Five Entry Points of the Mitochondrially Encoded Subunits in Mammalian Complex I Assembly

Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.Complex I (CI) (NADH-ubiquinone oxidoreductase; EC 1.6.5.3) is one of the main electron entry points in the mitochondrial respiratory electron transport chain catalyzing the oxidation of NADH to reduce ubiquinone to ubiquinol (31, 39, 40), contributing to the proton motive force to synthesize ATP by the process called oxidative phosphorylation (OXPHOS).CI assembly is a difficult problem to address due to the large size of the complex and its dual genomic nature, as 7 out of its 45 subunits are encoded by the mitochondrial DNA (mtDNA) (10, 11). Until very recently, mammalian CI assembly was explained using two different and apparently contradictory models. One model was proposed by following the time course of formation of CI intermediates in human cells in culture once mitochondrial protein synthesis had recovered after its inhibition by doxycycline (36). Based on these observations, human CI was proposed to be assembled through two different modules corresponding to the membrane and peripheral arms. The other model was proposed after analysis of a cohort of four CI-deficient patients in which seven putative assembly intermediates containing a combination of both peripheral- and membrane arm subunits were identified. Thus, an assembly pathway in which the peripheral- and membrane arm subassemblies came together before the completion of each of the arms was proposed (4). However, the most recent studies have refined the previous models and propose an overlapping view of the process. One study, by green fluorescent protein (GFP) tagging of the NDUFS3 subunit, identified six peripheral-arm intermediates. The second and third smaller NDUFS3-containing subassemblies were accumulated and could not advance into higher-molecular-mass species when mitochondrial protein synthesis was inhibited, thus determining the entry point of the mitochondrially encoded subunits in the CI assembly pathway (37). The most recent study analyzed the incorporation of the mitochondrial subunits in a time course to the fully assembled CI and, on the other hand, the incorporation of the nuclear subunits by importing them into isolated mitochondria (24). Although these two models differ in the order in which some subunits are incorporated, they agree on the general human CI assembly pathway, which takes place via evolutionarily conserved modular subassemblies (14, 25, 28, 37).However, the specific entry points of all the mtDNA-encoded CI subunits (ND subunits) in the CI assembly pathway and their roles in the stability of the complex remained to be clarified. Structural studies related to mutations in the ND subunits in pathological cases have given some hints as to the importance of each of them for CI assembly/stability. In this case, defects in specific ND subunits do not have the same effect: ND1, ND4, and ND6 seem to be fundamental to CI assembly, while ND3 and ND5 are important for its activity but not for assembly. On the other hand, mutations in ND2 alter CI assembly, with abnormal intermediate accumulation (19).In this article, we present new insights into the roles of the ND subunits by using mouse cells deficient for ND4, ND6, and a combination of ND6 and ND5. This study has allowed us to propose the five different entry points by which the mtDNA-encoded subunits are sequentially incorporated into the CI assembly pathway, completing the current view of the process. We conclude that ND4 and ND6 are required for the proper function and assembly of CI, although at different degrees due to their different entry points and roles in the CI assembly pathway.     

Abnormal 30S Ribosomal Subunits Determined by an Ochre Suppressor Mutation in Escherichia coli

The results demonstrate that an ochre suppressor mutant of Escherichia coli K-12 produces abnormal 30S ribosomes. The production of these abnormal 30S ribosomes is probably a secondary consequence of the suppressor mutation.     

Formation of an Inclusion Complex of Anthocyanin with Cyclodextrin

A new gallotannin named kurigalin was isolated from the bark of chestnut tree. The chemical structure of kurigalin was established as 2,5,6-tri-O-galloyl-α,β-d-hamamelose (I) on the basis of enzymatic hydrolyses and spectral analyses.     

The Ribosomal Stalk Plays a Key Role in IF2-Mediated Association of the Ribosomal Subunits

Ribosomal “stalk” protein L12 is known to activate translational GTPases EF-G and EF-Tu, but not much is known about its role in relation to other two translational G factors, IF2 and RF3. Here, we have clarified the role of L12 in IF2-mediated initiation of bacterial protein synthesis. With fast kinetics measurements, we have compared L12-depleted 50S subunits with the native ones in subunit association, GTP hydrolysis, P_i (inorganic phosphate) release and IF2 release assays. L12 depletion from 50S subunit slows the subunit association step significantly (∼ 40 fold) only when IF2·GTP is present on the 30S preinitiation complex. This demonstrates that rapid subunit association depends on a specific interaction between the L12 stalk on the 50S subunit and IF2·GTP on the 30S subunit. L12 depletion, however, did not affect the individual rates of the subsequent steps including GTP hydrolysis on IF2 and P_i release. Thus, L12 is not a GTPase activating protein (GAP) for IF2 unlike as suggested for EF-G and EF-Tu.     

Retention of the Native Epigenome in Purified Mammalian Chromatin

A protocol is presented for the isolation of native mammalian chromatin as fibers of 25–250 nucleosomes under conditions that preserve the natural epigenetic signature. The material is composed almost exclusively of histones and DNA and conforms to the structure expected by electron microscopy. All sequences probed for were retained, indicating that the material is representative of the majority of the genome. DNA methylation marks and histone marks resembled the patterns observed in vivo. Importantly, nucleosome positions also remained largely unchanged, except on CpG islands, where nucleosomes were found to be unstable. The technical challenges of reconstituting biochemical reactions with native mammalian chromatin are discussed.     

Effect of a Purified Initiation Factor F3 (B) on the Selection of Ribosomal Binding Sites on Phage MS2 RNA

STUDIES with T4 mRNA showed that initiation factor F2 (C) promotes the attachment of ribosomes to mRNA¹. On the 30S ribosomal subunit this effect is independent of the function of F2 in the binding of formylmethionyl tRNA², whereas formation of a 70S-mRNA complex depends on the binding of fMet-tRNA³. Template competition experiments⁴ showed that, with F2 (C), the ribosome seems to have the same affinity for synthetic polynucleotides as for natural mRNA. Addition of initiation factor F3 (B), however, leads to preferential binding of ribosomes to the natural mRNA. This suggests⁴ that while factor F2 (C) binds the ribosome to any site on the mRNA, the function of factor F3 (B) is to recognize some specific signal in natural mRNA corresponding, perhaps, to the beginning of a cistron. Fractionation of initiation factor F3 (B) into several species differing in their specificity for different mRNA templates⁵ gave further support to the hypothesis that this protein can select binding sites. An excellent system to demonstrate this effect of F3 (B) would be the binding of ribosomes to RNA from E. coli RNA bacteriophages, since Steitz⁶ has analysed and determined the nucleotide sequence of the three binding sites corresponding to the three cistrons of R17 mRNA. Experiments were thus undertaken to study the effect of a purified fraction of F3 (B) on the binding of ribosomes to the different sites of such a phage RNA.     

Heat Stabilities of Ribosomal Subunits and Reassociated Ribosomes from Bacillus stearothermophilus

Absorbance-temperature profiles reveal that both the 30S and 50S ribosomal subunits from Bacillus stearothermophilus are more thermostable than the comparable Escherichia coli particles. Thermophile ribosomes formed by the reassociation of subunits do not display functional heat stability.     

Initiation of Chromosomal DNA Replication in Mammalian Cell-Free Systems

Chromosomal DNA replication is a fundamental part of the cell division cycle of eukaryotes, and its disruption often leads to genome instability and cancer. A focus for regulation is the initiation of the first replication forks, marking the transition from G1 to S phase. Direct biochemical investigation of the establishment and further progression of chromosomal DNA replication in human somatic cell nuclei has become possible through a cell-free system that obeys cell cycle control. Since its development less than a decade ago, several modifications and adaptations of the original system have been reported, which have led to temporal resolution of replication complex assembly and to the identification of novel DNA replication factors. Here, I will review the different systems, highlight fundamental differences and unifying concepts, and discuss their potential for understanding chromosomal DNA replication in somatic mammalian cells.     

Translation Initiation Factor eIF3 Probably Binds with Microtubules in Mammalian Cells

Association of the translation apparatus with the cytoskeleton is essential for its transportation within the cell and probably also for translation regulation. Very little is known about the involvement of particular proteins of this association. A polypeptide homologous with the heavy chain of translation initiation factor eIF3 p170 was found earlier in a microtubule preparation from adrenal cells. Antibody A167 directed against the recombinant fragment of p170 has been generated to study eIF3 interaction with microtubules in mammalian cells. This antibody was shown to recognize a single 170-kDa polypeptide in eIF3 preparations as well as in homogenates of various cell types. A167 allowed detection of the 170-kDa polypeptide in microtubule preparation from bovine brain and confirmation of its presence in microtubule preparations from adrenal cells. As shown by immunofluorescence microscopy using A167, the 170-kDa polypeptide is mainly located in the endoplasm within numerous small and some large granules. Cell treatment with cycloheximide resulted in growth and clustering of the large granules, and partial antigen redistribution along intracellular microtubules. These new experimental data indicate that mammalian translation factor eIF3 may bind with microtubules.     

Molecular View of 43 S Complex Formation and Start Site Selection in Eukaryotic Translation Initiation

A central step to high fidelity protein synthesis is selection of the proper start codon. Recent structural, biochemical, and genetic analyses have provided molecular insights into the coordinated activities of the initiation factors in start codon selection. A molecular model is emerging in which start codon recognition is linked to dynamic reorganization of factors on the ribosome and structural changes in the ribosome itself.     

用于哺乳动物细胞转染的高纯度质粒DNA的制备

目的：建立简便高效、成本低廉和安全无污染的高纯度质粒提取方法。方法：在乙酸铵方法的基础上加以改进,主要改进之处在于增加了用聚乙二醇纯化质粒的步骤,并对溶液Ⅱ和溶液Ⅲ的成分和具体实验参数也做了合理的改进,以最少的步骤,充分去除了残存杂质,保证了质粒的超纯状态。结果：用本方法提取的质粒与用QIAGEN plasmid midi Kit提取的质粒在理化指标上没有差别,对哺乳动物胞具有同样的转染效率。结论：本方法可完全取代QIAGEN公司的试剂盒用于提取超纯质粒。     

Selective Dephosphorylation of the Subunits of Skeletal Muscle Calcium Channels by Purified Phosphoprotein Phosphatases

Abstract: Multiple sites on the α1 and β subunits of purified skeletal muscle calcium channels are phosphorylated by cyclic AMP-dependent protein kinase, resulting in three different tryptic phosphopeptides derived from each subunit. Phosphoprotein phosphatases dephosphorylated these sites selectively. Phosphoprotein phosphatase 1 (PP1) and phosphoprotein phosphatase 2A (PP2A) dephosphorylated both α1 and β subunits at similar rates, whereas calcineurin dephosphorylated β subunits preferentially. PP1 dephosphorylated phosphopeptides 1 and 2 of the α1 subunit more rapidly than phosphopeptide 3. In contrast, PP2A dephosphorylated phosphopeptide 3 of the α1 subunit preferentially. All three phosphoprotein phosphatases preferentially dephosphorylated phosphopeptide 1 of the β subunit and dephosphorylated phosphopeptides 2 and 3 more slowly. Mn²⁺ increased the rate and extent of dephosphorylation of all sites by calcineurin so that >80% dephosphorylation of both α1 and β sub-units was obtained. The results demonstrate selective dephosphorylation of different phosphorylation sites on the α1 and β subunits of skeletal muscle calcium channels by the three principal serine/threonine phosphoprotein phosphatases.     

On the Size of the DNA in the Mammalian Chromosome: Structural Subunits

Alkaline degradation of mammalian DNA indicates that the molecule exists in the chromosome as an array of structural subunits. The size of the subunit of single-stranded DNA is circa 5 × 10⁸ daltons, and it is of sufficient length to contain a number of synthetic units, replicons. The upper size limit of the multicomponent structure is in excess of 10¹⁰ daltons. Mammalian cells of three different origins have been shown to contain the same basic structural DNA components and these components exist throughout the cell cycle. The nature of the links between the subunits is not known.     

Defective 30S Ribosomal Subunits After Infection of Escherichia coli by T2 Ghosts

Ribosomes from ghost-treated Escherichia coli can form in vitro initiation complexes but cannot translate added template RNA because of a defect in the 30S subunit; 16S rRNA is degraded.     

Reversion of Ribosomal Helix Formation in Escherichia coli

After transfer into fresh medium, Escherichia coli cells containing ribosomal helices resume growth without a lag period. The helices disappear within 15 min after transfer, the number of 70S ribosomes decreases, and a steady-state ribosomal profile appears within one cell generation time. Subunits isolated from the helices support in vitro protein synthesis, but efficiency is optimal only when supplemented with an undetermined factor that is contained in the S-100 fraction of log-phase cells. The data suggest a possible role of helices as ribosomal reserve units.     

Complex Formation of Starch with Organic Substances

Cell free extracts of Hansenula miso IFO 0146 contained an enzyme which catalyzed acyloin condensation of acetaldehyde and α-ketoglutarate to form 5～hydroxy-4-ketohexanoic acid (HKH). The enzyme was specific for acetaldehyde and α-ketoglutarate. Condensation could not be demonstrated between α-ketoglutarate and other aldehydes tested (formaldehyde, propionaldehyde or butyraldehyde). No reaction occurred when boiled enzyme was used. The apparent Km values (at pH 7.5) for acetaldehyde and α-ketoglutarate are 24.4 mM and 3.2 mM, respectively. TPP and Mg²⁺ were not required for the reaction. The optimum pH of the reaction was 7.5～8.5. The reaction was inhibited by EDTA, PCMB and PMS. The enzyme forming HKH was different from that forming acetoin because the latter required TPP and was repressed when cells were grown in lactate medium while the former did not require TPP and was formed independently of its substrate. The product of this condensing reaction was isolated and identified as HKH from its chemical properties.