A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides

Depletion of a subset of 70K stress proteins in yeast mutants shows that they are involved in the post-translational import of precursor polypeptides into both mitochondria and the lumen of the endoplasmic reticulum. The identification of such a basic function may explain the remarkable evolutionary conservation of the gene family encoding these proteins.     

Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins

The mitochondrial import receptors MOM19 and MOM72 form a complex with two other proteins of the mitochondrial outer membrane, MOM38 and MOM22. This receptor complex is involved in recognition, membrane insertion and translocation of precursor proteins with MOM38 constituting (at least part of) the general insertion site GIP.     

Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis

Mitochondrial heat-shock protein hsp60 functions in the folding of proteins imported into mitochondria. Folding occurs at the surface of hsp60 in an ATP-mediated reaction, followed by release of the bound polypeptides. We propose that hsp60 catalyses protein folding.     

松材线虫与拟松材线虫hsp70和hsp90基因结构特征及其分子进化

转录组的多态性是探讨物种多样性与分子进化的重要方面。笔者选取物种间保守性较高的HSP70、HSP90蛋白作为研究对象,探讨二者在松材线虫和拟松材线虫基因组中的结构差异,探明他们在松材线虫和拟松材线虫中的特点,并试图揭示两种线虫的系统发育关系。结果表明二者内含子具有丰富的多态性,内含子中的G+C含量也显著不同。核酸序列gABG和gmc2分别编码松材线虫和拟松材线虫HSP70的642个氨基酸,相似性高达99%,仅存在9个氨基酸的差异,并且这种差异是由13个碱基的多态性造成的。gABO和gmc19分别编码松材线虫和拟松材线虫的HSP90,其氨基酸序列相似性为92%。对同义密码子的偏好性分析表明,17种氨基酸对应的58个密码子中,gABG和gmc2存在10个密码子差异,gABO和gmc19存在5个密码子差异。采用邻接法分别构建的系统进化树表明松材线虫与拟松材线虫hsp70和hsp90在系统发育关系上最为接近,并与其他线性动物门物种高度同源。     

三株弧菌热休克蛋白70基因的克隆和序列分析

 根据GenBank中同类蛋白序列设计特异PCR引物,从2株创伤弧菌Vibrio vulnificus和1株河弧菌Vibrio fluvialis中扩增出热休克蛋 白70（heat shock protein, hsp70）基因片段。对这3个片段进行克隆、测序和分析的结果表明,3个片段长均为1 911 bp,包含完整的 hsp70 ORF,编码636个氨基酸。它们的氨基酸序列与GenBank中其它物种hsp70的氨基酸序列比较发现,2株创伤弧菌hsp70基因序列 和同种其它菌株的同源性高,达98%以上;而河弧菌的hsp70序列属首次克隆;与多种原核和真核生物的hsp70氨基酸序列一起构建 了系统进化树,结果支持传统的分类结果。     

原位杂交检测hsp70 mRNA在黑腹果蝇胚胎中的表达

利用设计合成的地高辛标记的黑腹果蝇(Drosophila melanogaster)hsp70 DNA探针,整体原位杂交检测热休克反应中,hsp70 mRNA在胚胎发育各阶段的表达情况.实验发现,在正常温度下黑腹果蝇的胚胎检测不到hsp70 mRNA存在.在热激的果蝇胚胎中,可检测到hsp70 mRNA的广泛存在.hsp70 mRNA在热激的胚胎中,除早期细胞化囊胚的极细胞外,其余细胞均可检测到hsp70 mRNA的均一分布.随着胚胎发育,hsp70 mRNA仍广泛分布在各细胞中,晚期胚胎中神经细胞hsp70 mRNA比其余细胞的表达水平高,说明神经细胞具有更高的热诱导活性,在热激反应中hsp70 mRNA的表达水平更高.     

Interpreting the folding kinetics of helical proteins.

The detailed mechanism of protein folding is one of the major problems in structural biology. Its solution is of practical as well as fundamental interest because of its possible role in utilizing the many sequences becoming available from genomic analysis. Although the Levinthal paradox (namely, that a polypeptide chain can find its unique native state in spite of the astronomical number of configurations in the denatured state) has been resolved, the reasons for the differences in the folding behaviour of individual proteins remain to be elucidated. Here a Calpha-based three-helix-bundle-like protein model with a realistic thermodynamic phase diagram is used to calculate several hundred folding trajectories. By varying a single parameter, the difference between the strength of native and non-native contacts, folding is changed from a diffusion-collision mechanism to one that involves simultaneous collapse and partial secondary-structure formation, followed by reorganization to the native structure. Non-obligatory intermediates are important in the former, whereas there is an obligatory on-pathway intermediate in the latter. Our results provide a basis for understanding the range of folding behaviour that is observed in helical proteins.     

不同折叠类型蛋白编码基因的密码子使用

对195个编码不同折叠类型蛋白（50种全α结构蛋白，66种全β结构蛋白，37种α β结构蛋白，42种α/β结构蛋白）基因的密码子使用偏性的方差分析研究表明，不同折叠类型蛋白的密码子使用偏性有着显著的区别，特定折叠类型的蛋白有着特定的编码基因的密码子使用模式，这一结果表明，在蛋白质折叠类型和蛋白质二级结构的预测过程中，编码基因的密码子使用偏性可以作为蛋白质一级结构以外的一项重要的预测标准。     

线粒体膜间隙蛋白质释放与细胞凋亡研究进展

近年来,国内外有关线粒体和肿瘤的关系研究的越来越多,开发与线粒体相关的新型药物并诱导肿瘤细胞凋亡逐渐成为热点.该文主要综述了近年来关于几种线粒体膜间隙蛋白质在细胞凋亡中的作用和由线粒体中释放的可能机制及其在肿瘤药物治疗中的最新进展,指出线粒体膜间隙蛋白质在诱导肿瘤细胞凋亡中具有重要作用.     

Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria

A nuclear encoded mitochondrial heat-shock protein hsp60 is required for the assembly into oligomeric complexes of proteins imported into the mitochondrial matrix. hsp60 is a member of the 'chaperonin' class of protein factors, which include the Escherichia coli groEL protein and the Rubisco subunit-binding protein of chloroplasts.     

Trigger factor and DnaK cooperate in folding of newly synthesized proteins.

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro. Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality. Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins. In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.     

不同折叠类型蛋白中基于密码子的二级结构偏向性分析

在传统的Chou-Fasman蛋白质二级结构预测方法的基础上引入同义密码子使用的信息,计算了200个蛋白(49种全α结构蛋白,69种全β结构蛋白,38种仅α β结构蛋白,44种α/β结构蛋白)中不同密码子对应的氨基酸形成不同二级结构(α：螺旋,β：折叠,C：卷曲)的偏向性参数．通过对这些密码子对应氨基酸二级结构偏向性的分析,得到了氨基酸二级结构偏向性分析中所忽略的同义密码子的蛋白结构信息．这些新的信息量对于指导蛋白质设计以及提高蛋白质二级结构预测的准确率有着一定的作用．     

A family of mitochondrial proteins involved in bioenergetICS and biogenesis

The respiratory chain complexes of mitochondria consist of many different subunits, of which only a few partake directly in electron transport. The functions of the subunits that do not contain prosthetic groups are largely unknown. The cytochrome reductase complex of Neurospora crassa, for examine, consists of nine different subunits, of which the peripheral membrane proteins I and II (ref.3) that are located on the matrix side of the mitochondrial inner membrane are the largest subunits devoid of redox centres. Significantly, a cytochrome reductase fraction lacking these two subunits was inactive in electron transfer, and in yeast mutants with defective genes for either of the two subunits, assembly of the reductase is disrupted. Most mitochondrial proteins are imported into the mitochondrion as precursor proteins, and two proteins are necessary for cleaving their presequences, namely the matrix processing peptidase (MPP) and the processing enhancing protein (PEP), the latter strongly stimulating the activity of the former. Temperature-sensitive yeast mutants, which are affected in PEP or MPP, accumulate precursors at the nonpermissive temperature. We report here that subunit I of the cytochrome reductase can be grouped as members of the same protein family.     

Pumping of protons from the mitochondrial matrix by cytochrome oxidase

The stoichiometry and mechanism of redox-linked proton translocation by the mitochondrial respiratory chain is a major issue of debate in membrane bioenergetics. The function of cytochrome oxidase is a focal point of disagreement. In 1977 it was suggested that the terminal component of the respiratory chain, cytochrome oxidase, functions as a redox-linked proton pump. That and subsequent studies were based mainly on measurements of proton ejection from mitochondria or from vesicles reconstituted with isolated cytochrome oxidase, or on measurements of translocation of electrical charge equivalents across mitochondrial and vesicle membranes. This proton-translocating function of cytochrome oxidase is confirmed here by a quantitative determination of proton uptake from the inside (matrix) of intact mitochondria.     

Mapping of the protein import machinery in the mitochondrial outer membrane by crosslinking of translocation intermediates.

Mitochondria contain a complex machinery for the import of nuclear-encoded proteins. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein). Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were crosslinked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOM8, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.     

New variation on the translocation of proteins during early biogenesis of apolipoprotein B

Apolipoprotein B (apo B) is crucial for the transport of cholesterol in humans. It is a large secretory protein that mediates the uptake of low-density lipoproteins and renders several forms of lipid droplets soluble in the blood. The binding of lipid by apo B also prevents this hydrophobic protein from precipitating in aqueous solution. In the endoplasmic reticulum, nascent secretory proteins must be translocated through an aqueous channel in the membrane into the aqueous lumen, so some novel form of processing may be necessary to maintain the solubility of apo B during its translocation. We have discovered that the biogenesis of apo B in cell-free systems does indeed involve a new variation on protein translocation: unlike typical secretory proteins, apo B is synthesized as a series of transmembrane chains with large cytoplasmic domains and progressively longer amino-terminal regions that are protected against added proteases during the translocation process. In contrast to typical transmembrane proteins, these transmembrane chains are not integrated into the bilayer. Moreover, the transmembrane chains with the shortest protected domains are precursors of forms whose protection is progressively extended to cover the length of the protein. This stepwise conversion occurs post-translationally for the most part. We propose a model on the basis of these findings for the biogenesis of apo B.     

最小实体要求及其可逆要求的应用

本文简要介绍了一种新的公差原则，最小实体要求及其可逆要求，并结合镗模的精度设计，分析了它的应用特点，从而说明该公差原则确有提高技术经济效益的使用价值．     

Evidence for kinks in DNA folding in the nucleosome

The nucleosome subunit of chromatin consists of DNA folded around a histone core as a 1.8-turn left-handed solenoid. The crystal structure of the nucleosome core particle revealed that it has a dyad symmetry axis and that the minor helix groove faces outwards from the protein core. Richmond et al. noticed that the path traversed by the helix has severe bends at sites approximately one and four helix turns from the dyad axis. We have developed two photochemical methods to study the structure of DNA, and in particular that wrapped around the nucleosome core. One method depends on the sensitization of singlet oxygen production by an eosin analogue. We have monitored the rate at which excited state oxygen diffuses into contact with DNA base planes, and find that it attacks the nucleosome with high specificity. We have also mapped the DNA binding of the intercalating dye methylene blue, and conclude that it binds to the same sites accessible to oxygen by diffusion. On the basis of these results we suggest that the DNA in the nucleosome is bent or kinked at two sites, 1.5 helix turns from the dyad axis.     

Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex

Secretory-protein translocation into the endoplasmic reticulum (ER) is thought to be catalysed by integral membrane proteins. Genetic selections uncovered three Saccharomyces cerevisiae genes (SEC61, SEC62 and SEC63), mutations in which block import of precursor proteins into the ER lumen in vivo and in vitro. The DNA sequences of SEC62 and SEC63 predict multispanning membrane proteins, and biochemical characterization of the SEC62 protein (Sec62) confirms that it is an integral ER membrane protein. Here we show that Sec61, Sec62 and Sec63 are assembled with two additional proteins into a multisubunit membrane-associated complex. These results confirm previous predictions, based upon genetic interactions between the SEC genes, that Sec61, Sec62 and Sec63 act together to facilitate protein translocation into the ER.