Targeting efficiency of a mitochondrial pre-sequence is dependent on the passenger protein.

Steeg, Harry van; Oudshoorn, P.; Hell, B.; Polman, J. E. M.; Grivell, Leslie A.

doi:10.1002/j.1460-2075.1986.tb04694.x

Cited by 58 publications

(28 citation statements)

References 41 publications

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“…A probe specific for 5'-flanking sequences of the subunit-VIII gene reveals a 2.9-kb EcoRI fragment in the mutant (lane 2) and a 4.0-kb EcoRI fragment in wild type (lane 1). This result can be explained by an integration event at the subunit VIII gene locus and is confirmed by the results obtained with KpnI (lanes 7,9) or ClaI (lanes 8, 10) instead of EcoRI.…”

Section: Gene Inactivationsupporting

confidence: 80%

“…The 1447-bp Sall-SalI fragment carrying the 1 I-kDa-protein gene was subcloned from p285 [6] both in pEMBL-ye23R [7] and plA433 [8] to give respectively ye23R-SS2 and plA433-883 (Fig.1, lines 2 and 4). Plasmid plA433-274 (line 7) was obtained by subcloning the 839-bp HindIII -SalI fragment, containing the 11-kDa-protein gene (see line 1) in plA433 restricted with BarnHI and Sall.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

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Inactivation of the gene encoding the 11‐kDa subunit VIII of the ubiquinol–cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

Maarse¹,

Haan²,

Schoppink³

et al. 1988

European Journal of Biochemistry

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The single nuclear gene encoding the 1 I-kDa subunit VIII of the ubiquinol -cytochrome-c oxidoreductase (complex 111) in Succhuromyces cerevisiae has been inactivated by a one-step gene disruption procedure. Inactivation results in a loss of ubiquinol-cytochrome-c oxidoreductase activity (< 1% wild type) and respiratory deficiency. Cells lacking the 1 I-kDa protein also display lowered steady-state levels of other complex-I11 subunits encoded by nuclear genes including the 14-kDa subunit VII and the Rieske Fe-S protein and of the mitochondrially encoded cytochrome b. The steady-state levels of the transcripts from the genes encoding these proteins are however not reduced. The results strongly imply that the 11-kDa protein plays an important role in regulating the synthesis of complex I11 at the post-transcriptional level, most likely assembly. Separation of chromosomes by pulsed-field gel electrophoresis of DNA of wild-type and of the mutant lacking the 11-kDa-protein gene followed by Southern blot analysis reveals that the latter gene is located on chromosome X rather than on XI1 as reported by Van Loon et al.

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Section: Gene Inactivationsupporting

confidence: 80%

Section: Construction Of Plasmidsmentioning

confidence: 99%

Inactivation of the gene encoding the 11‐kDa subunit VIII of the ubiquinol–cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

Maarse¹,

Haan²,

Schoppink³

et al. 1988

European Journal of Biochemistry

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“…In the presence of valinomycin (Fig. 3A, B been observed in similar in vitro import studies (31,34). van Steeg et al (34) showed that the presequence of superoxide dismutase was unable to function effectively as a mitochondrial import signal for invertase but could perform this function for DHFR.…”

mentioning

confidence: 56%

Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases.

Ellis¹,

Hopper²,

Martín³

1989

Mol. Cell. Biol.

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Fusions between the TRMI gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRMI sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRMI and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.The TRMI gene of Saccharomyces cerevisiae codes for an enzyme that catalyzes the modification of a specific guanosine to N2,N2-dimethylguanosine in both cytoplasmic and mitochondrial tRNAs (17). In previous reports, we described the isolation and characterization of the TRMJ gene from S. cerevisiae (14,15 (24), FUMI (38), LEU4 (3), and VASI (6) loci of S. cerevisiae each code for two enzymes that differ by an amino-terminal extension, and in each case it has been shown that the amino-terminal extension functions to direct the enzyme to an intra-or extracellular location different from that of the enzyme lacking the aminoterminal extension.Since the amino-terminal extension of tRNA (m2G)

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“…Prehybridization was performed in hybridization buffer (50% formamide, 25 mM NaP i [pH 6.5], 5ϫ SSC [1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 5ϫ Denhardt's solution) containing 50 g of single-stranded salmon sperm DNA per ml. DNA fragments used as probes in this study include an 840-bp HindIII-SalI fragment from pJH1 (5), a 1.6-kb BamHI-KpnI fragment containing the yeast actin gene (19), a 2.5-kb HindIII-SalI fragment from YE23SH containing the QCR2 gene (22), a 1,333-bp NcoI-HindIII fragment from pAZ6 containing the yeast PDA1 gene (32), and a 1.2-kb BamHI-HindIII fragment from YE23R-SOD/SUC containing the SUC2 gene (31). Fragments were 32 P labeled by nick translation as described by Maniatis et al (18).…”

Section: Methodsmentioning

confidence: 99%

Redirection of the Respiro-Fermentative Flux Distribution in Saccharomyces cerevisiae by Overexpression of the Transcription Factor Hap4p

Blom

Mattos

Grivell

2000

Appl Environ Microbiol

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Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae. Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.Modification and control of metabolic fluxes is an important goal in most industrial applications of microorganisms. This is the case for baker's yeast, which has applications in production of heterologous proteins, brewing, and baking. Maximization of biomass yields of baker's yeast growing on sugars is hampered by the cell's strong tendency to produce ethanol, even under aerobic conditions when sugars are present in excess. This alcoholic fermentation might be prevented by manipulation of the carbon flux distribution between fermentation and respiration.Attempts at redirection of carbon fluxes by interference in expression levels of single enzymes of glycolytic or fermentative pathways have so far not been successful (24,25). Reduced activities of fermentative enzymes such as pyruvate decarboxylase result in impaired growth on glucose (8, 9) and deprive the cells of their fermentative capacity necessary for raising of dough. Increasing respiratory activity seems to be a better approach, but numerous studies have shown that overproduction of a single enzyme results in either little or no increase of flux through a metabolic pathway (20). This is because flux control is usually not exerted by only a single enzyme (2,20,28). A more rational approach would therefore be to manipulate the activity of a regulatory protein involved in control of all key enzymes of one or more specific metabolic pathways. The potential of this approach is illustrated by the partial alleviation of glucose control of sucrose and galactose metabolism as a result of disruption of the glucose repressor Mig1p alone or in combination with Mig2p (16,17). These modifications do not, however, result in a significant change in respiratory functions, ethanol production, or biomass yield.Aiming at redirection of fermentative flux toward oxidative carbon flux, we...

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Targeting efficiency of a mitochondrial pre-sequence is dependent on the passenger protein.

Cited by 58 publications

References 41 publications

Inactivation of the gene encoding the 11‐kDa subunit VIII of the ubiquinol–cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

Inactivation of the gene encoding the 11‐kDa subunit VIII of the ubiquinol–cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases.

Redirection of the Respiro-Fermentative Flux Distribution in Saccharomyces cerevisiae by Overexpression of the Transcription Factor Hap4p

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