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。

1. 缓冲液和储备溶液
<ol><li>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。</li>
	<li>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。</li>
	<li>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。</li>
	<li>在去离子水中制备2.5M蔗糖。高压灭菌后在室温下储存。</li>
	<li>在异丙醇中制备200mM苯甲基磺酰氟（PMSF）。储存在-20°C。 注意：PMSF 如果吞咽是有毒的，会导致严重的皮肤灼伤和眼睛损伤。不要吸入灰尘，并戴上防护手套和护目镜。</li>
	<li>在去离子水中制备72%的三氯乙酸（TCA）。储存在4°C。 注意：TCA 可能会导致呼吸道刺激、严重皮肤灼伤和眼睛损伤。不要吸入灰尘，并戴上防护手套和护目镜。</li>
	<li>准备SM缓冲液：20mM MOPS和0.6M山梨糖醇，pH 7.4。</li>
	<li>准备溶胀缓冲液：20 mM MOPS、0.5 mM EDTA、1 mM PMSF 和蛋白酶抑制剂混合物，pH 7.4。</li>
	<li>制备蔗糖梯度缓冲液：0.8 M、0.96 M、1.02 M、1.13 M 和 1.3 M 蔗糖，溶于 20 mM MOPS 和 0.5 mM EDTA，pH 7.4。 注意：所有缓冲液都可以储存在4°C;然而，建议将含蔗糖的缓冲液储存在-20°C以避免真菌生长。蛋白酶抑制剂必须在使用前新鲜添加。</li>
</ol>2. 软骨小泡的产生
<ol><li>根据既定方案新鲜分离粗酵母线粒体34,35。 注：在本实验中，从 酿酒酵母中分离出线粒体。没有必要产生没有其他细胞器的梯度纯线粒体。</li>
	<li>通过移液将10mg新鲜分离的线粒体重悬于1.6mL SM缓冲液（4°C）中（图2，步骤1）。</li>
	<li>将线粒体悬浮液转移到预冷的 100 mL 锥形瓶中。</li>
	<li>使用 20 mL 玻璃移液管缓慢加入 16 mL 溶胀缓冲液。在添加过程中，在冰上持续进行温和搅拌。这种渗透处理导致水吸收到基质空间中。线粒体内膜的肿胀会破坏外膜。然而，两层膜将在接触部位保持接触9（图2，步骤2）。</li>
	<li>将样品在恒定的温和搅拌下在冰上孵育30分钟。</li>
	<li>使用 5 mL 玻璃移液管缓慢加入 5 mL 的 2.5 M 蔗糖溶液，将样品中的蔗糖浓度增加到约 0.55 M。这种渗透处理导致水从基质36流出。内膜的收缩旨在最大限度地使其与外膜的残留碎片的距离最大化。这降低了通过超声处理产生内膜和外膜的人工杂化囊泡的可能性（图2，步骤3）。</li>
	<li>将样品在温和搅拌下在冰上孵育15分钟。</li>
	<li>使线粒体进行超声处理以产生提交软骨体膜囊泡（图2，步骤4）。<ol><li>将线粒体悬浮液转移到预冷的玫瑰花结细胞中。</li>
		<li>以10%振幅超声处理线粒体悬浮液30秒，同时在冰浴中冷却玫瑰花结细胞。</li>
		<li>将悬浮液在冰浴中静置30秒。</li>
		<li>重复步骤 2.8.2 和步骤 2.8.3 三次。 注意： 超声处理条件会根据所使用的超声仪而有所不同。对于单个机器，优化是必要的。过于苛刻的超声处理会导致人工形成由线粒体内膜和外膜组成的囊泡。</li>
	</ol></li>
</ol>3.软骨囊泡的分离
<ol><li>通过在4°C下以20,000× g 离心20分钟，将生成的囊泡与剩余的完整线粒体分离。 完整的线粒体将沉淀，而囊泡将留在上清液中。</li>
	<li>浓缩囊泡混合物。<ol><li>将上清液转移到新鲜的超速离心管中。</li>
		<li>使用配备 0.8 mm x 120 mm 套管的 1 mL 注射器在管底部加载 0.3 mL 的 2.5 M 蔗糖溶液垫。</li>
		<li>在4°C下以118,000× g 离心100分钟。 注意：这里使用了最大半径为 153.1 mm 的摆动铲斗转子。离心条件很大程度上取决于转子，当使用另一个转子时，必须进行调整。</li>
	</ol></li>
	<li>离心后，囊泡将在蔗糖垫的顶部显示为圆盘。弃去约90%的上清液。现在，通过上下移液将浓缩囊泡重悬于剩余的缓冲液中（包括2.5M蔗糖）来收获浓缩囊泡。将悬浮液转移到冰冷的 Dounce 均质器中。</li>
	<li>使用聚四氟乙烯陶器以至少10次冲程均质化悬浮液。</li>
	<li>准备蔗糖梯度。<ol><li>对于具有五个步骤（1.3 M、1.13 M、1.02 M、0.96 M和0.8 M蔗糖，溶于20 mM MOPS和0.5 mM EDTA，pH 7.4）的11 mL步进梯度，每层含有2.2 mL蔗糖溶液。 注意：建议使用折光仪来测量和调整蔗糖浓度;但是，这不是必需的。将 10 μL 缓冲液施加到折光仪上，并检测相应的折光率。蔗糖浓度的计算方法如下： <img alt="Equation 1" src="/files/ftp_upload/65444/65444eq01v2.jpg" style="margin: auto;"> 1.3333代表水的折射率。0.048403 是从摩尔折射率到水溶液中蔗糖浓度的线性缩放得到的蔗糖比转化指数 37。</li>
		<li>将最高浓度的蔗糖加入离心管中，并将管置于-20°C。 等到图层完全冻结后再应用下一个图层。相应地进行，直到添加最后一层，并将梯度储存在−20°C。 注意：请记住在开始实验时将冷冻梯度转移到4°C，以使梯度解冻。这大约需要3小时。对于这里的离心，使用了容量为 13.2 mL 的摆动吊篮转子。当使用不同的转子时，必须相应地调整梯度步进体积。</li>
	</ol></li>
	<li>使用折光仪测量样品的蔗糖浓度。如果无法使用折光仪，则可以假设蔗糖浓度为2 M。这个假定的蔗糖浓度将成为下一步的基础。</li>
	<li>通过加入适量的20mM MOPS，0.5mM EDTA，1mM PMSF和蛋白酶抑制剂混合物（pH 7.4）将蔗糖浓度调节至0.6M。如果蔗糖浓度估计为2M而不是测量，建议在调整后测试浓度是否足够低。在试管中将少量样品（约 50 μL）涂在 200 μL 的 0.8 M 蔗糖上。样品必须保持在0.8M蔗糖的顶部。</li>
	<li>将样品（约1mL）加载到蔗糖梯度的顶部（保留10%以备后用）。小心移液对于避免梯度干扰很重要（图2，步骤5）。</li>
	<li>通过在200,000× g 和4°C离心12小时来分离囊泡。如果可能，将离心机设置为缓慢的加速和减速，以避免梯度的干扰（图2，步骤6）。 注意：这里使用了最大半径为 153.1 mm 的摆动铲斗转子。离心条件很大程度上取决于转子，当使用另一个转子时，必须进行调整。</li>
	<li>使用 1 mL 移液器从 上到下收集 700 μL 馏分的梯度。这将产生 17 个分数，从而获得足够的分辨率。</li>
</ol>4. 提交软骨囊泡的分析
<ol><li>通过将每个级分置于两次连续进行的TCA沉淀38中来浓缩蛋白质，以制备SDS-PAGE样品。<ol><li>向各个馏分中加入 200 μL 72% TCA，并混合直至溶液均匀。将馏分在冰上孵育30分钟，并通过在20,000× g 和4°C下离心20分钟沉淀沉淀的蛋白质。弃去上清液，加入500μL的28%TCA溶液，充分混合，重复离心步骤。</li>
		<li>用 1 mL 丙酮 （-20°C） 洗涤沉淀，并在 20,000 x g 和 4 °C 下离心 10 分钟。 弃去上清液，让沉淀风干。将沉淀重悬于60μLSDS样品缓冲液中，并将样品在95°C下孵育5分钟。 注：在第一次TCA沉淀后，大量的蔗糖将残留，特别是在高密度馏分中。这将通过额外的 TCA 沉淀来消除。</li>
	</ol></li>
	<li>通过 SDS-PAGE39 和免疫印迹 40、41、42、43 分析每个级分的20 μL。</li>
</ol>

分离线粒体接触位点

作者声明不存在利益冲突。

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?...

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、Mic19、Mic12 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、12、13、14、15、16、Fzo1-Ugo1 复合物9、Por1 10、OM45 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19、20。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、25、26 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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>13.2 mL, Open-Top Thinwall Ultra-Clear Tube, 14 x 89mm</td>
			<td>Beckman Instruments, Germany</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL, Open-Top Thickwall Polycarbonate Open-Top Tube, 29 x 104mm</td>
			<td>Beckman Instruments, Germany</td>
			<td>363647</td>
			<td></td>
		</tr>
		<tr>
			<td>A-25.50 Fixed-Angle Rotor- Aluminum, 8 x 50 mL, 25,000 rpm, 75,600 x g</td>
			<td>Beckman Instruments, Germany</td>
			<td>363055</td>
			<td></td>
		</tr>
		<tr>
			<td>Abbe refractometer</td>
			<td>Zeiss, Germany</td>
			<td></td>
			<td>discontinued, 
			any pipet controller will suffice</td>
		</tr>
		<tr>
			<td>accu-jet pro Pipet Controller</td>
			<td>Brandtech, USA</td>
			<td>BR26320</td>
			<td>discontinued, 
			any pipet controller will suffice</td>
		</tr>
		<tr>
			<td>Beaker 1000 mL</td>
			<td>DWK Life Science, Germany</td>
			<td>C118.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson&#160; Digital Sonifier W-250 D</td>
			<td>Branson Ultrasonics, USA</td>
			<td>FIS15-338-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson Ultrasonic 3mm TAPERED MICROTIP</td>
			<td>Branson Ultrasonics, USA</td>
			<td>101-148-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson Ultrasonics 200- and 400-Watt Sonifiers: Rosette Cooling Cell</td>
			<td>Branson Ultrasonics, USA</td>
			<td>15-338-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Avanti JXN-26</td>
			<td>Beckman Instruments, Germany</td>
			<td>B37912</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Optima XPN-100 ultra</td>
			<td>Beckman Instruments, Germany</td>
			<td>8043-30-0031</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Proteaseinhibtor-Cocktail</td>
			<td>Roche, Switzerland</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbit</td>
			<td>Roth, Germany</td>
			<td>6213</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (Ethylendiamin-tetraacetic acid disodium salt dihydrate)</td>
			<td>Roth, Germany</td>
			<td>8043</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask, 100 mL</td>
			<td>Roth, Germany</td>
			<td>X747.1</td>
			<td></td>
		</tr>
		<tr>
			<td>graduated pipette, Kl. B, 25:0, 0.1</td>
			<td>Hirschmann, Germany</td>
			<td>1180170</td>
			<td></td>
		</tr>
		<tr>
			<td>graduated pipette, Kl. B, 5:0, 0.05</td>
			<td>Hirschmann, Germany</td>
			<td>1180153</td>
			<td></td>
		</tr>
		<tr>
			<td>ice bath</td>
			<td>neoLab, Germany</td>
			<td>&#160;S12651</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer RCT basic</td>
			<td>IKA-Werke GmbH, Germany</td>
			<td>Z645060GB-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS (3-(N-Morpholino)propanesulphonic acid)</td>
			<td>Gerbu, Germany</td>
			<td>1081</td>
			<td></td>
		</tr>
		<tr>
			<td>MyPipetman Select P1000</td>
			<td>Gilson, USA</td>
			<td>FP10006S</td>
			<td></td>
		</tr>
		<tr>
			<td>MyPipetman Select P20</td>
			<td>Gilson, USA</td>
			<td>FP10003S</td>
			<td></td>
		</tr>
		<tr>
			<td>MyPipetman Select P200</td>
			<td>Gilson, USA</td>
			<td>FP10005S</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnifix 1 mL</td>
			<td>Braun, Germany</td>
			<td>4022495251879</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Serva, Germany</td>
			<td>32395.03</td>
			<td></td>
		</tr>
		<tr>
			<td>STERICAN cannula 21 Gx4 4/5 0.8x120 mm</td>
			<td>Braun, Germany</td>
			<td>4022495052414</td>
			<td></td>
		</tr>
		<tr>
			<td>stirring bar, 15 mm</td>
			<td>VWR, USA</td>
			<td>442-0366</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck, Germany</td>
			<td>S8501</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Instruments, Germany</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tubes</td>
			<td>Eppendorf, Germany</td>
			<td>3810X</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue grinders, Potter-Elvehjem type, 2 mL glass vessel</td>
			<td>VWR, USA</td>
			<td>432-0200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue grinders, Potter-Elvehjem type, 2 mL plunger with serrated tip</td>
			<td>VWR, USA</td>
			<td>432-0212</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid (TCA)</td>
			<td>Sigma Aldrich, Germany</td>
			<td>33731</td>
			<td>discontinued, 
			any TCA will suffice (CAS: 73-03-9)</td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Roth, Germany</td>
			<td>4855</td>
			<td></td>
		</tr>
	</tbody>
</table>

an improved method to isolate mitochondrial contact sites

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。

Mitochondria perform a variety of different functions in eukaryotes, with the most well-known being the production of ATP through oxidative phosphorylation. Other functions include the production of iron-sulfur clusters, lipid synthesis, and in higher eukaryotes, Ca2+ signaling, and the induction of apoptosis1,2,3,4. These functions are inseparably linked to their complex ultrastructure.
The mitochondrial ultrastructure was first described by electron microscopy5. It was shown that mitochondria are rather complex organelles consisting of two membranes: the mitochondrial outer membrane and the mitochondrial inner membrane. Thus, two aqueous compartments are formed by these membranes: the intermembrane space and the matrix. The mitochondrial inner membrane can be even further divided into different sections. The inner boundary membrane stays in close proximity to the outer membrane, and the cristae form invaginations. So-called crista junctions connect the inner boundary membrane and the cristae (Figure 1). Furthermore, electron micrographs of osmotically shrunken mitochondria reveal that sites exist at which the mitochondrial membranes are tightly connected6,7. These so-called contact sites are formed by protein complexes spanning the two membranes (Figure 1). It is thought that these interaction sites are essential for cell viability due to their importance for the regulation of mitochondrial dynamics and inheritance, as well as the transfer of metabolites and signals between the cytosol and the matrix8.
The MICOS complex in the mitochondrial inner membrane is probably the best characterized and the most versatile contact site-forming complex. MICOS was described in yeast in 2011, and it consists of six subunits9,10,11: Mic60, Mic27, Mic26, Mic19, Mic12, and Mic10. These form a complex of approximately 1.5 MDa that localizes to the crista junctions9,10,11. The deletion of either core subunit, Mic10 or Mic60, leads to the absence of this complex9,11, meaning these two subunits are essential for the stability of MICOS. Interestingly, MICOS forms not only one but multiple contact sites with various mitochondrial outer membrane proteins and complexes: the TOM complex11,12, the TOB/SAM complex9,12,13,14,15,16, the Fzo1-Ugo1 complex9, Por110, OM4510, and Miro17. This strongly indicates that the MICOS complex is involved in various mitochondrial processes, such as protein import, phospholipid metabolism, and the generation of the mitochondrial ultrastructure18. The latter function is probably the major function of&#160;MICOS, as the absence of the MICOS complex induced through the deletion of MIC10&#160;or MIC60&#160;leads to an abnormal mitochondrial ultrastructure that virtually completely lacks regular cristae. Instead, internal membrane vesicles without connection to the inner boundary membrane accumulate19, 20. Importantly, MICOS is conserved in form and function from yeast to&#160;human21. The association of mutations in MICOS subunits with severe human diseases also emphasizes its importance for higher eukaryotes22,23. Although MICOS is highly versatile, additional contact sites must exist (based on our unpublished observations). Indeed, several other contact sites have been identified, for instance, the mitochondrial fusion machineries Mgm1-Ugo1/Fzo124,25,26&#160;or Mdm31-Por1, which is involved in the biosynthesis of the mitochondrial-specific phospholipid cardiolipin27. Recently, we improved the method that led us to the identification of MICOS to identify Cqd1 as part of a novel contact site formed with the outer membrane complex Por1-Om1428. Interestingly, this contact site also seems to be involved in multiple processes such as mitochondrial membrane homeostasis, phospholipid metabolism, and the distribution of coenzyme Q28,29.
Here, we used a variation of the previously described fractionation of mitochondria9,30,31,32,33. Osmotic treatment of mitochondria leads to the disruption of the mitochondrial outer membrane and to a shrinkage of the matrix space, leaving the two membranes only in close proximity at contact sites. This allows for the generation of vesicles that consist exclusively of mitochondrial outer membrane or mitochondrial inner membrane or at contain contact sites of both membranes through mild sonication. Due to the mitochondrial inner membrane possessing a much higher protein-to-lipid ratio, mitochondrial inner membrane vesicles exhibit a higher density compared to mitochondrial outer membrane vesicles. The difference in density can be used to separate the membrane vesicles through sucrose buoyant density gradient centrifugation. Thus, the mitochondrial outer membrane vesicles accumulate at low sucrose concentrations, while the mitochondrial inner membrane vesicles are enriched at high sucrose concentrations. The vesicles containing contact sites concentrate at intermediate sucrose concentrations (Figure 2). The following protocol describes this improved method, which requires less specialized equipment, time, and energy compared to our previously established one32, in detail and provides a useful tool for the identification of possible contact site proteins.

作者聚焦：揭开线粒体接触位点和架构见解 

一种分离线粒体接触位点的改进方法

We aim to understand the molecular function of mitochondrial contact sites, allowing the communication between the two mitochondrial membranes. This actually is a prerequisite to grasp the function for mitochondrial biogenesis and thus, cell viability. We discovered a new contact site between the mitochondrial inner and outer membrane created by Cqd1 and Om14-Por1.Our data suggests that this site might assist in lipid import like phosphatidic acid. Based on the identification of the MICOS complex, we contributed to get a deeper understanding of how mitochondrial membrane architecture is generated. This allowed us to develop a working model that explains how the two forms of mitochondrial cristae, lamellar and tubular cristae are formed.Our research was done in yeast in the past. However, we are convinced that the formation of correct mitochondrial architecture is important for higher eukaryotes too. So the next step in our research is analyzing the role of mitochondrial architecture elements in more complex models such as primary neurons.

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?...

Watch this Scientific Journal Video about Unveiling Mitochondrial Contact Sites and Architectural Insights at JoVE.com

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。

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

我们的目标是了解线粒体接触位点的分子功能，允许两个线粒体膜之间的交流。这实际上是掌握线粒体生物发生功能的先决条件，从而掌握细胞活力。我们发现了由 Cqd1 和 Om14-Por1 产生的线粒体内膜和外膜之间的新接触位点。我们的数据表明，该位点可能有助于磷脂酸等脂质输入。基于对MICOS复合物的鉴定，我们有助于更深入地了解线粒体膜结构是如何产生的。这使我们能够开发一个工作模型来解释线粒体嵴的两种形式，层状嵴和管状嵴是如何形成的。我们的研究过去是在酵母中完成的。然而，我们相信正确的线粒体结构的形成对于高等真核生物也很重要。因此，我们研究的下一步是分析线粒体结构元件在更复杂的模型（如初级神经元）中的作用。

an-improved-method-to-isolate-mitochondrial-contact-sites

Research

JoVE Journal

Biochemistry

An Improved Method to Isolate Mitochondrial Contact Sites

1.0K 次观看.  Ludwig-Maximilians University Munich. 我们的目标是了解线粒体接触位点的分子功能，允许两个线粒体膜之间的交流。这实际上是掌握线粒体生物发生功能的先决条件，从而掌握细胞活力。我们发现了由 Cqd1 和 Om14-Por1 产生的线粒体内膜和外膜之间的新接触位点。我们的数据表明，该位点可能有助于磷脂酸等脂质输入。基于对MICOS复合物的鉴定，我们有助于更深入地了解线粒体膜结构是如何产生的。这使我们...

视频: 作者聚焦：揭开线粒体接触位点和架构见解 

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

科研

生物化学

Generating Submitochondrial Vesicles from Saccharomyces cerevisiae Mitochondria to Isolate Mitochondrial Contact Sites

Generating Submitochondrial Vesicles from  Saccharomyces cerevisiae Mitochondria to Isolate Mitochondrial Contact Sites  

To begin, take 10 milligrams of freshly-isolated crude yeast mitochondria and re-suspend them in 1.6 milliliters of SM buffer at four degrees celsius by pipetting. Then, transfer the mitochondrial suspension to a pre-cooled 100-milliliter Erlenmeyer flask. Slowly, add 16 milliliters of the swelling buffer, using a 20-milliliter glass pipette while applying constant mild stirring on ice.Incubate the samples under constant mild stirring for 30 minutes on ice. Then, slowly add five milliliters of 2.5-molar sucrose solution using a five-milliliter glass pipette, allowing the inner membrane to shrink. Incubate the samples under mild stirring for 15 minutes on ice.To generate sub-mitochondrial membrane vesicles, transfer the mitochondrial suspension to a pre-cool rosette cell, and sonicate the suspension at 10%amplitude for 30 seconds while cooling the rosette cell in an ice bath. Rest the suspension for 30 seconds in an ice bath.

从酿酒酵母线粒体中生成提交 软 体囊泡以分离线粒体接触位点

Separation and Analysis of Submitochondrial Vesicles to Identify Mitochondrial Contact Site Proteins

To begin, take the prepared yeast sub-mitochondrial vesicles and centrifuge the generated vesicles and remaining intact mitochondria at 20, 000 G for 20 minutes at four degrees Celsius. The intact mitochondria formed the pellet while the vesicles stay in the supernatant. Next, transfer the supernatant to fresh ultra centrifugation tubes.Load a cushion of 0.3 milliliters of 2.5 molar sucrose solution at the bottom of the tube using a one milliliter syringe equipped with a cannula, and centrifuge at 118, 000 G for 100 minutes at four degrees Celsius. After centrifugation, the vesicles will appear as a disc on the top of the sucrose cushion. Discard approximately 90%of the supernatant.To harvest the concentrated vesicles, resuspend them in the remaining buffer, including the 2.5 molar sucrose by pipetting. Transfer the suspension to an ice cold Dounce homogenizer and homogenize the suspension using a polytetrafluoroethylene potter with at least 10 strokes. Next, prepare an 11 milliliter step gradient with each layer containing 2.2 milliliters of sucrose solution.Calculate the sucrose concentration using this formula. Add the highest sucrose concentration to the centrifugation tube and place it at minus 20 degrees Celsius until the layer is completely frozen. Measure the sucrose concentration of the samples using a refractometer.If a refractometer is unavailable, assume the sucrose concentration is 2 molar. To adjust the sucrose concentration to 0.6 molars, add the appropriate MOPS, E-D-T-A, P-M-S-F, and protease inhibitor cocktail at pH 7.4. Carefully load the samples on the sucrose gradient to avoid the disturbance of the gradient.Centrifuge the vesicles at 200, 000 G and four degree Celsius for 12 hours. If possible, set slow acceleration and deceleration to avoid disruption of the gradient. Then harvest the gradient from top to bottom in 700 microliters fractions, using a one milliliter pipette.Resulting in 17 fractions, which provides a sufficient resolution. Next, perform two sequentially executed trichloroacetic acid precipitations by adding 200 microliters of 72%trichloroacetic acid to the individual fractions and mixing until the solution is homogeneous. Incubate the fractions for 30 minutes on ice and pellet the precipitated proteins by centrifuging at 20, 000 G and four degrees Celsius for 20 minutes.Discard the supernatant. Add 500 microliters of 28%trichloroacetic acid solution. Mix well and repeat the centrifugation step.Finally, analyze the fractions by SDS-PAGE and immunoblotting. The importance of mild sonication is presented here. The mitochondrial outer membrane marker Tom40 was enriched in the early low density fractions and was virtually absent in the later ones.The mitochondrial inner membrane marker Tim17 was concentrated in high density fractions. In contrast, the contact site protein Mic60 accumulated infractions of intermediate sucrose concentration, indicating the successful generation and subsequent separation of the various membrane vesicles. In contrast, with harsher sonication conditions, the mitochondrial outer membrane marker Tom40 could be detected in lower fractions of the gradient.Additionally, there was a significant amount of Tim17 infractions of intermediate density.