细胞亚器文献阅读之酵母液泡与线粒体的动态互作A Dynamic Interface between Vacuoles and Mitochondria in Yeast

细胞亚器文献阅读之酵母液泡与线粒体的动态互作A Dynamic Interface between Vacuoles and Mitochondria in Yeast

本文和前一篇阅读的文献之间的关联，本文发现线粒体依赖于两个接触位点中的一个，ERMES或vCLAMP。前一天的文章也谈到这两个位点。

细胞的生命依赖于脂质和小分子在线粒体和内膜系统之间的连续运输。近年来，内质网-线粒体接触结构（ERMES）被认为是这种转运的一种重要但非必需的接触。利用酵母中的高含量筛选，作者在酵母溶酶体小室（Vam6/Vps39）和线粒体（vCLAMP）之间发现了一个接触位点。vCLAMP富含离子和氨基酸转运体，在内膜系统和线粒体之间的脂质传递中起作用。关键的是，作者发现线粒体依赖于两个接触位点中的一个，ERMES或vCLAMP。缺少一个会导致另一个的膨胀，而消除这两个都是致命的。鉴别vCLAMP增加了我们对细胞器间的互作复杂性的理解能力。

本文作者通过在不同的营养状态下，比如说葡糖糖和正常培养基培养，在三种参与两个膜接触位点的蛋白质的敲除的情况下，通过薄层层析的(TLC)的方法检测线粒体内的磷脂(包括心磷脂CL，磷脂酰乙醇胺，磷脂酰丝氨酸，磷脂酰肌醇)的含量的变化，与此同时，通过H3标记ser来检测ptdser向PtdEtn，进一步向PtdCho转化的过程，即线粒体中合成的磷脂酰乙醇胺和胆碱的情况。这些实验，在单敲除上述三种蛋白的情况下，各种磷脂的减少并不可见。这表明两种膜接触位点ERMES vCLAMP共同调节线粒体内的脂质与ER和Vacuoles囊泡之间的合成和原料的转运，以及不同的细胞器之间功能的协调。

引言部分

Mitochondria generate the majority of cellular energy and house enzymes required for the synthesis, breakdown, and intercon version of various species of amino acids, lipids, iron/sulfur clusters, and other small molecules. Due to their diverse functions and essential roles in cellular metabolism, mitochondria serve as hubs for signaling in events such as growth, differentiation, or cell death. Loss of optimal mitochondrial activity is therefore, not surprisingly, implicated in a growing number of human diseases as well as in aging。

线粒体产生合成、分解和相互转化各种氨基酸、脂质、铁/硫簇和其他小分子所需的大部分细胞能量和酶。由于线粒体在细胞代谢中的不同功能和重要作用，线粒体在诸如生长、分化或细胞死亡等事件中充当信号中枢。因此，毫不奇怪，线粒体最佳活性的丧失与越来越多的人类疾病和衰老有关。

The central tasks of mitochondria in cells necessitate constant communication and transport of small molecules with other organelles. However, mitochondria are not connected to the endomembrane system via the vesicular pathway. Instead, mitochondria have been shown to communicate with the endomembrane system by virtue of a membrane contact site (MCS) where membranes of mitochondria come into close proximity to membranes of the endoplasmic reticulum (ER). This MCS, also termed mitochondria-associated-membranes (Achleitner et al.,1999; Vance, 1990), enables ions and lipids to be rapidly transported in a nonvesicular manner (Elbaz and Schuldiner, 2011; Levine and Loewen, 2006; Tatsuta et al., 2014). Understanding the molecular machineries creating and regulating this MCS has been the arena of intense investigations in the past decade.

细胞内线粒体的中心任务需要小分子与其他细胞器的持续通讯和运输。然而，线粒体并不通过囊泡途径与内膜系统相连。相反，线粒体通过膜接触位点（MCS）与内膜膜系统进行通信，其中线粒体膜接近内质网（ER）的膜。这种MCS也称为线粒体相关膜（Achleitner等人，1999年；Vance，1990年），使离子和脂质以非特异方式快速运输（Elbaz和Schuldiner，2011年；Levine和Loewen，2006年；Tatsuta等人，2014年）。在过去的十年里，了解产生和调节这种MCS的分子机制一直是研究的热点。

In yeast, the molecular identity of the ER-mitochondria tethering complex was recently uncovered and is mediated by a four-protein complex termed the ER-mitochondria encounter structure (ERMES) (Kornmann et al., 2009). One of its hypothesized functions was to enable phospholipid transport (Kopec et al., 2010; Kornmann and Walter, 2010) required for building mitochondrial membranes as well as for the three-step biosynthetic pathway of aminoglycerophospholipids. Therefore, it was of great surprise when the loss of ERMES subunits had very little effect on cellular levels of aminoglycerophospholipids (Kornmann et al., 2009; Nguyen et al., 2012; Voss et al., 2012).
Hence, it became clear that alternate routes of phospholipid transport must exist in the cell, and uncovering them should shed light on novel modes of communication between mitochondria and the endomembrane system. We report here our findings of an MCS between mitochondria and vacuoles (the yeast lysosomal compartment) and its functional significance in lipid transport between the endomembrane system and mitochondria.

在酵母中，内质网线粒体系链复合物的分子特性最近被发现，并由一种称为内质网线粒体接触结构（ERMES）的四蛋白复合物介导（Kornmann等人，2009）。其假设功能之一是使磷脂运输（Kopec等人，2010年；Kornmann和Walter，2010年）成为构建线粒体膜(主要的是心磷脂的合成和转运，之前的文章总综述的4或5)以及氨基甘油磷脂三步生物合成途径所必需的。因此，当ERMES亚单位的缺失对细胞内氨基甘油磷脂水平的影响非常小时，这是非常令人惊讶的（Kornmann等人，2009；Nguyen等人，2012；Voss等人，2012）。因此，很显然，在细胞中必须存在磷脂转运的替代途径，而揭示它们的途径应该阐明线粒体和内膜膜系统之间的新的通讯模式。作者在此报告线粒体和液泡（酵母溶酶体室）之间MCS的发现及其在内膜系统和线粒体之间脂质转运中的功能意义。

The screen resulted in identification of over 100 different genetic backgrounds in which the Mdm34-GFP signal was altered (Table S1 available online). Strains that altered Mdm34-GFP morphology included deletion strains of ERMES components themselves that caused loss of the junction altogether, reduction in proteasomal subunits that caused an increase in protein levels, and loss of a large repertoire of mitochondrial proteins that caused a decrease in the intensity of foci. We focused on the four mutant backgrounds that displayed elevated number of foci per cell: Ddnm1, Dfis1 (Figure S1A), Dvam6/vps39 (hereinafter referred to as vps39), and Dvam7 (Figures 1B; Figures S1B and S1C).

Dnm1 and Fis1 are essential components of the mitochondrial fission machinery (Bleazard et al., 1999; Mozdy et al.,
A B Figure 1. A Genome-wide Screen to Uncover Mutants Affecting the Number of ERMES-Mediated Mitochondria-ER Contact Sites (A) Schematic representation of the screen flow. 1. A strain harboring a GFP-tagged ERMES subunit (Mdm34) was crossed against yeast KO and DAmP collections, and a library was created so that each strain harbors a tagged ERMES on the background of a single mutation. 2. Live cells were imaged using an automated image acquisition system. 3. Images were manually inspected for an increase in the number of ERMES foci. (B) Fluorescence images of the increase in Mdm34-GFP foci in deletion of either vps39 or vam7 relative to a WT background. Scale bars, 5 mm. See also Figure S1. 2000). Interestingly, it has recently been shown that mitochondrial/ER contact sites mark the sites for fission to occur (Friedman et al., 2011; Murley et al.,2013). We therefore reasoned that the increase in ERMES foci on these backgrounds is most probably a result
of an indirect effect on mitochondrial morphology or due to crosstalk with the ERMES complex.

通过高内涵的遗传筛选，发现Mdm34-GFP信号被改变。改变Mdm34-GFP形态的菌株包括导致连接完全丧失的ERMES组分本身的缺失菌株、导致蛋白质水平升高的蛋白酶体亚单位的减少以及导致病灶强度降低的线粒体蛋白的大量储备的丢失。我们聚焦于显示每个细胞聚焦数目增加的四个突变背景：Ddnm1、Dfis1（图S1A）Dvam6/vps39（以下称为vps39）和Dvam7。有趣的是，最近有研究表明，线粒体/ER接触位点标志着裂变发生的位点（Friedman等人，2011年；Murley等人，2013年）。因此，作者推断，在这些背景下，ERMES聚焦的增加很可能是间接影响线粒体形态或与ERMES复合体交叉对话的结果。

Vps39 and Vam7 have been previously characterized as components of the vacuolar fusion process (Price et al., 2000; Stroupe et al., 2006). These hits were surprising because no apparent direct link was reported between vacuolar morphology and ER-mitochondria connections or function. It has, however, previously been reported that the absence of Vps39 leads to impaired respiration capacity (Merz and Westermann, 2009). Therefore, we decided to
examine the connection between the absence of Vps39 to the increase in ERMES foci.

Vps39和Vam7先前被描述为空泡融合过程的组成部分（Price等人，2000；Stroupe等人，2006）。这些结果令人惊讶，因为在液泡形态和内质网线粒体连接或功能之间没有明显的直接联系。然而，此前有报道称，缺乏Vps39会导致呼吸能力受损（Merz和Westermann，2009）。因此，作者决定研究Vps39的缺失与ERMES病灶增加之间的关系。

Vps39 has been studied in depth for many years as part of the homotypic fusion and vacuole protein sorting (HOPS) tethering complex (Stroupe et al., 2006). In order to start and characterize its function at the vCLAMP, we decided to identify proteins interacting with GFP-Vps39 specifically at the contact site. To do that, we isolated cellular fractions enriched for mitochondria and performed pull-downs with an anti-GFP antibody. We reasoned that this should enrich for proteins that lie at the interface of the two organelles. Enriched proteins were identified by mass spectrometry analysis. (The complete list of significantly enriched proteins is given in Table S2.) The strongest interactions observed were among Vps39 and the additional five HOPS complex proteins (Figure S2A), as would be expected
from the established role of Vps39. Interestingly, in addition to these known interactions, we found nine small molecule transporters that were enriched in our samples in a statistically significant manner (false discovery rate [FDR] = 0.05; Table S3). We tagged all nine transporters with a GFP tag in order to verify their exact cellular localization. Five of the eight vacuolar proteins were not homogenously distributed over the vacuolar membrane but rather localized to specific patches (Figure S2B). Further colocalization with a mitochondrial marker showed that GFP-Mnr2 and GFP-Pho91 partially colocalized with mitochondria. (Figures S2C and S2D). This could suggest that the vCLAMP, marked by GFP-Vps39, could serve as a hub for interorganelle transport of small molecules.

Vps39作为同型融合和液泡蛋白分类（HOPS）系链复合物的一部分已经深入研究了多年。为了启动和表征其在vCLAMP中的功能，作者决定鉴定与GFP-Vps39特别是在接触位点相互作用的蛋白质。为此，作者分离了富含线粒体的细胞组分，并用抗GFP抗体进行了下拉。我们认为这应该富集于两个细胞器界面的蛋白质。富集蛋白经质谱分析鉴定。察到的最强相互作用是在Vps39和额外的五个啤酒花复合蛋白之间（图S2A），这是从Vps39的既定作用可以预料到的。有趣的是，除了这些已知的相互作用外，我们还发现了9种小分子转运体，它们以统计显著的方式富集在我们的样本中（错误发现率[FDR]=0.05；表S3）。我们用一个GFP标签标记了所有9个转运蛋白，以验证它们的准确细胞定位。八种液泡蛋白中有五种不是均匀分布在液泡膜上，而是定位在特定的斑块上（图S2B）。进一步的线粒体标记共定位显示GFP-Mnr2和GFP-Pho91部分与线粒体共定位。（图S2C和S2D）。这可能表明，以GFP-Vps39为标记的vCLAMP可以作为小分子在生物体内运输的枢纽。

Although 5%of PtdEtn is created in the late endomembrane system by the paralogous enzyme Psd2 (Trotter and Voelker, 1995), the mitochondrial Psd1 is the major enzyme, and so a large lipid flux between the ER and mitochondria should exist. If indeed vCLAMP and ERMES are coregulated to sustain constant contact area between the endomembrane system and mitochondria, then this would explain the lack of phenotype on PtdEtn levels caused by losing ERMES alone (Kornmann et al., 2009; Nguyen et al., 2012; Voss et al., 2012). It would also predict that concomitant loss of both MCSs would result in a more dramatic decrease in the phospholipids that require such transport events. To measure this effect, we created a conditional strain that is deleted for vps39 and expresses MDM34 under the control of a repressible GalS promoter. We eliminated additional pathways for de novo PtdEtn synthesis by growing cells in synthetic media depleted for ethanolamine, so as to limit the Kennedy pathway (Daum et al., 1998), and by deleting the late endomembrane localized PtdSer decarboxylase, Psd2 (Trotter and Voelker, 1995). Under these conditions, the only existing pathway for de novo PtdEtn synthesis involves the mitochondrial Psd1 and the obligation of transporting ER synthesized PtdSer into mitochondria.

两个线粒体接触位点的缺失导致内膜系统和线粒体之间的磷脂转运缺陷
磷脂在内膜系统和线粒体之间的转运是产生氨基甘油磷脂的三步酶促途径的关键（见图4H中的模型）。在这一途径中，内质网合成的磷脂酰丝氨酸（PtdSer）被线粒体膜内酶PtdSer脱羧酶1（Psd1）转运到线粒体转化为磷脂酰乙醇胺（PtdEtn）。一些PtdEtn然后被运送回ER，在ER中它可以作为磷脂酰胆碱的来源。5%的磷脂酰乙醇胺是由晚期内膜系统中的同源性酶PSD2（Trutter和Voelk，1995），线粒体PSD1催化产生的，而且后者是主要的酶，因此存在ER和线粒体之间的大的脂质流。如果vCLAMP和ERMES被证实确实共同调节以维持内膜系统和线粒体之间的恒定接触面积，那么这将解释单独丢失ERMES导致PtdEtn水平缺乏的表型。它还可以预测，伴随着两种mcs的丢失将导致需要这种转运事件的磷脂的更大幅度的减少。为了测量这种效应，我们创建了一个条件菌株，在抑制性GalS启动子的控制下，它被vps39删除并表达MDM34。我们通过在乙醇胺耗尽的培养基中培养细胞来消除从头合成PtdEtn的额外途径，从而限制Kennedy pathway（Daum等人，1998），并通过删除晚期内膜定位的PtdSer脱羧酶Psd2（Trotter和Voelker，1995）。在这些条件下，新的磷脂酰乙醇胺合成的唯一途径包括线粒体PSD1的合成和将ER合成的PTDSER转运到线粒体中这两种方式。

The phospholipid composition of the triple mutant compared to control cells was examined after growth for 24 hr in glucose (to repress GalSp-MDM34 expression), at which point the cells are still alive (Figure S4B) but mitochondrial shape is already affected (Figure S4C), indicating a depletion in Mdm34 levels. Thin-layer chromatography (TLC) of phospholipids extracted from whole cells (Figure 4A, independent quadruplicate quantified in Figure 4B) or enriched mitochondrial samples (Figure 4C, independent triplicate quantifiedinFigure 4D) demonstrated that the mutant has up to 40% decrease in the amounts of PtdEtn and cardiolipin (CL) (CL observed only in the enriched mitochondrial samples) concomitantly with near doubling in phosphatidylinositol (PtdIns) levels. Specifically, the decrease in PtdEtn could only be seen in this condition, whereas in the single mutants (Dvps39 or GalSp-MDM34 in glucose; data not shown) or after short-term repression of GalSp-MDM34 in the triple mutant (Figures S4D and S4E), this reduction was not seen. This demonstrates that only the concomitant loss of both MCSs causes a halt in shuttling of PtdSer. More generally, PtdEtn, CL, and PtdIns are all synthesized from a common precursor, phosphatidic acid (PA); however, PtdEtn and CL are synthesized in mitochondria, whereas PtdIns is synthesized in the ER. Thus, accumulation of PtdIns and reduction of CL most probably also reflect a defect in PA transport from ER to mitochondria (Figure 4H)

作者通过提取线粒体，从富集的线粒体中在用薄层色谱TLC的方法检测各种磷脂的含量，发现在有葡萄糖培养的情况下，三突变体中的PtdEtn和心磷脂（CL）的量减少了40%，而磷脂酰肌醇的含量反而加倍。具体来说，PtdEtn的减少仅在这种情况下可见，而在单突变体（在敲除vps39或GalSp-MDM34；数据未显示）或在三突变体中的GalSp-MDM34短期抑制（图S4D和S4E）后，未见这种减少。因此作者推测，这表明，只有同时失去两个MCS时，PtdSer才会停止穿梭。更普遍的是，PtdEtn、CL(心磷脂)和PtdIns都是由一种常见的前体磷脂酸（PA）合成的；然而，PtdEtn和CL是在线粒体中合成的，而PtdIns是在内质网中合成的。因此，PtdIns的积累和CL的减少很可能也反映了PA从内质网到线粒体的转运缺陷（图4H）。

本文总的机制图如下

In order to monitor the direct conversion of PtdSer to PtdEtn and, consequently, to PtdCho, we metabolically labeled the cells using3H-serine. The radioactive serine was incorporated in the ER to PtdSer, and, following phospholipid extraction and separation by TLC, we could follow its fate in control versus mutant cells. In the mutant cells, where vCLAMP is absent and ERMES is gradually depleted, there is a massive accumulation of PtdSer (PtdSer content mutant/wild-type = 1.75), suggesting that as both contacts between mitochondria and the endomembrane system are gradually collapsing, more and more PtdSer, which cannot be transported to mitochondria, is accumulating in the ER (Figure 4E and independent quadruplicate quantified in Figure 4F; see also Figure 4G). Quantification of the various lipid species demonstrates a large difference in the amounts of de novo synthesized PtdCho. While PtdCho accumulates in control cells, it is hardly synthesized in the mutant. This difference probably reflects the inability of PtdEtn that was synthesized in the mitochondria to be transported back to the ER for further processing. Taken together, the results of steady-state and de novo phospholipid amounts demonstrate the role of both ERMES and vCLAMP in phospholipid transport. More importantly, they highlight how well coordinated these back-up path ways are, as only elimination of both yields a dramatic change in cellular phospholipid levels.

为了监测PtdSer直接转化为PtdEtn，进而转化为PtdCho，我们用H3丝氨酸对细胞进行代谢标记。在ER-PtdSer中加入放射性丝氨酸，经磷脂提取和薄层色谱分离，我们可以观察其在对照细胞和突变细胞中的命运。在突变细胞中，vCLAMP缺失，ERMES逐渐耗尽，PtdSer大量积累,（PtdSer含量突变型/野生型=1.75），表明随着线粒体和内膜系统之间的接触逐渐塌陷，越来越多的不能运输到线粒体的PtdSer在内质网中积聚（图4E和图4F中量化的独立四倍体；另见图4G）。不同脂质种类的定量显示，从头合成的PtdCho的量存在很大差异。虽然PtdCho在对照细胞中积累，但在突变体中几乎不合成。这种差异可能反映了线粒体合成的PtdEtn不能被转运回内质网进行进一步的处理。总而言之，稳态和从头磷脂量的结果显示ERMES和vCLAMP在磷脂转运中都起作用。更重要的是，它们强调了这些备用途径的协调性，因为只有消除这两种途径，才能产生细胞磷脂水平的显著变化
.

讨论部分
Functional cooperation between mitochondria and other organelles is essential. Since mitochondria is not connected to the endomembrane system through vesicular trafficking, the ER mitochondria MCS serves as an important interorganellar interaction, allowing for fast and direct transport of phospholipids and Ca+2. We show here that, in yeast, mitochondria maintain a second MCS, vCLAMP, linking it to the endomembrane system through the vacuole. Since vacuoles are linked to the ER—both directly, through the nuclear vacuolar junction (NVJ), and indirectly, through vesicular traffic—this MCS can serve as a bypass for flow of information and nutrients between the ER and mitochondria. Indeed, we show that ERMES and vCLAMP are coregulated and that vCLAMP serves as a backup for ERMES, as only in the case where both are missing do yeast suffer from dramatic alternations in phospholipid levels and death.

线粒体与其他细胞器之间的功能合作是必不可少的。由于线粒体不通过囊泡运输与内膜系统相连，线粒体MCS起着重要的细胞间相互作用，使磷脂和Ca+2快速直接转运。我们在这里显示，在酵母中，线粒体维持第二个MCS，vCLAMP，通过液泡将其连接到内膜系统。由于液泡与内质网直接相连，通过核液泡连接（NVJ）和间接相连，通过囊泡交通，MCS可以作为内质网和线粒体之间信息和营养流动的旁路。事实上，我们证明了ERMES和vCLAMP是相互调节的，vCLAMP可以作为ERMES的备份，因为只有在两者都缺失的情况下，酵母才会遭受磷脂水平的剧烈变化和死亡。

The dynamic nature of vCLAMP and its apparent regulation through metabolic cues (see Ho ¨nscher et al., 2014) marks yet another link between intracellular organelle positioning, morphology, and degree of contact as determinants in the cell’s adaptation to changing metabolic requirements (Liesa and Shirihai, 2013) and cell fate (Csorda ´s et al., 2006).

vCLAMP的动态特性及其通过代谢线路的明显调节（见Ho–nscher等人，2014年）标志着细胞内细胞器的定位、形态和接触程度之间的另一个联系，作为细胞适应不断变化的代谢需求的决定因素（Liesa和Shirihai，2013年）和细胞命运.

To summarize, we demonstrate an MCS between vacuoles and mitochondria. This contact site, which we term vCLAMP, is in dynamic equilibrium with the ERMES mediated junction between mitochondria and the ER and works in parallel to enable the shuttling of small molecules (such as lipids) between the endomembrane system and mitochondria (model in Figure 4I). More generally, identification of the vCLAMP demonstrates the true complexity of interorganellar crosstalk: not only must an interface between two organelles be formed, but bypass systems must also be in place, and their size should be coregulated to serve the changing needs of the cell. Future efforts should be directed to elucidate the nature of the vCLAMP tether. Our proteomic analysis did not yield a definite answer as to how Vps39 is tethered to the mitochondria; whether through an adaptorproteinorayet-to-be discovered mitochondrial partner.Future studies aimed at understanding the coregulation of the vCLAMP and ERMES should provide novel insights into what the cells sense to ensure optimal MCS surface area. Having visual markers and the genetic capacity to alter junction size should now give a platform for in depth investigations of this new cellular organization module.

总而言之，我们证明在液泡和线粒体之间有一个MCS。这个接触点，我们称之为vCLAMP，与线粒体和内质网之间的ERMES介导的连接处处于动态平衡状态，并平行工作，使小分子（如脂质）能够在内膜系统和线粒体之间穿梭（图4I中的模型）。更一般地，识别vCLAMP证明了细胞间交叉对话的真实复杂性：不仅必须形成两个细胞器之间的接口，而且旁路系统也必须到位，并且它们的大小应该被调节以适应细胞的变化需要。今后的工作应该是阐明vCLAMP栓带的性质。我们的蛋白质组分析并没有给出一个明确的答案，即Vps39是如何与线粒体相连的；是否通过一种适应性或蛋白射线被发现为线粒体伴侣。未来的研究旨在了解vCLAMP和ERMES的共同调节作用，这将为细胞的感觉提供新的见解，以确保最佳MCS表面积。有了视觉标记和改变连接大小的遗传能力，现在应该为深入研究这个新的细胞组织模块提供一个平台。