A cytosolic protein contains a cryptic mitochondrial targeting signal

Cytosolic dihydrofolate reductase from mouse contains a cryptic mitochondrial targeting sequence. If this sequence is attached to the amino terminus of 'passenger' proteins which by themselves cannot enter mitochondria, the resulting fusion proteins are transported into yeast mitochondria.     

Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.     

Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids

Fusions between the mouse mammary tumour virus long terminal repeat and a mouse dihydrofolate reductase cDNA have been constructed in a SV40 vector. When these plasmids are transferred into recipient cells, the production of dihydrofolate reductase is regulated by glucocorticoid hormones. These results define a hormonally responsive region of the viral genome.     

Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase.

The construction and analysis of bacterial plasmids that contain and phenotypically express a mammalian genetic sequence are described. Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the antimetabolic drug trimethoprim.     

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.     

Exclusion of linkage to 5q11-13 in families with schizophrenia and other psychiatric disorders

Recently a linkage study on five Icelandic and two English pedigrees has provided evidence for a dominant gene for schizophrenia on 5q11-13 (ref. 1). In that study, families with bipolar illness were not included. Using the same probes, two similar but independent investigations on one Swedish pedigree and on fifteen Scottish families excluded linkage to schizophrenia. To evaluate whether the susceptibility gene on 5q11-13 is a common cause of schizophrenia in other populations, we examined five affected North American pedigrees using probes to the D5S39, D5S76 and dihydrofolate reductase loci. Two families in the present series had cases of bipolar disorder. We found that linkage can be excluded by multipoint analysis. These results, taken together, suggest that the disease gene on 5q11-13 does not account for most cases of familial schizophrenia.     

Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria

Only five mitochondrial proteins are known to be essential for viability of the yeast Saccharomyces cerevisiae; all of them are key components of the mitochondrial protein import system. Other components of this system are not essential for life; they include functionally redundant import receptors on the mitochondrial surface and enzymes acting upon only a few precursor proteins.     

A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site

An engineered precursor protein that sticks in the import site of isolated yeast mitochondria can be specifically photo-crosslinked to a mitochondrial outer-membrane protein of relative molecular mass 42,000 (42K). This protein (termed import-site protein 42 or ISP 42) is exposed on the mitochondrial surface; antibodies against it block protein import into mitochondria. ISP 42 is the first identified component of the putative transmembrane machinery that imports proteins into mitochondria.     

Gene targeting in normal and amplified cell lines

Targeted recombination in mammalian cells is rare compared with non-homologous integration. In Saccharomyces cerevisiae the reverse is true. Differences in targeting efficiency could arise because a target of unique DNA is 200 times more dilute in mammalian genomes than it is in yeast. We tested this possibility by measuring gene targeting in normal CHO cells with two copies of the dihydrofolate reductase (DHFR) gene and in amplified CHOC 400 cells, which carry 800 copies. If the concentration of the target gene is critical, amplified cells should show an enhanced frequency of targeted recombination relative to non-homologous integration. Using a positive/negative selection protocol, we demonstrated that the efficiency of targeting into DHFR genes is indistinguishable in normal and amplified CHO cells. As targeting does not depend on the number of targets, the search for homology is not a rate-limiting step in the mammalian pathway of gene targeting. Thus, the difference in genome size is not the basis for the different outcomes of targeting experiments in S. cerevisiae and mammals.     

DNA-protein conjugates can enter mitochondria via the protein import pathway

Mitochondria import most of their proteins and small molecules from the cytoplasm. There is some tentative evidence that they import some of their RNAs, but it is not known how nucleic acids could enter mitochondria. Here, we show that isolated yeast mitochondria can import a single-stranded or double-stranded 24-base pair piece of DNA whose 5' end is covalently linked to the C-terminus of a mitochondrial precursor protein.     

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.     

Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.     

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.     

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.     

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.     

突变型大肠杆菌二氢叶酸还原酶基因的合成

利用PCR定点突变技术，以野生型大肠杆菌二氢叶酸还原酶基因为模板，获取3种突变型(Cys85Ser．Cysl51Ser，Cys85／151Ser)二氢叶酸还原酶(DHFR)基因．用限制性内切酶BamH Ⅰ与Pst Ⅰ将3种突变基因片段插入到克隆载体pUC18上，进行蓝白筛选，将筛选的克隆进行DNA序列测定．     

Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.

Introduction of Ca2+ indicators (photoproteins, fluorescent dyes) that can be trapped in the cytosolic compartment of living cells has yielded major advances in our knowledge of Ca2+ homeostasis. Ca2+ however regulates functions not only in the cytosol but also within various organelles where indicators have not yet been specifically targeted. Here we present a novel procedure by which the free Ca2+ concentration of mitochondria, [Ca2+]m, can be monitored continuously at rest and during stimulation. The complementary DNA for the Ca2+ sensitive photoprotein aequorin was fused in frame with that encoding a mitochondrial presequence. The hybrid cDNA was transfected into bovine endothelial cells and stable clones were obtained expressing variable amounts of mitochondrially targeted apoaequorin. The functional photoprotein could be reconstituted in intact cells by incubation with purified coelenterazine and [Ca2+]m could thus be monitored in situ. This allowed the unprecedented direct demonstration that agonist-stimulated elevations of cytosolic free Ca2+, [Ca2+]i, (measured in parallel with Fura-2) evoke rapid and transient increases of [Ca2+]m, which can be prevented by pretreatment with a mitochondrial uncoupler. The possibility of targeting aequorin to cellular organelles not only offers a new and powerful method for studying aspects of Ca2+ homeostasis that up to now could not be directly approached, but might also be used in the future as a tool to report in situ a variety of apparently unrelated phenomena of wide biological interest.     

Characterization and expression of a cDNA, AmphiSDHD , encoding the amphioxus cytochrome b small subunit in mitochondrial succinate ubiquinone oxidoreductase

Succinate ubiquinone oxidoreductase ,alsoknownascomplexII ,isthesmallestenzymecomplexfunctioninginboththetricarboxylicacidcycle(TAC)andtheaerobicrespiratorychainofeukaryoticcellmitochondriaandprokaryoticcells[1] .Itiscom posedoffournuclear encoded proteins ,SDHAorflavoprotein (70kD) ,SDHBoriron sulfurprotein(2 7kD) ,SDHCorCII 3(15kD)andSDHDorCII 4 (7— 9kD)inmosteukaryotes[2 ] .Thetwoproteins ,SDHAandSDHB ,comprisethesuccinatedehydroge nase (SDH) ,anenzymeoftheTACandaperipher…     

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.