蛋白质跨线粒体膜运送的研究进展

线粒体拥有约1000种蛋白质,其中98％以上系由细胞核编码,在细胞质核糖体上以前体形式合成,之后再运至线粒体,经跨膜运送并分选定位于各部分。现对定位于外膜、基质和内膜的蛋白质的运送途径的研究进展作一扼要介绍。脱血红素细胞色素c是细胞色素c的前体,它不含导肽,对其转运的研究概况也作了评述。     

磷脂酸(PA)-磷脂酰乙醇胺(PE)相互作用有利于脱血红素细胞色素c跨脂质体的运送

细胞色素c的前体 -脱血红素细胞色素c(Apocyt.c)在细胞质中核糖体合成 ,之后跨线粒体膜运送 ,在线粒体内、外膜间隙中经酶催化与血红素Heme结合形成成熟型的细胞色素c定位于线粒体内膜外侧     

植物呼吸及代谢的研究 Ⅷ.水稻幼苗线粒体电子传递的多条途径

由差异光谱的测定结果表明,水稻幼苗的线粒体呼吸链存在细胞色素系统的所有电子载体:黄素蛋白、细胞色素 b、细胞色素 c、细胞色素 a 和细胞色素 a_3。用氧电极测定及铁氰化物还原的结果表明,在水稻线粒体中至少存在四条电子传递途径:第一条定位于线粒体内膜内侧,对鱼藤酮、抗霉素 A 及氰化物敏感;第二条也定位于线粒体内膜内侧,但对鱼藤酮不敏感;第三条能氧化外源的 NADH,对鱼藤酮不敏感,而受抗霉素 A抑制,其 NADH 脱氢酶定位于线粒体内膜外侧;第四条途径在氧化外源 NADH 时,如有外源细胞色素 c 存在,可通过外膜上对抗霉素 A 不敏感的 NADH 脱氢酶进行电子传递。由此可以得出结论,高等植物以水稻为代表,其线粒体中的电子传递途径也是多条的。     

线粒体与细胞凋亡机制

细胞凋亡是生理性的细胞死亡过程,受到多种基因的精确调节,一类被统称为ｃａｓｐａｓｅ的半胱氨酸蛋白酶是细胞凋亡程序的执行者,综们被激活后作用于细胞内的一些蛋白质,经起细胞凋亡。线粒体中含有许多凋亡相关因子,在凋亡信号转导中起着重要作用。细胞受到凋亡刺激后,细胞色素ｃ、ＡＩＦ、ｃａｓｐａｓｅ－９等凋亡相关因子从线粒体中释放出来。细胞色素ｃ通过和Ａｐａｆ－１、ｃａｓｐａｓｅ－９相互作用,激活ｃａｓｐａｓ     

灵芝孢子和L-NNA对脊髓损伤后背核线粒体细胞色素氧化酶活性的影响

目的探讨灵芝孢子和一氧化氮合酶 (NOS)抑制剂L-NNA联合应用对大鼠脊髓半横断后受损伤的背核线粒体细胞色素氧化酶活性的影响.方法将20只SD成年雌性大鼠(200-250g)行右侧T11脊髓半横断30d后,对受损伤脊髓做细胞色素氧化酶酶组化染色;用图像分析方法检测L1脊髓段背核线粒体细胞色素氧化酶活性的变化,用酶组化电镜技术观察L1脊髓段背核细胞色素氧化酶活性的分布位置.结果与对照组相比,L-NNA组和灵芝孢子组L1脊髓损伤侧背核线粒体细胞色素氧化酶活性有所提高,灵芝孢子 L-NNA组损伤侧背核线粒体细胞色素氧化酶活性最大.各组L1脊髓背核细胞色素氧化酶活性均出现在线粒体内,具有细胞色素氧化酶活性的线粒体存在所有神经元胞体及其树突和轴突内,也存在于神经胶质细胞胞体及其突起内.结论灵芝孢子和L-NNA均可提高大鼠脊髓半横断后受损伤的脊髓背核线粒体细胞色素氧化酶的活性,两者联合应用更能提高受损伤的背核线粒体细胞色素氧化酶的活性.     

凋亡诱导因子与细胞凋亡

凋亡诱导因子 (apoptosisinducefactor,AIF)是定位于线粒体膜间隙中的一种氧化还原酶 ,含有线粒体定位信号和核定位信号序列 ,具有很强的促凋亡活性 ,在类胚体成腔和胚胎早期分化过程中具有重要作用。在死亡信号或细胞胁迫的刺激下 ,线粒体通透性转变孔开放 ,释放AIF及细胞色素c至细胞质溶质中 ,具有核定位信号序列的AIF便进入细胞核内 ,引起染色质的初步凝集和DNA大规模断片化 (约 5 0kb) ,进而引发不依赖于胱冬肽酶的细胞凋亡途径 ;线粒体膜间隙释放出来的细胞色素c则可引起染色质的进一步凝集和DNA的寡核小体断片化 ,从而引发依赖于胱冬肽酶的细胞凋亡途径 ;与此同时 ,从线粒体膜间隙释放出来的AIF又可反馈放大线粒体通透性转变孔的渗透性 ,引起AIF与细胞色素c的进一步释放从而加快细胞死亡的进程。此外 ,细胞胁迫还可激活由多聚 (ADP 核糖 )聚合酶 1(PARP 1)所引发的细胞凋亡途径 ,通过AIF和细胞色素c引发细胞凋亡。最新研究结果表明 ,AIF同源线粒体关联死亡诱导者 (AIF homologousmitochondria associatedinducerofdeath ,AMID)与p5 3应答基因的编码产物 (p5 3 responsivegene 3,PRG3)均为AIF的同源蛋白质 ,可直接诱导人类细胞的凋亡。线虫的凋亡诱导因子WAH 1所诱导的细胞凋亡途径依赖于胱冬肽酶     

肺细胞色素氧化酶体视学测量法在高压氧研究中的应用

采用超微结构细胞化学方法,用计算机图象分析仪测量计算了小鼠肺毛细血管和Ⅱ型肺泡线粒体、线粒体膜和嵴上细胞色素氧化酶活性变化的二维形态学、三维立体学定量参数。结果表明,毛细血管线粒体面积较Ⅱ型肺泡细胞小,但体密度却大;毛细血管线粒体细胞色素氧化酶阳性反应面积比正型肺泡细胞小,但酶反应体密度却大、暴露于高压氧（０．５ＭＰａ）后小鼠Ⅱ型肺泡细胞线粒体细胞色素氧化酶活性降低。     

蛋白质亚叶绿体和亚线粒体定位预测研究进展

蛋白质合成后被转运到特定的细胞器中,只有转运到正确的部位才能参与细胞的各种生命活动,有效地发挥功能,因此蛋白质的功能与其亚细胞定位有着密切的联系,通过确定蛋白质在细胞中的位置可以获取蛋白质功能和结构的信息。在近二十年中,蛋白质亚细胞定位预测算法研究已经取得很大的成绩,在此基础上,蛋白质在细胞器内亚结构的定位预测研究,如对蛋白质亚线粒体和亚叶绿体定位的研究成为更深层次的问题,本文简要介绍国内外在蛋白质亚叶绿体和亚线粒体定位预测方面的研究进展。     

线粒体蛋白质的转运

线粒体含有约1000种蛋白质,其中99%由细胞核DNA编码,在细胞质核糖体上合成后被分别转运至线粒体的内膜或外膜上、基质或膜间隙中。由众多分子机器组成的线粒体蛋白质转运系统参与了该生物学过程的执行。线粒体DNA编码的13种蛋白质也由该系统转运至线粒体内膜。本文就线粒体蛋白质转运系统中线粒体前体蛋白质的定位分选信号、转运复合物和转运途径作简要介绍。     

细胞色素c与细胞凋亡

在哺乳动物细胞凋亡的发生过程中,位于线粒体内膜外侧的细胞色素ｃ被释放进入胞质,作为辅助因子参与死亡蛋白酶－３的激活,后者在凋亡过程中起到重要作用。Ｂｃｌ－２、Ｂｃｌ－ＸＬ,Ｂａｘ可阻止或诱导线粒体细胞色素ｃ释放入胞质,这可能是Ｂｃｌ－２,Ｂｃｌ－ＸＬ,Ｂａｘ调节凋亡的机制之一。     

An unusual mitochondrial import pathway for the precursor to yeast cytochrome c oxidase subunit Va

We have studied the import of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. Like the majority of mitochondrial precursor proteins studied thus far, import of presubunit Va was dependent upon both a membrane potential (delta psi) and the hydrolysis of ATP. However, the levels of ATP necessary for the import of presubunit Va were significantly lower than those required for the import of a different mitochondrial precursor protein, the beta subunit of the F1-ATPase. The rate of import of presubunit Va was found to be unaffected by temperature over the range 0 to 30 degrees C, and was not facilitated by prior denaturation of the protein. These results, in conjunction with those of an earlier study demonstrating that presubunit Va could be efficiently targeted to mitochondria with minimal presequences, suggest that the subunit Va precursor normally exists in a loosely folded conformation. Presubunit Va could also be imported into mitochondria that had been pretreated with high concentrations of trypsin or proteinase K (1 mg/ml and 200 micrograms/ml, respectively). Furthermore, the rate of import into trypsin-treated mitochondria, at both 0 and 30 degrees C, was identical to that observed with the untreated organelles. Thus, import of presubunit Va is not dependent upon the function of a protease-sensitive surface receptor. When taken together, the results of this study suggest that presubunit Va follows an unusual import pathway. While this pathway uses several well-established translocation steps, in its entirety it is distinct from either the receptor-independent pathway used by apocytochrome c, or the more general pathway used by a majority of mitochondrial precursor proteins.     

Erv1 mediates the Mia40-dependent protein import pathway and provides a functional link to the respiratory chain by shuttling electrons to cytochrome c

Unlike matrix-targeted or inner membrane proteins, those that are targeted to the mitochondrial intermembrane space (IMS) do not require ATP or the inner membrane electrochemical potential. Their import is mediated primarily by the essential IMS protein Mia40/Tim40. Here, we show that the mitochondrial flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidase Erv1 (essential for respiration and vegetative growth 1) plays a central role in the biogenesis of small, cysteine proteins of the IMS that are import substrates for Mia40. In a temperature-sensitive strain of Erv1, steady-state levels of small translocases of the inner membrane (Tims) are specifically affected when cells are grown at the non-permissive temperature. Furthermore, mitochondria isolated from the erv1-ts show a specific import and assembly defect for the small Tims but not in any other protein import pathway. Erv1 does not directly oxidise the small Tims, as thiol trapping assays show that the small Tims can still be oxidised in erv1-ts cells grown at the non-permissive temperature and in isolated mitochondria from this strain. Moreover, addition of pure Erv1 into erv1-ts mitochondria lacking the endogenous protein restores import and assembly of the small Tims only to an extent, arguing for a cascade of interactions with Erv1 rather than for a direct interaction of Erv1 with the small Tims. Cytochrome c (cyt c) is the in vivo oxidase for Erv1, as yeast cells mutated in cyt c cannot grow under anaerobic conditions. Therefore, Erv1 functionally links the Mia40-dependent import pathway to the Mia40-independent cyt c import pathway transferring electrons from the incoming precursors to cyt c as an acceptor. In this context, the protein import process is linked to the respiratory chain via the communication of Erv1 with cyt c.     

The Mitochondrial Protein Import Pathway: Are Precursors Imported through Membrane Channels?

Mitochondrial biogenesis requires the import of hundreds of different proteins from the cytosol. Protein import into mitochondria is a multistep pathway that includes recognition of precursor proteins by machinery both in the cytoplasm and on the mitochondrial surface, translocation of the precursor across one or both mitochondrial membranes, and folding of the protein after its import into the organelle. Over the past several years, many components of the import machinery have been identified using both biochemical and genetic methods. Recently, significant progress has been made determining the function of some of these import proteins. One purpose of this minireview is to summarize our current understanding of the import pathway, and to introduce the topics of the minireviews that will follow. The other goal of this minireview is to discuss recent findings suggesting that proteins are translocated across both the mitochondrial inner and outer membranes through aqueous channels.     

Apocytochrome c requires the TOM complex for translocation across the mitochondrial outer membrane

The import of proteins into the mitochondrial intermembrane space differs in various aspects from the classical import pathway into the matrix. Apocytochrome c defines one of several pathways known to reach the intermembrane space, yet the components and pathways involved in outer membrane translocation are poorly defined. Here, we report the reconstitution of the apocytochrome c import reaction using proteoliposomes harbouring purified components. Import specifically requires the protease-resistant part of the TOM complex and is driven by interactions of the apoprotein with internal parts of the complex (involving Tom40) and the 'trans-side receptor' cytochrome c haem lyase. Despite the necessity of TOM complex function, the translocation pathway of apocytochrome c does not overlap with that of presequence-containing preproteins. We conclude that the TOM complex is a universal preprotein translocase that mediates membrane passage of apocytochrome c and other preproteins along distinct pathways. Apocytochrome c may provide a paradigm for the import of other small proteins into the intermembrane space such as factors used in apoptosis and protection from stress.     

Apocytochrome c: an exceptional mitochondrial precursor protein using an exceptional import pathway

The cytochrome c import pathway differs markedly from the general route taken by the majority of other imported proteins, which is characterized by the import involvement of namely, surface receptors, the general insertion protein (GIP), contact sites and by the requirement of a membrane potential (delta psi). Unique features of both the cytochrome c precursor (apocytochrome c) and of the mechanism that transports it into mitochondria, have contributed to the evolution of a distinct import pathway that is not shared by any other mitochondrial protein analysed thus far. The cytochrome c pathway is particularly unique because i) apocytochrome c appears to have spontaneous membrane insertion-activity; ii) cytochrome c heme lyase seems to act as a specific binding site in lieu of a surface receptor and; iii) covalent heme addition and the associated refolding of the polypeptide appears to provide the free energy for the translocation of the cytochrome c polypeptide across the outer mitochondrial membrane.     

The mitochondrial intermembrane space: the most constricted mitochondrial sub-compartment with the largest variety of protein import pathways

The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.     

Ancestral and derived protein import pathways in the mitochondrion of Reclinomonas americana

The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or "translocase in the inner mitochondrial membrane complex." Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.     

Trypanosoma brucei cytochrome c1 is imported into mitochondria along an unusual pathway

In most eukaryotic organisms, cytochrome c(1) is encoded in the nucleus, translated on cytosolic ribosomes, and directed to its final destination in the mitochondrial inner membrane by a bipartite, cleaved, amino-terminal presequence. However, in the kinetoplastids and euglenoids, the cytochrome c(1) protein has been shown to lack a cleaved presequence; a single methionine is removed from the amino terminus upon maturation, and the sequence upstream of the heme-binding site is generally shorter than that of the other eukaryotic homologs. We have used a newly developed mitochondrial protein import assay system from Trypanosoma brucei to demonstrate that the T. brucei cytochrome c(1) protein is imported along a non-conservative pathway similar to that described for the inner membrane carrier proteins of other organisms. This pathway requires external ATP and an external protein receptor but is not absolutely dependent on a membrane potential or on ATP hydrolysis in the mitochondrial matrix. We propose the cytochrome c(1) import in T. brucei is a two-step process first involving a membrane potential independent translocation across the outer mitochondrial membrane followed by heme attachment and a membrane potential-dependent insertion into the inner membrane.