域（TMDs）聚集5。因此，专门的机器已经发展到可以保护疏水性TMD免受细胞质基质的影响，并促进靶向和插入适当的细胞膜6，7，8，9，10，11，12，13，14，15。

线粒体是细胞的代谢中心，参与许多必要的细胞过程，例如：氧化磷酸化，铁硫簇生成和凋亡调节16，17。这些内共生细胞器包含两个膜，称为内线粒体膜（IMM）和外线粒体膜（OMM）。超过99%的1，500种人类线粒体蛋白编码在核基因组中，需要在一个或两个不同的膜上易位18，19。因此，正确的线粒体功能取决于强大的蛋白质稳态网络来纠正蛋白质靶向或易位中的任何错误。

我们的实验室专注于称为尾锚（TA）蛋白的线粒体膜蛋白的子集，它们在C端20，21，22，23，24处具有单个跨膜结构域。TA蛋白参与许多基本过程，例如细胞凋亡，囊泡转运和蛋白质易位25。TA蛋白的独特拓扑需要翻译后插入，其发生在内质网（ER）中，通过尾锚（GET）或内质网膜蛋白复合物（EMC）途径的引导进入或通过表征不良的途径进入OMM 20，26，27，28。TMD的生物物理特性是必要且足以将TA蛋白引导到正确的膜29。对生物物理特征而不是定义的序列基序的识别限制了靶向途径的保真度5。因此，TA蛋白的错位是蛋白质稳态网络的常见压力。细胞应激，例如GET途径的抑制，导致OMM和线粒体功能障碍的蛋白质错位增加，除非这些蛋白质被迅速去除30，31。

膜蛋白平衡的一个共同主题是使用AAA+（ATP酶A与细胞Activities结合）蛋白从脂质双层中去除旧的，受损的或错位的蛋白质1，32，33，34，35，36，37，38 .AAA +蛋白是形成六聚环并经历ATP依赖性运动以重塑底物的分子马达，通常通过狭窄的轴向孔39，40易位。尽管在研究AAA+ATPases提取膜蛋白方面投入了大量精力，但重构是复杂的或涉及脂质和洗涤剂41，42的混合物，这限制了从脂质双层中提取底物的机制的实验能力。

Msp1是一种高度保守的AAA + ATP酶，锚定在OMM和过氧化物酶体中，通过去除错位的TA蛋白43，44，45，46，47在膜蛋白平衡中起关键作用。最近还显示，Msp1通过去除在OMM48易位期间停滞的膜蛋白来缓解线粒体蛋白导入应激。失去Msp1或人类同源物ATAD1导致线粒体碎片化，氧化磷酸化失败，癫痫发作，中风后损伤增加和早期死亡31，49，50，51，52，53，54，55，56。

我们已经证明，有可能与Msp1共同重建TA蛋白并检测从脂质双层57中提取。这个简化的系统使用完全纯化的蛋白质重组成定义的脂质体，模仿OMM（图1）58，59。这种水平的实验控制可以解决底物提取的详细机制问题，这些问题在实验上难以解决，涉及其他AAA +蛋白的更复杂的重构。在这里，我们提供了实验方案，详细介绍了我们的脂质体制备，膜蛋白重构和提取测定的方法。我们希望这些实验细节将有助于进一步研究膜蛋白平衡的基本但知之甚少的过程。

Protocol

1. 脂质体制备 以适当的比例混合脂质的氯仿储备，以模拟外线粒体膜。 准备25毫克脂质混合物。我们使用先前建立的模仿线粒体膜的脂质混合物，由48：28：10：10：4摩尔比的鸡蛋磷脂酰胆碱（PC），鸡蛋磷脂酰乙醇胺（PE），牛肝磷脂酰肌醇（PI），合成1，2-二油酰基-sn-甘油-3-磷酸-L-丝氨酸（DOPS）和合成1’，3′-双[1，2-二油酰基-sn-甘油-3-磷酸]-甘油（TOCL）58，59组成。</sup…

Representative Results

为了正确解释结果，必须同时观察无污渍凝胶和蛋白质印迹。无污渍凝胶可确保所有样品的上样量相等。当观察无污渍凝胶时，陪同人员（GST-钙调蛋白和GST-SGTA）将在输入（I）和洗脱（E）通道中可见。仔细检查这些波段的强度在所有 INPUT 样本中是否均匀。同样，确保洗脱样品的强度均匀。洗脱液的浓度是INPUT的5倍，这种强度差异在凝胶中是可见的。 使用无?…

Discussion

正确的线粒体功能取决于强大的蛋白质质量控制系统。由于TA蛋白靶向途径的保真度的固有限制，错位的TA蛋白是线粒体的持续应激源。线粒体蛋白质平衡网络的一个关键组成部分是Msp1，它是一种膜锚定的AAA + ATP酶，可从OMM中去除错位的TA蛋白。在这里，我们描述了如何制备蛋白脂质体，共同重建Msp1和模型TA蛋白，并进行提取测定。我们之前使用该测定来证明Msp1直接识别错位的TA蛋白，并且能够在…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

MLW在芝加哥大学与Robert Keenan博士的博士后研究期间开发了该协议的一部分。

这项工作由NIH资助1R35GM137904-01给MLW。

Materials

Biobeads Bio-Rad 1523920
Bovine liver phosphatidyl inositol Avanti 840042C PI
Chicken egg phosphatidyl choline Avanti 840051C PC
Chicken egg phosphatidyl ethanolamine Avanti 840021C PE
ECL Select western blotting detection reagent GE RPN2235
Filter supports Avanti 610014
Glass vial VWR 60910L-1
Glutathione spin column Thermo Fisher PI16103
Goat anti-rabbit Thermo Fisher NC1050917
Mini-Extruder Avanti 610020
Polycarbonate membrane Avanti 610006 200 nM
PVDF membrane Thermo Fisher 88518 45 µM
Rabbit anti-FLAG Sigma-Aldrich F7245
Synthetic 1,2-dioleoyl-sn-glycero-3-phospho-L-serine Avanti 840035C DOPS
Synthetic 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol Avanti 710335C TOCL
Syringe, 1 mL Norm-Ject 53548-001
Syringe, 1 mL, gas-tight Avanti 610017
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Riferimenti

  1. Song, J., Herrmann, J. M., Becker, T. Quality control of the mitochondrial proteome. Nature Reviews Molecular Cell Biology. 22, 54-70 (2021).
  2. Phillips, B. P., Miller, E. A. Membrane protein folding and quality control. Current Opinion in Structural Biology. 69, 50-54 (2021).
  3. Jiang, H. Quality control pathways of tail-anchored proteins. Biochimica et Biophysica Acta – Molecular Cell Research. 1868, 118922 (2020).
  4. McKenna, M. J., et al. The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase. Science. 369, (2020).
  5. Hegde, R. S., Zavodszky, E. Recognition and Degradation of Mislocalized Proteins in Health and Disease. Cold Spring Harbor Perspectives in Biology. 11, 033902 (2019).
  6. Shao, S., Hegde, R. S. A calmodulin-dependent translocation pathway for small secretory proteins. Cell. 147, 1576-1588 (2011).
  7. Samuelson, J. C., et al. YidC mediates membrane protein insertion in bacteria. Nature. 406, 637-641 (2000).
  8. Anghel, S. A., McGilvray, P. T., Hegde, R. S., Keenan, R. J. Identification of Oxa1 Homologs Operating in the Eukaryotic Endoplasmic Reticulum. Cell Reports. 21, 3708-3716 (2017).
  9. Aviram, N., et al. The SND proteins constitute an alternative targeting route to the endoplasmic reticulum. Nature. 540, 134-138 (2016).
  10. Voorhees, R. M., Hegde, R. S. Structure of the Sec61 channel opened by a signal sequence. Science. 351, 88-91 (2016).
  11. Cichocki, B. A., Krumpe, K., Vitali, D. G., Rapaport, D. Pex19 is involved in importing dually targeted tail-anchored proteins to both mitochondria and peroxisomes. Traffic. 19, 770-785 (2018).
  12. Mateja, A., et al. Protein targeting. Structure of the Get3 targeting factor in complex with its membrane protein cargo. Science. 347, 1152-1155 (2015).
  13. Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T., Pfanner, N. Importing mitochondrial proteins: machineries and mechanisms. Cell. 138, 628-644 (2009).
  14. Chitwood, P. J., Hegde, R. S. An intramembrane chaperone complex facilitates membrane protein biogenesis. Nature. , (2020).
  15. Chitwood, P. J., Juszkiewicz, S., Guna, A., Shao, S., Hegde, R. S. EMC Is Required to Initiate Accurate Membrane Protein Topogenesis. Cell. 175, 1-30 (2018).
  16. Bock, F. J., Tait, S. W. G. Mitochondria as multifaceted regulators of cell death. Nature Reviews Molecular Cell Biology. 21, 85-100 (2020).
  17. Pfanner, N., Warscheid, B., Wiedemann, N. Mitochondrial proteins: from biogenesis to functional networks. Nature Reviews Molecular Cell Biology. 20, (2019).
  18. Bykov, Y. S., Rapaport, D., Herrmann, J. M., Schuldiner, M. Cytosolic Events in the Biogenesis of Mitochondrial Proteins. Trends in Biochemical Sciences. 45, 650-667 (2020).
  19. Pfanner, N., Warscheid, B., Wiedemann, N. Mitochondrial proteins: from biogenesis to functional networks. Nature Reviews Molecular Cell Biology. 427, 1135 (2019).
  20. Borgese, N., Coy-Vergara, J., Colombo, S. F., Schwappach, B. The Ways of Tails: the GET Pathway and more. The Protein Journal. , 1-17 (2019).
  21. Mateja, A., Keenan, R. J. A structural perspective on tail-anchored protein biogenesis by the GET pathway. Current Opinion in Structural Biology. 51, 195-202 (2018).
  22. Chio, U. S., Cho, H., Shan, S. Mechanisms of Tail-Anchored Membrane Protein Targeting and Insertion. Annual review of cell and developmental biology. 33, 417-438 (2017).
  23. Denic, V. A portrait of the GET pathway as a surprisingly complicated young man. Trends in biochemical sciences. , (2012).
  24. Hegde, R. S., Keenan, R. J. Tail-anchored membrane protein insertion into the endoplasmic reticulum. Nature Reviews Molecular Cell Biology. 12, 787-798 (2011).
  25. Kalbfleisch, T., Cambon, A., Wattenberg, B. W. A bioinformatics approach to identifying tail-anchored proteins in the human genome. Traffic. 8, 1687-1694 (2007).
  26. Doan, K. N., et al. The Mitochondrial Import Complex MIM Functions as Main Translocase for α-Helical Outer Membrane Proteins. Cell Reports. 31, (2020).
  27. McDowell, M. A., et al. Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex. Molecular Cell. 80, (2020).
  28. Guna, A., Volkmar, N., Christianson, J. C., Hegde, R. S. The ER membrane protein complex is a transmembrane domain insertase. Science. 591, 3099 (2017).
  29. Rao, M., et al. Multiple selection filters ensure accurate tail-anchored membrane protein targeting. eLife. 5, 21301 (2016).
  30. Schuldiner, M., et al. The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell. 134, 634-645 (2008).
  31. Chen, Y. -. C., et al. Msp1/ATAD1 maintains mitochondrial function by facilitating the degradation of mislocalized tail-anchored proteins. The EMBO journal. 33, 1548-1564 (2014).
  32. Wu, X., Rapoport, T. A. Translocation of Proteins through a Distorted Lipid Bilayer. Trends in Cell Biology. , (2021).
  33. Phillips, B. P., Gomez-Navarro, N., Miller, E. A. Protein quality control in the endoplasmic reticulum. Current Opinion in Cell Biology. 65, 96-102 (2020).
  34. van de Weijer, M. L., et al. Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex. Molecular Cell. 79, (2020).
  35. Weir, N. R., Kamber, R. A., Martenson, J. S., Denic, V. The AAA protein Msp1 mediates clearance of excess tail-anchored proteins from the peroxisomal membrane. eLife. 6, 28507 (2017).
  36. Gardner, B. M., et al. The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading. Nature communications. 9, 135 (2018).
  37. Puchades, C., et al. Unique Structural Features of the Mitochondrial AAA+ Protease AFG3L2 Reveal the Molecular Basis for Activity in Health and Disease. Molecular Cell. , (2019).
  38. Castanzo, D. T., LaFrance, B., Martin, A. The AAA+ ATPase Msp1 is a processive protein translocase with robust unfoldase activity. Proceedings of the National Academy of Sciences of the United States of America. 117, 14970-14977 (2020).
  39. Wang, L., Myasnikov, A., Pan, X., Walter, P. Structure of the AAA protein Msp1 reveals mechanism of mislocalized membrane protein extraction. eLife. 9, (2020).
  40. Puchades, C., Sandate, C. R., Lander, G. C. The molecular principles governing the activity and functional diversity of AAA+ proteins. Nature Reviews Molecular Cell Biology. , 1-16 (2019).
  41. Yang, Y., et al. Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH. Journal of the American Chemical Society. , 10 (2018).
  42. Baldridge, R. D., Rapoport, T. A. Autoubiquitination of the Hrd1 Ligase Triggers Protein Retrotranslocation in ERAD. Cell. 166, 394-407 (2016).
  43. Fresenius, H. L., Wohlever, M. L. Sorting out how Msp1 maintains mitochondrial membrane proteostasis. Mitochondrion. 49, 128-134 (2019).
  44. Wang, L., Walter, P. Msp1/ATAD1 in Protein Quality Control and Regulation of Synaptic Activities. Annual Review of Cell and Developmental Biology. 36, 1-24 (2020).
  45. Dederer, V., et al. Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins. eLife. 8, 1126 (2019).
  46. Matsumoto, S., et al. Msp1 Clears Mistargeted Proteins by Facilitating Their Transfer from Mitochondria to the ER. Molecular Cell. , (2019).
  47. Li, L., Zheng, J., Wu, X., Jiang, H. Mitochondrial AAA-ATPase Msp1 detects mislocalized tail-anchored proteins through a dual-recognition mechanism. EMBO Reports. 20, (2019).
  48. Weidberg, H., Amon, A. MitoCPR – a surveillance pathway that protects mitochondria in response to protein import stress. Science. 360, (2018).
  49. Okreglak, V., Walter, P. The conserved AAA-ATPase Msp1 confers organelle specificity to tail-anchored proteins. Proceedings of the National Academy of Sciences of the United States of America. 111, (2014).
  50. Piard, J., et al. A homozygous ATAD1 mutation impairs postsynaptic AMPA receptor trafficking and causes a lethal encephalopathy. Brain. , (2018).
  51. Zhang, J., et al. The AAA+ ATPase Thorase regulates AMPA receptor-dependent synaptic plasticity and behavior. Cell. 145, 284-299 (2011).
  52. Prendergast, J., et al. Ganglioside regulation of AMPA receptor trafficking. The Journal of Neuroscience. 34, 13246-13258 (2014).
  53. Umanah, G. K. E., et al. Thorase variants are associated with defects in glutamatergic neurotransmission that can be rescued by Perampanel. Science Translational Medicine. 9, 4985 (2017).
  54. Pignatelli, M., et al. Synaptic Plasticity onto Dopamine Neurons Shapes Fear Learning. Neuron. 93, 425-440 (2017).
  55. Zhang, J., et al. The AAA Thorase is neuroprotective against ischemic injury. Journal of Cerebral Blood Flow and Metabolism. , 271678 (2018).
  56. Umanah, G. K. E., et al. AMPA Receptor Surface Expression Is Regulated by S-Nitrosylation of Thorase and Transnitrosylation of NSF. Cell Reports. 33, 108329 (2020).
  57. Wohlever, M. L., Mateja, A., McGilvray, P. T., Day, K. J., Keenan, R. J. Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins. Molecular Cell. 67, 194-202 (2017).
  58. Lovell, J. F., et al. Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell. 135, 1074-1084 (2008).
  59. Leshchiner, E. S., Braun, C. R., Bird, G. H., Walensky, L. D. Direct activation of full-length proapoptotic BAK. Proceedings of the National Academy of Sciences of the United States of America. 110, 986-995 (2013).

Tags

Keywords: Msp1AAA-ATPaseProtein Quality ControlReconstitutionLipid BilayerTA ProteinsLiposomesBioBeadsGlutathione Spin ColumnsExtraction AssaySDS-PAGE
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Fresenius, H. L., Wohlever, M. L. Reconstitution of Msp1 Extraction Activity with Fully Purified Components. J. Vis. Exp. (174), e62928, doi:10.3791/62928 (2021).
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