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Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria. 总被引:10,自引:1,他引:9
Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane. 相似文献
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High-level expression of a mitochondrial enzyme, ornithine transcarbamylase from rat liver, in a baculovirus expression system 总被引:3,自引:0,他引:3
The mitochondrial enzyme, ornithine transcarbamylase (OTC) from rat liver was expressed in Spodoptera frugiperda (Sf) insect cells using a baculovirus vector. When insect cells were infected with recombinant Autographica californica nuclear polyhedrosis virus (AcNPV) containing a cDNA encoding the precursor form of OTC (pOTC) inserted into the polyhedrin gene, they expressed catalytically active enzyme at levels of approximately 2.5 micrograms/10(6) cells. About 25% of the active enzyme was a novel, partially processed product of pOTC containing four extra amino acids at the amino terminus of OTC. The most abundant protein found in mitochondria from infected insect cells was the normal processing intermediate iOTC, which contains 8 extra amino acids at the amino terminus of OTC. Whereas this species, present at 20 micrograms/10(6) cells, was not active and did not bind the transition-state analog inhibitor of OTC, delta-PALO, the novel processing product did bind and was affinity-purified, along with mature OTC, on a PALO-affinity column. The OTC expressed in insect cells was located in the same compartment of the mitochondrion as in rat liver. The incomplete processing occurred in vitro in both noninfected and infected insect cells. The high level of expression of iOTC using the baculoviral expression system provides a means of overproducing an obligatory intermediate in the mitochondrial import process. 相似文献
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Muriel JM Brannan M Taylor K Johnstone IL Lithgow GJ Tuckwell D 《Developmental biology》2003,260(2):339-351
The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6. 相似文献
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N-acyl-homoserine lactone inhibition of rhizobial growth is mediated by two quorum-sensing genes that regulate plasmid transfer 下载免费PDF全文
Wilkinson A Danino V Wisniewski-Dyé F Lithgow JK Downie JA 《Journal of bacteriology》2002,184(16):4510-4519
The growth of some strains of Rhizobium leguminosarum bv. viciae is inhibited by N-(3-hydroxy-7-cis tetradecenoyl)-L-homoserine lactone (3OH-C(14:1)-HSL), which was previously known as the small bacteriocin before its characterization as an N-acyl homoserine lactone (AHL). Tn5-induced mutants of R. leguminosarum bv. viciae resistant to 3OH-C(14:1)-HSL were isolated, and mutations in two genes were identified. These genes, bisR and triR, which both encode LuxR-type regulators required for plasmid transfer, were found downstream of an operon containing trb genes involved in the transfer of the symbiotic plasmid pRL1JI. The first gene in this operon is traI, which encodes an AHL synthase, and the trbBCDEJKLFGHI genes were found between traI and bisR. Mutations in bisR, triR, traI, or trbL blocked plasmid transfer. Using gene fusions, it was demonstrated that bisR regulates triR in response to the presence of 3OH-C(14:1)-HSL. In turn, triR is then required for the induction of the traI-trb operon required for plasmid transfer. bisR also represses expression of cinI, which is chromosomally located and determines the level of production of 3OH-C(14:1)-HSL. The cloned bisR and triR genes conferred 3OH-C(14:1)-HSL sensitivity to strains of R. leguminosarum bv. viciae normally resistant to this AHL. Furthermore, bisR and triR made Agrobacterium tumefaciens sensitive to R. leguminosarum bv. viciae strains producing 3OH-C(14:1)-HSL. Analysis of patterns of growth inhibition using mutant strains and synthetic AHLs revealed that maximal growth inhibition required, in addition to 3OH-C(14:1)-HSL, the presence of other AHLs such as N-octanoyl-L-homoserine lactone and/or N-(3-oxo-octanoyl)-L-homoserine lactone. In an attempt to identify the causes of growth inhibition, a strain of R. leguminosarum bv. viciae carrying cloned bisR and triR was treated with an AHL extract containing 3OH-C(14:1)-HSL. N-terminal sequencing of induced proteins revealed one with significant similarity to the protein translation factor Ef-Ts. 相似文献
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Oxidative stress in Caenorhabditis elegans: protective effects of superoxide dismutase/catalase mimetics 总被引:3,自引:1,他引:2
The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superoxide dismutase/catalase mimetics (SCMs) without any effects on development or fertility. Here we demonstrate that the mimetics, Euk-134 and Euk-8, confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress. The protective effects of the compounds are apparent with treatments either during development or during adulthood and are independent of an insulin/IGF-I-like signalling pathway also known to affect thermal and oxidative stress resistance. Worms exposed to the compounds do not induce a cellular stress response and no detrimental effects are observed. 相似文献
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Lithgow T 《FEBS letters》2000,476(1-2):22-26
A clear picture has emerged over the past years on how a 'classic' mitochondrial protein, like subunit IV of cytochrome c oxidase, might be targeted to mitochondria. The targeting and subsequent import process involves the commitment of the TOM (translocase in the outer mitochondrial membrane) receptor complex on the mitochondrial surface, a TIM (translocase in the inner mitochondrial membrane) translocation complex in the mitochondrial inner membrane, and assorted chaperones and processing enzymes within the organelle. Recent work suggests that while very many mitochondrial precursor proteins might follow this basic targeting pathway, a large number have further requirements if they are to be successfully imported. These include ribosome-associated factors and soluble factors in the cytosol, soluble factors in the mitochondrial intermembrane space, an additional TIM translocase in the inner membrane and a range of narrow specificity assembly factors in the inner membrane. This review is focused on the targeting of proteins up to the stage at which they enter the TOM complex in the outer membrane. 相似文献