Summary

一种分离线粒体接触位点的改进方法

Published: June 16, 2023
doi:

Summary

线粒体接触位点是与线粒体内膜和外膜蛋白相互作用的蛋白质复合物。这些位点对于线粒体膜之间的通信以及胞质溶胶和线粒体基质之间的通信至关重要。在这里,我们描述了一种识别符合这一特定类别蛋白质条件的候选者的方法。

Abstract

线粒体几乎存在于所有真核细胞中,其基本功能远远超出了能量产生,例如,铁硫簇、脂质或蛋白质的合成、Ca2+ 缓冲和诱导细胞凋亡。同样,线粒体功能障碍会导致严重的人类疾病,如癌症、糖尿病和神经退行性疾病。为了执行这些功能,线粒体必须通过其包膜与细胞的其余部分进行通信,该包膜由两个膜组成。因此,这两种膜必须不断相互作用。在这方面,线粒体内膜和外膜之间的蛋白质接触位点是必不可少的。到目前为止,已经确定了几个接触点。在此描述的方法中, 酿酒酵 母线粒体用于分离接触位点,从而确定符合接触位点蛋白条件的候选物。我们使用这种方法鉴定了线粒体接触位点和嵴组织系统(MICOS)复合物,这是线粒体内膜中主要的接触位点形成复合物之一,从酵母到人类都是保守的。最近,我们进一步改进了这种方法,以鉴定由 Cqd1 和 Por1-Om14 复合物组成的新接触位点。

Introduction

线粒体在真核生物中发挥着各种不同的功能,其中最著名的是通过氧化磷酸化产生ATP。其他功能包括铁硫簇的产生、脂质合成,以及在高等真核生物中,Ca2+ 信号传导和诱导细胞凋亡 1,2,3,4。这些功能与其复杂的超微结构密不可分。

线粒体超微结构首先通过电子显微镜5 描述。结果表明,线粒体是由两层膜组成的相当复杂的细胞器:线粒体外膜和线粒体内膜。因此,这些膜形成了两个水室:膜间空间和基质。线粒体内膜可以进一步分为不同的部分。内边界膜与外膜保持紧密联系,嵴形成内陷。所谓的嵴连接连接内边界膜和嵴(图1)。此外,渗透收缩的线粒体的电子显微照片显示存在线粒体膜紧密连接的位点 6,7。这些所谓的接触位点是由跨越两个膜的蛋白质复合物形成的(图1)。人们认为,这些相互作用位点对于细胞活力至关重要,因为它们对线粒体动力学和遗传的调节以及胞质溶胶和基质之间的代谢物和信号的转移非常重要8。

线粒体内膜中的MICOS复合物可能是特征最好、用途最广泛的接触位点形成复合物。MICOS 于 2011 年在酵母中描述,它由六个亚基9、10、1 1 组成:Mic60、Mic27、Mic26、Mic19Mic12 和 Mic10。它们形成一个大约 1.5 MDa 的复合物,定位于嵴连接 9,10,11。核心亚基 Mic10 或 Mic60 的缺失导致该复合物 9,11 的缺失,这意味着这两个亚基对 MICOS 的稳定性至关重要。有趣的是,MICOS 不仅与各种线粒体外膜蛋白和复合物形成一个接触位点,而且与多个接触位点形成:TOM 复合物 11,12、TOB/SAM 复合物 9、1213、14、15、16、Fzo1-Ugo1 复合物9Por1 10OM45 10 和 Miro 17.这强烈表明 MICOS 复合物参与各种线粒体过程,例如蛋白质输入、磷脂代谢和线粒体超微结构的产生18。后一种功能可能是MICOS的主要功能,因为通过MIC10或MIC60缺失诱导的MICOS复合物的缺失导致线粒体超微结构异常,几乎完全缺乏规则的嵴。相反,与内边界膜不相连的内膜囊泡积聚1920。重要的是,MICOS 在形式和功能上从酵母到人都是保守的21。MICOS 亚基突变与严重人类疾病的关联也强调了其对高等真核生物的重要性22,23。尽管MICOS具有高度的通用性,但必须存在其他接触点(基于我们未发表的观察结果)。事实上,已经确定了其他几个接触位点,例如,线粒体融合机制 Mgm1-Ugo1/Fzo1 24、2526 或 Mdm31-Por1,它们参与线粒体特异性磷脂心磷脂 27 的生物合成。最近,我们改进了导致我们鉴定 MICOS 的方法,以将 Cqd1 鉴定为与外膜复合物 Por1-Om1428 形成的新接触位点的一部分。有趣的是,这个接触位点似乎也参与了多个过程,例如线粒体膜稳态、磷脂代谢和辅酶 Q28,29 的分布。

在这里,我们使用了前面描述的线粒体分馏 9,30,31,32,33 的变体。线粒体的渗透处理导致线粒体外膜的破坏和基质空间的收缩,使两个膜仅在接触部位靠近。这允许通过温和的超声处理产生仅由线粒体外膜或线粒体内膜组成的囊泡,或在两个膜的接触部位产生囊泡。由于线粒体内膜具有更高的蛋白质脂质比,因此与线粒体外膜囊泡相比,线粒体内膜囊泡表现出更高的密度。密度的差异可用于通过蔗糖浮力密度梯度离心分离膜囊泡。因此,线粒体外膜囊泡在低蔗糖浓度下积累,而线粒体内膜囊泡在高蔗糖浓度下富集。含有接触位点的囊泡浓缩在中间蔗糖浓度(图2)。以下方案详细描述了这种改进的方法,与我们先前建立的方法32相比,它需要更少的专业设备,时间和能量,并为鉴定可能的接触位点蛋白提供了有用的工具。

Protocol

1. 缓冲液和储备溶液 在去离子水中制备 1 M 3-吗啉基丙烷-1-磺酸 (MOPS) 溶液,pH 7.4。储存在4°C。 在去离子水中制备500mM乙二胺四乙酸(EDTA),pH 8.0。在室温下储存。 在去离子水中制备2.4M山梨糖醇。高压灭菌后在室温下储存。 在去离子水中制备2.5M蔗糖。高压灭菌后在室温下储存。 在异丙醇中制备200mM苯甲基磺酰氟(PMSF)。储存在-20°C。注?…

Representative Results

分离线粒体内膜和外膜相对容易。然而,含有接触位点的囊泡的产生和分离要困难得多。在我们看来,有两个步骤是关键和必不可少的:超声处理条件和使用的梯度。 通常,与阶梯梯度相比,线性梯度被认为具有更好的分辨率。然而,它们的可重复生产是乏味的,需要特殊的设备。因此,我们建立了一种方法来生成各个步骤之间差异相对较小的阶梯梯度(参见步骤3.5)。我?…

Discussion

线粒体亚分割是一项复杂的实验,具有几个高度复杂的步骤。因此,我们的目标是进一步改进并在一定程度上简化我们既定的方法32。在这里,挑战是对复杂和高度专业化设备的要求,这些设备通常是单独的结构,以及巨大的时间和能源消耗。为此,我们尝试移除用于铸造和收获线性梯度的泵和单个结构,并将其更改为阶梯梯度。重要的是,我们意识到,至少在按此处所述制备?…

Declarações

The authors have nothing to disclose.

Acknowledgements

M.E.H. 感谢 Deutsche Forschungsgemeinschaft (DFG),项目编号 413985647,提供财政支持。作者感谢慕尼黑路德维希-马克西米利安大学的Michael Kiebler博士的慷慨和广泛的支持。我们感谢 Walter Neupert 的科学投入、有益的讨论和持续的灵感。J.F.感谢慕尼黑生命科学研究生院(LSM)的支持。

Materials

13.2 mL, Open-Top Thinwall Ultra-Clear Tube, 14 x 89mm Beckman Instruments, Germany 344059
50 mL, Open-Top Thickwall Polycarbonate Open-Top Tube, 29 x 104mm Beckman Instruments, Germany 363647
A-25.50 Fixed-Angle Rotor- Aluminum, 8 x 50 mL, 25,000 rpm, 75,600 x g Beckman Instruments, Germany 363055
Abbe refractometer Zeiss, Germany discontinued,
any pipet controller will suffice
accu-jet pro Pipet Controller Brandtech, USA BR26320 discontinued,
any pipet controller will suffice
Beaker 1000 mL DWK Life Science, Germany C118.1
Branson  Digital Sonifier W-250 D Branson Ultrasonics, USA FIS15-338-125
Branson Ultrasonic 3mm TAPERED MICROTIP Branson Ultrasonics, USA 101-148-062
Branson Ultrasonics 200- and 400-Watt Sonifiers: Rosette Cooling Cell Branson Ultrasonics, USA 15-338-70
Centrifuge Avanti JXN-26 Beckman Instruments, Germany B37912
Centrifuge Optima XPN-100 ultra Beckman Instruments, Germany 8043-30-0031
cOmplete Proteaseinhibtor-Cocktail Roche, Switzerland 11697498001
D-Sorbit Roth, Germany 6213
EDTA (Ethylendiamin-tetraacetic acid disodium salt dihydrate) Roth, Germany 8043
Erlenmeyer flask, 100 mL Roth, Germany X747.1
graduated pipette, Kl. B, 25:0, 0.1 Hirschmann, Germany 1180170
graduated pipette, Kl. B, 5:0, 0.05 Hirschmann, Germany 1180153
ice bath neoLab, Germany  S12651
Magnetic stirrer RCT basic IKA-Werke GmbH, Germany Z645060GB-1EA
MOPS (3-(N-Morpholino)propanesulphonic acid) Gerbu, Germany 1081
MyPipetman Select P1000 Gilson, USA FP10006S
MyPipetman Select P20 Gilson, USA FP10003S
MyPipetman Select P200 Gilson, USA FP10005S
Omnifix 1 mL Braun, Germany 4022495251879
Phenylmethylsulfonyl fluoride (PMSF) Serva, Germany 32395.03
STERICAN cannula 21 Gx4 4/5 0.8×120 mm Braun, Germany 4022495052414
stirring bar, 15 mm VWR, USA 442-0366
Sucrose Merck, Germany S8501
SW 41 Ti Swinging-Bucket Rotor Beckman Instruments, Germany 331362
Test tubes Eppendorf, Germany 3810X
Tissue grinders, Potter-Elvehjem type, 2 mL glass vessel VWR, USA 432-0200
Tissue grinders, Potter-Elvehjem type, 2 mL plunger with serrated tip VWR, USA 432-0212
Trichloroacetic acid (TCA) Sigma Aldrich, Germany 33731 discontinued,
any TCA will suffice (CAS: 73-03-9)
TRIS Roth, Germany 4855

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Khosravi, S., Frickel, J., Harner, M. E. An Improved Method to Isolate Mitochondrial Contact Sites. J. Vis. Exp. (196), e65444, doi:10.3791/65444 (2023).

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