骨骼肌线粒体蛋白质导入能力的测定
Summary
线粒体是关键的代谢细胞器，在骨骼肌中表现出高水平的表型可塑性。从细胞质基质中导入蛋白质是细胞器生物发生的关键途径，对于网状物的扩增和线粒体功能的维持至关重要。 因此，蛋白质进口是细胞健康的晴雨表。
Abstract
线粒体是关键的代谢和调节细胞器，决定能量供应以及细胞的整体健康状况。在骨骼肌中，线粒体存在于一系列复杂的形态中，从小的椭圆形细胞器到广泛的网状网络。 了解线粒体网如何随着各种刺激（如能量需求的变化）而膨胀和发展，长期以来一直是研究的主题。 这种生长或生物发生的一个关键方面是前体蛋白的导入，这些前体蛋白最初由核基因组编码，在细胞质基质中合成，并转移到各种线粒体亚室中。线粒体已经为这种导入过程开发了一种复杂的机制，涉及许多选择性的内膜和外膜通道，称为蛋白质导入机制（PIM）。导入线粒体取决于活膜电位和通过氧化磷酸化获得的细胞器衍生的ATP的可用性。因此，它的测量可以作为细胞器健康的衡量标准。PIM还在骨骼肌中表现出高水平的适应性可塑性，与细胞的能量状态紧密耦合。 例如，运动训练已被证明可以增加导入能力，而肌肉使用可以减少导入能力，这与线粒体含量标志物的变化相吻合。虽然蛋白质导入是线粒体生物发生和扩张的关键步骤，但该过程在骨骼肌中并未得到广泛研究。 因此，本文概述了如何使用骨骼肌中分离和功能齐全的线粒体来测量蛋白质导入能力，以促进对所涉及的方法的更多理解，并了解细胞器更新途径在运动，健康和疾病中的重要性。
Introduction
线粒体是存在于不同细胞类型复杂形态中的细胞器，并且被认为具有对细胞健康至关重要的越来越多的功能。因此，它们不能再仅仅被削减为产生能量的细胞器。线粒体是关键的代谢调节因子，细胞命运的决定因素和信号传导中枢，其功能可以作为整体细胞健康的有用指标。在骨骼肌细胞中，电子显微镜研究揭示了地理上不同的构体下（SS）和肌原纤维间（IMF）线粒体的存在，它们表现出一定程度的连接性1，2，3，4现在被认为是高度动态的，并且能够适应骨骼肌活动水平的变化，以及年龄和疾病。可以通过多种方式评估肌肉中的线粒体含量和功能5，6，并且已经应用了传统的细胞器分离方法来更好地了解线粒体的呼吸和酶促能力（Vmax），这与细胞环境的影响不同7，8。特别是，这些传统方法揭示了从构肌下和肌原纤维间区域分离的线粒体之间的微妙生化差异，掩盖了这些亚细胞区域代谢的可能功能意义8，9，10，11。
线粒体的生物发生是独特的，需要来自核和线粒体DNA的基因产物的贡献。然而，其中绝大多数来自细胞核，因为mtDNA转录仅导致13种蛋白质的合成。由于线粒体通常由参与不同代谢途径的>1000蛋白组成，因此细胞器的生物发生需要严格调节的方式将前体蛋白从细胞质基质导入和组装到各种线粒体亚室中，以维持适当的化学计量学和功能12，13。用于线粒体的核编码蛋白通常携带线粒体靶向序列（MTS），该序列将它们靶向细胞器并促进其亚区室定位。大多数基质结合蛋白含有可切割的N末端MTS，而那些发往线粒体膜外或内侧的蛋白通常具有内部靶向结构域14。导入过程由一组不同的通道进行，这些通道为进入细胞器提供了多种途径13。外膜（TOM）复合物的转座将前体从细胞质基质运送到膜间空间，在那里它们被内膜（TIM）复合物的转座体识别。该复合物负责将核编码的前体导入基质中，其中蛋白酶切割N端靶向预序列。发往外膜的蛋白质可以通过TOM复合物直接插入该膜中，而那些发往内膜的蛋白质则由TIM蛋白插入，特别是TIM22。在导入之后，蛋白质由常驻蛋白酶和伴侣进一步处理，并且通常结合形成更大的复合物，例如在电子传递链中发现的复合物。
线粒体蛋白导入本身也可以作为线粒体健康的测量，因为该过程依赖于膜电位的存在和ATP15形式的能量来源。例如，当膜电位消散时，蛋白激酶PINK1不能被细胞器吸收，这导致磷酸化信号，通过称为线粒体自噬的途径触发细胞器降解的开始16，17。在类似情况下，当导入受阻时，蛋白质ATF5不能进入细胞器，随后易位到细胞核，在那里它作为UPR基因表达上调的转录因子18，19。因此，测量蛋白质导入效率可以提供对细胞器健康状况的全面洞察，而基因表达响应可用于指示向细胞核的逆行信号传导的程度。
尽管它对线粒体的生物发生和一般的细胞健康具有明显的重要性，但哺乳动物线粒体的导入途径研究不足。在本报告中，我们描述了测量前体蛋白导入骨骼肌线粒体所涉及的具体步骤，并提供数据来说明导入系统对肌肉变化和废弃的适应性反应，说明蛋白质导入对骨骼肌适应性可塑性的贡献。
Protocol
这些实验中使用的所有动物都保存在约克大学的动物护理设施中。这些实验是根据加拿大动物护理委员会指南进行的，并得到了约克大学动物护理委员会的批准（许可证：2017-08）。 1. 从骨骼肌中分离出构肌下和肌原纤维间线粒体的功能分离 试剂制备： 按照 表1所述准备所有缓冲液和培养基。 将缓冲液设置为pH 7.4并储存在4°C（长达2周），?…
Representative Results
我们已经广泛地说明了该协议是一种有效的测定方法，用于确定导入功能性和完整分离的骨骼肌线粒体的速率。与未经处理的条件相比，将典型的前体蛋白（如苹果酸脱氢酶（MDH））导入基质对膜电位敏感，因为它可以被一种呼吸链解聚剂valinomycin抑制（图2A）。当线粒体内膜和外膜在洗涤剂Triton X-100存在下溶解时，进口也会受到阻碍。导入过程对外部ATP的存在?…
Discussion
线粒体独特地依赖于核基因组和线粒体基因组的表达和协调，以在细胞内合成和扩增。然而，核基因组编码绝大多数（99%）的线粒体蛋白质组，这强调了蛋白质导入机制在支持线粒体生物发生方面的重要性。导入也是一个重要的信号传导事件，因为导入失败会促进未折叠蛋白质反应和/或线粒体自噬的启动15，16，26。由于导入?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢麦吉尔大学的G.C. Shore博士，华盛顿医学院的A. Strauss博士和拉筹伯大学的.M.T. Ryan博士最初捐赠了用于这项研究的表达质粒。这项工作得到了加拿大自然科学和工程研究委员会（NSERC）对D. A. Hood的资助。D. A. Hood也是加拿大细胞生理学研究主席的持有人。
Materials
| 0.2% BSA | Sigma | A2153 | |
| 35S-methionine | Perkin Elmer | NEG709A500UC | Purchase requires a valid radioisotope permit |
| ATP | Sigma | A7699 | |
| Blotting paper; Whatman 3MM CHR Paper | Thermo Fisher | 05-714-5 | |
| Cassette for film | Kodak | Kodak Xomatic | |
| Centrifugation Tube | Thermo Fisher | 3138-0050 | |
| Chloroform | Thermo Fisher | C298-4 | |
| DTT | Sigma | D9779-5G | |
| EDTA | BioShop | EDT002 | |
| EGTA | Sigma | E4378 | |
| Gel Dryer | BioRad | Model 583 | |
| Gel Drying Kit | Sigma or BioRad | Z377570-1PAK or OW-GDF-10 | Various options are commercially available through many companies, these are just as few examples. |
| Glycerol | Caledon Laboratory Chemicals | 5350-1-40 | |
| HEPES | Sigma | H3375 | |
| High Speed Centrifuge | Beckman Coulter | Avanti J-25 Centrifuge | |
| Homogenizer | IKA | T25 Digital Ultra Turrex | |
| Isoamylalcohol, or 3-methylbutanol | Sigma | I9392 | |
| KAc | BioShop | POA301.500 | |
| KCl | Sigma | P3911 | |
| M7G | New England Biolab | S1404S | Dilute with 1000ul 20mM HEPES to make 1mM stock |
| MgCl | BioShop | MAG510 | |
| MgSO4 | Thermo Fisher | M65-500 | |
| MOPS | BioShop | MOP001 | |
| NaCl | BioShop | SOD001 | |
| NTP | Thermo Fisher | R0191 | |
| OCT Plasmid | – | – | Donated from Dr. G. C. Shore, McGill University, Montreal, Canada; alternative available through Addgene, plasmid #71877 |
| pGEM4Z/hTom40 Plasmid | – | – | Donated from Dr. M. T. Ryan, La Trobe University, Melbourne, Australia |
| pGMDH Plasmid | – | – | Donated from Dr. A. Strauss, Washington University School of Medicine |
| Phenol | Sigma | P4557 | |
| Phenol:Chloroform:Isoamyalcohol | Sigma | P3803 | Can also be made with the ratio provided |
| Phosphorus Film | Fujifilm | BAS-IP MS 2025 | |
| Rabbit reticulocyte lysate | Promega | L4960 | Avoid freeze-thaw; aliquot lysate upon arrival; amino acids are provided in the kit as well |
| RNAsin | Promega | N2311 | |
| Rotor for High Speed Centrifuge | Beckman Coulter | JA-25.50 | |
| SDS | BioShop | SDS001.500 | Caution: harmful if ingested or inhaled, wear a mask. |
| Sodium acetate | Bioshop | SAA 304 | |
| Sodium Carbonate | VWR | BDH9284 | |
| Sodium salicylate | Millipore Sigma | 106601 | |
| Sorbitol | Sigma | S6021 | |
| SP6 RNA Polymerase | Promega | P1085 | |
| Spectrophotometer | Thermo Fisher | Nanodrop 2000 | |
| Spermidine | Sigma | S-2626 | |
| Sucrose | BioShop | SUC507 | |
| T7 RNA Polymerase | Promega | P2075 | |
| Tabletop Centrifuge | Thermo Fisher | AccuSpin Micro 17 | |
| Trichloroacetic acid | Thermo Fisher | A322-500 | |
| Tris | BioShop | TRS001 | |
| β-mercaptoethanol | Sigma | M6250-100ML |
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Citer Cet Article
Oliveira, A. N., Richards, B. J., Hood, D. A. Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria. J. Vis. Exp. (179), e63055, doi:10.3791/63055 (2022).