Functional cooperation of mitochondrial protein import receptors in yeast.

We have identified a 20 kDa yeast mitochondrial outer membrane protein (termed MAS20) which appears to function as a protein import receptor. We cloned, sequenced and physically mapped the MAS20 gene and found that the protein is homologous to the MOM19 import receptor from Neurospora crassa. MAS20 and MOM19 contain the sequence motif F-X-K-A-L-X-V/L, which is repeated several times with minor variations in the MAS70/MOM72 receptors. To determine how MAS20 functions together with the previously identified yeast receptor MAS70, we constructed yeast mutants lacking either one or both of the receptors. Deletion of either receptor alone had little or no effect on fermentative growth and only partially inhibited mitochondrial protein import in vivo. Deletion of both receptors was lethal. Deleting only MAS70 did not affect respiration; deleting only MAS20 caused loss of respiration, but respiration could be restored by overexpressing MAS70. Import of the F1-ATPase beta-subunit into isolated mitochondria was only partly inhibited by IgGs against either MAS20 or MAS70, but both IgGs inhibited import completely. We conclude that the two receptors have overlapping specificities for mitochondrial precursor proteins and that neither receptor is by itself essential.     

基于线粒体细胞色素c氧化酶亚基I基因序列的帘蛤科贝类分子系统发育研究

对21种帘蛤科贝类线粒体细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunit I,COI)基因核苷酸序列进行了分析,以探讨这一序列在种质鉴定、分子系统发生研究中的应用价值。测序结果表明,所有物种扩增片段长度均为707 bp(含引物),序列A+T含量(62.4%—67.8%)明显高于G+C含量。物种间共有变异位点379个,其中简约信息位点334个;此区段共编码235个氨基酸,种间共有氨基酸变异位点100个。以COI基因片段序列为标记,用中国蛤蜊(Mactra chinensis)作外群,构建了35种帘蛤科贝类(其中14种贝类COI序列从GenBank下载)的系统发生树,结合拓扑结构分析和序列比对分析,结果表明:支持将短文蛤(Meretrix petechinalis)和丽文蛤(M.lusoria)订为文蛤(M.meretrix)的同物异名的观点,建议将丽文蛤和短文蛤订为文蛤的地理亚种;支持将薄片镜蛤(Dosinia corrugata)和D.angulosa订为2个独立种的观点;认为将波纹巴非蛤(Paphia undulata)和织锦巴非蛤(P.textile)订为2个独立种是合适的。COI基因序列含有丰富的遗传信息,适合作为帘蛤科贝类种群遗传结构和系统发生研究的分子标记。     

Mitochondrial DNA analysis of the introduced fall webworm, showing its shift in life cycle in Japan

The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), was introduced from North America into Japan in 1945. For the first three decades after its introduction, its life cycle was bivoltine. Thereafter, its life cycle shifted to trivoltine in south‐western areas of Japan. Two hypotheses have been proposed for the process that led to the shift in voltinism: one based on a single and the other on multiple independent colonizations. To test these hypotheses, mitochondrial (mt)DNA sequences were analyzed in the black‐headed type of 14 Japanese, one Korean and two North American populations of H. cunea. In addition, the same regions of mtDNA were compared with the red‐headed type of two North American populations. In the black‐headed type, mtDNA sequences were the same in all Japanese populations and in the Korean population, but sequences of the North American populations differed from each other and from those of the other populations. These results suggest that the process of the shift in voltinism occurred originally in Japan, and that the Japanese and the Korean population of H. cunea originated from a relatively small area in North America.     

Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences

Abstract.  Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b ) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed.     

Identification of elmid larvae (Coleoptera: Elmidae) from Sanin District of Honshu,Japan, based on mitochondrial DNA sequences

Larvae of Elmidae from the Sanin District, Honshu, Japan, were classified into 14 types based on morphological features, of which 11 types were unidentified for species. Species or genus of the unidentified types were determined by comparing their mitochondrial cytochrome oxidase subunit I gene sequences with those of identified adult specimens. A new key to species/genera of elmid larvae was proposed.     

Brazilian populations of Drosophila maculifrons (Diptera: Drosophilidae): low diversity levels and signals of a population expansion after the Last Glacial Maximum

The Quaternary period was marked by considerable changes in climate. Such palaeoclimatic changes affected the population dynamics of many species, both in the Northern and in the Southern Hemisphere. However, the extent of these impacts on the demographic patterns of Neotropical species presenting different ecological requirements remains unclear. Drosophila maculifrons DUDA 1947 belongs to the guaramunu group of Drosophila and represents a potential indicator of the genetic consequences caused by the climatic fluctuations of the Quaternary, because it seems to be sensitive to temperature and humidity shifts. The aim of this study was to evaluate the evolutionary processes subjacent to the patterns of intraspecific diversity and structure of different populations of D. maculifrons. In total, 152 individuals were collected in the south and south‐east Brazil. Phylogenetic and phylogeographical analyses were performed based on sequences of COI and COII mitochondrial genes. In general, the results pointed to Brazilian populations of D. maculifrons being extremely impoverished in terms of mitochondrial diversity and population structure, which could be explained by a recent population expansion event dated to approximately 12 000 years ago. In fact, with the assistance of species palaeo‐distribution modelling strategies, it was possible to infer that most of the sampled region did not present the D. maculifrons environmental suitability requirements at least during the period of the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 55–66.     

Phylogeographic analysis of Pimoidae (Arachnida: Araneae) inferred from mitochondrial cytochrome c oxidase subunit I and nuclear 28S rRNA gene regions

Using mitochondrial DNA cytochrome c oxidase subunit I and nuclear DNA 28S rRNA data, we explored the phylogenetic relationships of the family Pimoidae (Arachnida: Araneae) and tested the North America to Asia dispersal hypothesis. Sequence data were analysed using maximum parsimony and Bayesian inference. A phylogenetic analysis suggested that vicariance, instead of dispersal, better explained the present distribution pattern of Pimoidae. Times of divergence events were estimated using penalized likelihood method. The dating analysis suggested that the emergence time of Pimoidae was approximately 140 million years ago (Ma). The divergence time of the North American and Asian species of Pimoa was approximately 110 Ma. Our phylogenetic hypothesis supports the current morphology‐based taxonomy and suggests that the cave dwelling might have played an important role in the speciation of pimoids in arid areas.     

Polymorphism observed in mitochondrial genes of red flour beetle,Tribolium castaneum (Coleoptera: Tenebrionidae) of different origin in laboratory cultures

Tribolium castaneum, a pest of stored grain, has a uniform morphology, preventing the visual identification of strains from different areas. Polymorphisms in the nucleotide sequences of the mitochondrial genes of this species were examined, and combined into seven haplotypes among the test insect specimens originating from Japan, Thailand, and Canada. These results suggested the potential for geographical differentiation.     

Site directed mutagenesis of subunit 8 of yeast mitochondrial ATP synthase: Functional and import properties of a series of C-terminally truncated forms

The function of the positively charged C-terminal region of mitochondrially encoded subunit 8 of yeast mitochondrial ATP synthase was investigated using derivatives truncated at each of the 3 positively charged residues (Arg³⁷, Arg⁴² and Lys⁴⁷). Each construct, allotopically expressed in the nucleus, was tested for its ability to import and assemble functionally into ATP synthase in yeast cells unable to synthesize mitochondrial subunit 8. The efficiency of import of each construct into isolated wild-type yeast mitochondria was also determined. One construct truncated at the penultimate residue of subunit 8 (Lys⁴⁷) functions in vivo and shows efficient import in vitro. Thus subunit 8 can function with only two positively charged residues. The remainder of the subunit 8 variants failed to rescue in vivo. Since they all show greatly reduced or undetectable import in vitro, presumably because of the increased hydrophobic character of the subunit 8 moiety in the chimaeric precursors, the status of these variants as regards assembly and function is not clear.     

Morphological and mitochondrial DNA variation revealed an undescribed lineage under the genus Sperata (Bagridae) in Bangladesh

Catfishes Sperata are popular, known for its taste and nutritional value, and are found naturally in wide variety of freshwaters in South Asia. The taxonomy of Sperata spp., sampled from Hakaluki Haor in Bangladesh, was re‐evaluated based on morphological variation and DNA barcoding. The collective variation in morphometric characters and mitochondrial DNA revealed an undescribed old and well‐separated lineage under the genus Sperata along with two previously known Sperata aor and Sperata seenghala in Bangladesh. Analyses of morphological traits suggested significant differentiation among Sperata species. The variation in mitochondrial DNA supported the distinct lineage and taxonomical discrimination. Sperata aor diverged earlier from the new lineage and Sperata seenghala with a divergence of 5.39 (CI: 3.91–7.19) Mya (PP > 90). Sperata seenghala and the new lineage shared a most recent common ancestry, which diverged from each other around 3.41 (CI: 2.24–4.62) Mya (PP > 90). Thus, the newly identified lineage could be a subspecies of S. seenghala or even a species under the genus Sperata. The information of the study will be useful for conservation, sustainable management and selective breeding of the putative species, including previously reported S. aor and S. seenghala in Bangladesh.     

Molecular systematics of the Chrysoperla carnea group (Neuroptera: Chrysopidae) in Europe

The green lacewing Chrysoperla carnea is a complex of cryptic species whose differentiation has been so far based upon morphology, ecophysiology, behaviour and preliminary mitochondrial DNA data using cytochrome oxidase subunit II (COII) and NADH dehydrogenase subunit II. In this work we extended the DNA data by screening nucleotide sequences of COII, cytochrome oxidase I, cytochrome b and the large ribosomal subunit of the mtDNA. These new data suggest that C. carnea s.s. is a well-supported, separate taxon, but that other taxa of the complex are not consistently differentiated by the current DNA data.     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

Mutations restoring import of a yeast mitochondrial protein with a nonfunctional presequence

An internal deletion in the presequence of the precursor to yeast cytochrome oxidase subunit IV blocks import of the protein into mitochondria. We have identified two mechanisms by which yeast cells can suppress the defect of the plasmid-borne defective gene. One mechanism creates a new functional presequence by point mutations, or local DNA rearrangements, within the open reading frame or its 5'-untranslated region. The second mechanism compensates for the defective presequence by recessive mutations in single nuclear genes. The plasmid-linked mutations may mimic the mechanism(s) by which mitochondrial presequences arose during evolution whereas the chromosomal mutations may help to identify components of the mitochondrial import machinery.     

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F₁ subunit of the ATP synthase (pF₁) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu²⁺ were inhibitory. Inhibition by Cu²⁺ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondria than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu²⁺ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF₁ with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu²⁺ were inhibitory. NEM, MPB, DTNB and Cu²⁺ inhibited import of the NEM-modified pF₁ into intact mitochondria. Import of pF₁ through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu²⁺ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.     

Historical demography and genetic differentiation of the giant freshwater prawn Macrobrachium rosenbergii in Bangladesh based on mitochondrial and ddRAD sequence variation

Macrobrachium rosenbergii, the giant freshwater prawn, is an important source of high quality protein and occurs naturally in rivers as well as commercial farms in South and South‐East Asia, including Bangladesh. This study investigated the genetic variation and population structure of M. rosenbergii sampled from four rivers in Bangladesh (sample size ranged from 19 to 20), assessing sequence variation, both in the mitochondrial cytochrome oxidase subunit 1 (CO1) gene and in 106 single nucleotide polymorphisms (SNPs) sampled randomly from the genome with double digest RAD sequencing (ddRADseq). The mitochondrial variation presented a shallow genealogy with high haplotype diversity (h = 0.95), reflecting an expansion in population size for the last ~82 kyr. Based on the CO1 variation the current effective population size (N_e) was 9.7 × 10⁶ (CI: 1.33 × 10⁶ – 35.84 × 10⁶) individuals. A significant population differentiation was observed with the mitochondrial CO1 sequence variation and based on the ddRADseq variation, which could be traced to the divergence of the population in the Naf River in the South‐East border with Myanmar from the other populations. A differentiation in mtDNA haplotype frequencies was also observed between the Biskhali River and the Karnaphuli Rivers in eastern Bangladesh. This study demonstrated the use of high‐throughput genotyping based on the ddRADseq method to reveal population structure at a small geographical scale for an important freshwater prawn. The information from this study can be utilized for management and conservation of this species in Bangladesh.     

Membrane protein import in yeast mitochondria

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn(2+)-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous intermembrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.