Isolation and characterization of the gene for a yeast mitochondrial import receptor

We have previously identified an integral membrane protein (p32) from Saccharomyces cerevisiae as a receptor for protein import into mitochondria, and have localized it to the mitochondrial outer membrane at contact sites. Here we report isolation of the corresponding mitochondrial import receptor gene, termed MIR1. The deduced amino-acid sequence of p32 shows roughly 40% identity with proteins of bovine heart and rat liver that have been suggested to be mitochondrial phosphate carriers. Haploid cells carrying a disrupted MIR1 allele were unable to grow on a non-fermentable carbon source but grew in media containing glucose, indicating that the MIR1 protein is essential for mitochondrial function. Compared with wild type, amounts of some mitochondrial proteins were markedly reduced in cells containing a disrupted MIR1 allele, whereas levels of others were unchanged. This indicates that yeast contains more than one pathway for protein import into mitochondria.     

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.     

Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.

Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains. Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections. The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown. Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig. 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base. Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine. The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells.     

Single gene circles in dinoflagellate chloroplast genomes.

Photosynthetic dinoflagellates are important aquatic primary producers and notorious causes of toxic 'red tides'. Typical dinoflagellate chloroplasts differ from all other plastids in having a combination of three envelope membranes and peridinin-chlorophyll a/c light-harvesting pigments. Despite evidence of a dinoflagellete satellite DNA containing chloroplast genes, previous attempts to obtain chloroplast gene sequences have been uniformly unsuccessful. Here we show that the dinoflagellate chloroplast DNA genome structure is unique. Complete sequences of chloroplast ribosomal RNA genes and seven chloroplast protein genes from the dinoflagellate Heterocapsa triquetra reveal that each is located alone on a separate minicircular chromosome: 'one gene-one circle'. The genes are the most divergent known from chloroplast genomes. Each circle has an unusual tripartite non-coding region (putative replicon origin), which is highly conserved among the nine circles through extensive gene conversion, but is very divergent between species. Several other dinoflagellate species have minicircular chloroplast genes, indicating that this type of genomic organization may have evolved in ancestral peridinean dinoflagellates. Phylogenetic analysis indicates that dinoflagellate chloroplasts are related to chromistan and red algal chloroplasts and supports their origin by secondary symbiogenesis.     

A yeast mitochondrial outer membrane protein essential for protein import and cell viability

The gene encoding ISP42, an integral outermembrane protein located at the yeast mitochondrial protein import site was cloned, sequenced and modified. Yeast cells depleted of ISP42 accumulate uncleaved mitochondrial precursor proteins and then die. ISP42 is the first mitochondrial membrane protein shown to be indispensable for protein import and cell viability.     

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.     

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.     

线粒体蛋白转运系统的序列相似性分析

通过在27个不同进化层次物种的基因组和蛋白组中搜索酵母线粒体蛋白转运系统亚基的同源序列, 并进一步分析了同源亚基序列相似性与其所在线粒体位置的关系. 结果表明, 位于线粒体相同位置的模块有类似的序列相似性曲线, 相似性曲线在模块内部一般有波峰和波谷. 从线粒体外膜到基质, 序列相似性整体升高. 线粒体蛋白转运系统亚基与一些功能不相关的蛋白也表现出序列相似关系, 且这些亚基多集中在线粒体的内膜和外膜.     

真蓝甲藻(Gymnodinium eucyanochroum Tseng Sp.nov.)的亚微结构、色素性质及其在藻类进化中的意义

本文报告了在南京发现的蓝色裸甲藻,测定了它的藻胆色素。证明蓝色藻胆蛋白不是山“胞内蓝藻”(Cyanellen)所提供。电子显微镜的扫描表明细胞表面有众多的突起,不是光滑的;横沟内的鞭毛不是“带状”而是由细纤维丝膜状物拉着的螺旋形。透视电子显微镜观察表明,该藻有两个类型的细胞核,即“甲藻核结构”(Dinocaryotic structure)和“真核结构”,(Eucaryotic Structure),真核与叶绿体有一个共同的膜的包被,有一个与原生动物相近似的伸缩泡系统。叶绿体是分枝状,在细胞的边缘位,但也有其它形态。有淀粉颗粒而无“造粉核”或称”“蛋白核”(Pyrenoid),多数位于叶绿体外或之间,类囊体与一般甲藻不同,不是三个排成一条“带”而是两个排列成“带”。有发达的线粒体,和高尔基体;鞭毛不论纵沟内的或横沟内的,其横切面,均为9+2的形式,其纵切面是由纤维丝成束的结构。蓝色色素提取物,可见光最大吸收峰为456nm,与隐藻藻胆色素十分相似。从细胞亚微结构及其色素性质,作者认为它是藻类演化过程中的一个中间类型的甲藻共生体。     

Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

The ε subunit of the chloroplast ATP synthase and the truncated ε mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coil When the ε subunit or the truncated ε proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of ε-deficient membrane reconstituted with the ε subunit or the truncated ε proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type ε protein. These results suggested that: (a) the N-terminal domain of the ε subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of ε subunit; and (b) the C-terminal domain of the ε subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.     

Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter

The complementary DNA sequence encoding the cell-surface receptor for ecotropic host-range murine retroviruses (ecoR) shows that it contains 622 amino acids and 14 hydrophobic potentially membrane-spanning sequences. Because this receptor occurs on many or all murine cells and is probably essential for viability of cultured fibroblasts, its normal function might be to transport an essential metabolite. We expressed ecoR in Xenopus laevis oocytes by injecting RNA transcribed from the cloned cDNA. These oocytes specifically bound the gp70 envelope glycoprotein from an ecotropic murine leukaemia virus. An inward current was recorded electrophysiologically when a mixture of amino-acids was applied: this resulted from a stereoselective, saturable uptake of lysine, arginine and ornithine; it was independent of sodium and not substantially altered by gp70. Cysteine and homoserine were also taken up, but sodium was necessary for their transport. These properties of ecoR correspond to those of the y+ amino-acid transporter. Our results demonstrate the subversion of a ubiquitous cell membrane protein, in this case a basic amino acid transporter, for use as a retroviral receptor.     

菜豆锈病菌侵染对寄主生理代谢的影响

从细胞膜透性、呼吸强度、光合作用及游离氨基酸和蛋白质代谢等几方面,对菜豆锈病菌(Uromyces appendiculatus)侵染与菜豆生理代谢变化的关系进行了研究.结果表明U.appendiculatus侵染可导致被侵染菜豆叶片细胞膜透性的增加,但这种增加在抗感程度不同的品种中出现的早晚和程度不同; 抗感品种在接种后呼吸强度都增加,但不同品种呼吸强度升高的时间和程度有异;U.appendiculatus对寄主光合作用的影响体现在叶绿体含量的降低及光合产物(糖和淀粉)的异常积累;接种前后菜豆叶片中蛋白质含量无明显变化,但个别游离氨基酸的含量在抗病品种中有变化.     

唐古特虎耳草叶绿体和线粒体超微结构的研究

通过对生长在青藏高原东北部高海拔地区的唐古特虎耳草（Ｓａｘｉｆｒａｇａｔａｎｇｕｔｉｃａ）叶绿体和线粒体超微结构的分析表明：叶绿体长度缩短,厚度增加,局部有损伤,基粒垛叠程度小,类囊体有肿胀现象。线粒体的形态由棒状变为球状,被膜不整齐,局部有损伤,嵴的数目少而且有肿胀现象。这些特征的出现,是与青藏高原强辐射,低气压,高寒的生态环境密切相关。     

Structural changes of envelope proteins during alphavirus fusion

Alphaviruses are enveloped RNA viruses that have a diameter of about 700?? and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.     

Identification of a receptor for protein import into mitochondria

Anti-idiotypic antibodies, prepared using a chemically synthesized signal peptide of a mitochondrial precursor protein, recognized a mitochondrial integral membrane protein (p32). Fab fragments derived from both anti-idiotypic antibodies and monospecific antibodies against purified p32 inhibited protein import into mitochondria. Moreover, anti-p32 antibodies specifically immunoprecipitated a precursor-p32 complex after detergent solubilization of mitochondria. Immunoelectron microscopy and subfractionation of mitochondria indicate that p32 is located in contact sites between the outer and inner mitochondrial membranes.     

Rapid regulation of steroidogenesis by mitochondrial protein import

Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and imported into mitochondria. The imported proteins are directed to one of four submitochondrial compartments--the outer mitochondrial membrane, the inner mitochondrial membrane, the intramembraneous space, or the matrix--where the protein then functions. Here we show that the steroidogenic acute regulatory protein (StAR), a mitochondrial protein required for stress responses, reproduction, and sexual differentiation of male fetuses, exerts its activity transiently at the outer mitochondrial membrane rather than at its final resting place in the matrix. We also show that its residence time at this outer membrane and its activity are regulated by its speed of mitochondrial import. This may be the first example of a mitochondrial protein exerting its biological activity in a compartment other than that to which it is finally targeted. This system enables steroidogenic cells to initiate and terminate massive levels of steroidogenesis within a few minutes, permitting the rapid regulation of serum steroid hormone concentrations.     

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.