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1.
Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing rho(0)-lethality are limited to the ATP1, 2 and 3 genes coding for the alpha-, beta- and gamma- subunits of mitochondrial F(1)-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F(1)F(0)-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress rho(0)-lethality. Positioning of the seven amino acids subject to mutation on the bovine F(1)-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F(1) in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F(1)-ATPase in the maintenance of mitochondrial inner membrane potential.  相似文献   

2.
Loss of mtDNA by the petite-negative yeast Kluyveromyces lactis is lethal (rho(o)-lethality). However, mutations in the alpha, beta and gamma subunits of F(1)-ATPase can suppress lethality by increasing intramitochondrial hydrolysis of ATP. Increased hydrolysis of ATP can also occur on inactivation of Inh1, the natural inhibitor of F(1)-ATPase. However, not all strains of K. lactis show suppression of rho(o)-lethality on inactivation of INH1. Genetic analysis indicates that one or more alleles of modifying factors are required for suppression. Papillae showing enhanced resistance to ethidium bromide (EB) in INH1 disruptants have mutations in the alpha, beta and gamma subunits of F(1)-ATPase. Increased growth of double mutants on EB has been investigated by disruption of INH1 in previously characterized atp suppressor mutants. Inactivation of Inh1, with one exception, results in better growth on EB and increased F(1)-ATPase activity, indicating that suppression of rho(o)-lethality is not due to atp mutations preventing Inh1 from interacting with the F(1)-complex. By contrast, in suppressor mutants altered in Arg435 of the beta subunit, disruption of INH1 did not change the kinetic properties of F(1)-ATPase or alter growth on EB. Consequently, Arg435 appears to be required for interaction of Inh1 with the beta subunit. In a previous study, a mex1-1 allele was found to enhance mgi(atp) expression. In accord with results from double mutants, it has been found that mex1-1 is a frameshift mutation in INH1 causing inactivation of Inh1p.  相似文献   

3.
Cytokines such as interleukin-3 (IL-3) promote the survival of hematopoietic cells through mechanisms that are not well characterized. Withdrawal of IL-3 from an IL-3-dependent pro-B cell line induced early stress-related events that preceded cell death by more than 40 h. Intracellular pH rose above pH 7.8, peaking 2-3 h post-IL-3 withdrawal, and induced a transient increase in mitochondrial membrane potential (Delta Psi(m)) detected using several different dyes. Similar events were observed following IL-7 withdrawal from a different dependent cell line. Bcl-2, Bax, and caspases were unrelated to these early events. Intracellular alkaline pH inhibited the mitochondrial import of ADP, which would limit ATP synthesis. Total cellular ATP sharply declined during this early period, presumably as a consequence of suppressed ADP import. This was followed by an increase in reduced pyridine nucleotides. The transient increase in Delta Psi(m) was blocked by oligomycin, an inhibitor of F(0)F(1-)ATPase that may have undergone reversal caused by the abnormal ADP-ATP balance within mitochondria. These findings suggest a novel sequence of early events following trophic factor withdrawal in which alkaline pH inhibits ADP import into mitochondria, reversing the F(0)F(1-)ATPase, which in turn consumes ATP and pumps out protons, raising Delta Psi(m).  相似文献   

4.
The atp6 gene, encoding the ATP6 subunit of F(1)F(0)-ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomonas reinhardtii. Analysis of C. reinhardtii expressed sequence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5'- and 3'-RACE were used to obtain the complete cDNA and genomic sequences of C. reinhardtii atp6. The atp6 gene exhibited characteristics of a nucleus-encoded gene: Southern hybridization signals consistent with nuclear localization, the presence of introns, and a codon usage and a polyadenylation signal typical of nuclear genes. The corresponding ATP6 protein was confirmed as a subunit of the mitochondrial F(1)F(0)-ATP synthase from C. reinhardtii by N-terminal sequencing. The predicted ATP6 polypeptide has a 107-amino acid cleavable mitochondrial targeting sequence. The mean hydrophobicity of the protein is decreased in those transmembrane regions that are predicted not to participate directly in proton translocation or in intersubunit contacts with the multimeric ring of c subunits. This is the first example of a mitochondrial protein with more than two transmembrane stretches, directly involved in proton translocation, that is nucleus-encoded.  相似文献   

5.
By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.  相似文献   

6.
We have shown in previous research that the loss of phosphatidylglycerol and cardiolipin caused by disruption of the PGS1 gene is lethal for the petite-negative yeast Kluyveromyces lactis . This present study demonstrates the role and mechanism of atp2.1 in the suppression of pgs1 lethality in K. lactis cells. Phenotypic characterization has shown that a strain lacking the phosphatidylglycerolphosphate synthase (atp2.1pgs1Δ) possessed a markedly impaired respiratory chain, very low endogenous respiration, and uncoupled mitochondria. As a result the mutant strain was unable to generate a sufficient mitochondrial membrane potential via respiration. The atp2.1 suppressor mutation enabled an increase in the affinity of F(1)-ATPase for ATP in the hydrolytic reaction, resulting in the maintenance of sufficient membrane potential for the biogenesis of mitochondria and survival of cells lacking anionic phospholipid biosynthesis.  相似文献   

7.
Saccharomyces cerevisiae strains expressing a single type of ADP/ATP carrier (AAC) protein were prepared from a mutant in which all AAC genes were disrupted, by transformation with plasmids containing a chosen AAC gene. As demonstrated by measurements of [14C]ADP specific binding and transport, all three translocator proteins, AAC1, AAC2 and AAC3 when present in the mitochondrial membrane, exhibited similar translocation properties. The disruption of some AAC genes, however, resulted in phenotypes indicating that the function of these proteins in whole cells can be quite different. Specifically, we found that the disruption of AAC1 gene, but not AAC2 and AAC3, resulted in a change in colony phenotype.  相似文献   

8.
9.
Smith CP  Thorsness PE 《Genetics》2008,179(3):1285-1299
AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.  相似文献   

10.
Genetic and biochemical analysis of Saccharomyces cerevisiae containing a disruption of the nuclear gene (AAC1) encoding the mitochondrial ADP/ATP carrier has revealed a second gene for this protein. The second gene, designated AAC2, has been isolated by genetic complementation and sequenced. AAC2 contains a 954-base pair open reading frame coding for a protein of 318 amino acids which is highly homologous to the AAC1 gene product except that it is nine amino acids longer at the NH2 terminus. The two yeast genes are highly conserved at the level of DNA and protein and share identity with the ADP/ATP carriers from other organisms. Both genes complement an ADP/ATP carrier defect (op1 or pet9). However, the newly isolated gene AAC2 need be present only in one or two copies while the previously isolated AAC1 gene must be present in multiple copies to support growth dependent on a functional carrier protein. This gene dosage-dependent complementation combined with the high degree of conservation suggest that these two functionally equivalent genes may be differentially expressed.  相似文献   

11.
12.
Adenine nucleotide translocator (ANT) is a mitochondrial inner membrane protein involved in the ADP/ATP exchange and is a component of the mitochondrial permeability transition pore (PTP). In mammalian apoptosis, the PTP can mediate mitochondrial outer membrane permeabilization (MOMP), which is suspected to be responsible for the release of apoptogenic factors, including cytochrome c. Although release of cytochrome c in yeast apoptosis has previously been reported, it is not known how it occurs. Herein we used yeast genetics to investigate whether depletion of proteins putatively involved in MOMP and cytochrome c release affects these processes in yeast. While deletion of POR1 (yeast voltage-dependent anion channel) enhances apoptosis triggered by acetic acid, H(2)O(2) and diamide, CPR3 (mitochondrial cyclophilin) deletion had no effect. Absence of ADP/ATP carrier (AAC) proteins, yeast orthologues of ANT, protects cells exposed to acetic acid and diamide but not to H(2)O(2). Expression of a mutated form of Aac2p (op1) exhibiting very low ADP/ATP translocase activity indicates that AAC's pro-death role does not require translocase activity. Absence of AAC proteins impairs MOMP and release of cytochrome c, which, together with other mitochondrial inner membrane proteins, is degraded. Our findings point to a crucial role of AAC in yeast apoptosis.  相似文献   

13.
T Drgon  L Sabová  N Nelson  J Kolarov 《FEBS letters》1991,289(2):159-162
All three genes (AAC1, AAC2 and AAC3) encoding the mitochondrial ADP/ATP translocator, were inactivated in a haploid yeast strain by a gene disruption technique. The triple mutant was still able to grow on fermentable carbon sources but only in the presence of oxygen. Under aerobic conditions neither translocator-protein nor carrier-mediated transport was detected in all mutants in which the AAC2 gene was disrupted. It was further shown that a functional AAC genes product is essential only for anaerobic growth of Saccharomyces cerevisiae but not for growth under derepressed conditions. Under anaerobic conditions a non-detectable amount of AAC3 gene product is sufficient to ensure the cell growth and multiplication.  相似文献   

14.
In Saccharomyces cerevisiae, SAL1 encodes a Ca2+ -binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+ -binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.  相似文献   

15.
A mechanism for hypoxia survival by eukaryotic cells is suggested from studies on the petite mutation of yeasts. Previous work has shown that mutations in the alpha, beta and gamma subunit genes of F1-ATPase can suppress lethality due to loss of the mitochondrial genome from the petite-negative yeast Kluyveromyceslactis. Here it is reported that suppressor mutations appear to increase the affinity of F1-ATPase for ATP. Extension of this study to other yeasts shows that petite-positive species have a higher affinity for ATP in the hydrolysis reaction than petite-negative species. Possession of a F1-ATPase with a low K(m) for ATP is considered to be an adaptation for hypoxic growth, enabling maintenance of the mitochondrial inner membrane potential, deltapsi, by enhanced export of protons through F1F0-ATP synthase connected to increased ATP hydrolysis at low substrate concentration.  相似文献   

16.
L Bonen  S Bird 《Gene》1988,73(1):47-56
The nucleotide sequence of the wheat mitochondrial gene for subunit 6 (atp6) of the F1F0 ATPase complex has been determined. Unlike bacterial, chloroplast or animal/fungal mitochondrial atp6 counterparts, which encode proteins of about 230-270 amino acids, the wheat mitochondrial atp6 homologue comprises the latter part of an open reading frame (ORF) of 386 codons. The ATP6 protein may therefore by synthesized with a long N-terminal presequence. This is supported by the finding that the ORF is preceded by a conserved sequence block closely related to ones preceding several other actively transcribed wheat mitochondrial protein-coding genes. The fused upstream ORF is similar in length, but unrelated in sequence, to those preceding the maize and tobacco mitochondrial atp6 genes. In wheat, the atp6 gene is located on a recombinationally active repeated DNA element, whose length of 1.4 kb corresponds approximately to that of the atp6 mRNA. A comparison of the wheat and maize ATP6 sequences reveals unexpectedly high divergence in the region corresponding to the mature N-terminal domain and may reflect mitochondrial DNA rearrangements during atp6 gene evolution in monocotyledonous plants.  相似文献   

17.
Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ~ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ~ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.  相似文献   

18.
A third ADP/ATP translocator gene in yeast   总被引:14,自引:0,他引:14  
The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.  相似文献   

19.
Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed rho degrees cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in rho degrees cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4- for ADP3- by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1-ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian rho degrees cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in rho degrees cells and an incomplete FoF1-ATP synthase was also required. The maximum value of this potential was approximately 110 mV in permeabilized cells and approximately 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1-ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but approximately 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of rho degrees mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1-ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1-ATP synthase in maintaining the mitochondrial membrane potential in rho degrees cells.  相似文献   

20.
Pollen formation is a complex process that is strictly controlled by genetic factors. Although many novel mitochondrial genes have been implicated in the dysfunction of mitochondrial enzymes and the cytoplasmic male sterility (CMS), there is little empirical evidence to show that CMS-related genes actually result in the dysfunction of enzyme and little is known about the regulatory mechanisms of the aberrant mitochondrial enzymes in male sterility in CMS lines. Here, we report the characterization of a novel mitochondrial gene, Ψatp6-2, which is hypothesized to play a role in male sterility in pepper. Using virus-induced gene silencing (VIGS), we observed that silencing the atp6-2 gene in the maintainer line resulted in an increase in ATP hydrolysis activity of the mitochondrial F1Fo-ATP synthase along with pollen abortion, while silencing the truncated Ψatp6-2 gene in the CMS line resulted in an inhibition of ATP hydrolysis activity and restoration of fertility. Altered ATP hydrolysis also affected the tolerance of the gene-silenced plants to abiotic stresses. Localization experiments showed that premature ATP hydrolysis occurred at the tetrad stage of pollen development in the CMS line, but no ATPase activity was observed in the microspores at the later stage. These results suggest that the Ψatp6-2 gene causes the alteration in ATP hydrolysis activity of the mitochondrial F1Fo-ATP synthase during pollen development, which eventually leads to male sterility in pepper.  相似文献   

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