In vivo import of yeast adenylate kinase into mitochondria affected by site‐directed mutagenesis

Magdolen, Viktor; Schricker, Roland; Strobel, Gertrud; Germaier, Herbert; Bandlow, Wolfhard

doi:10.1016/0014-5793(92)80129-5

Cited by 25 publications

(19 citation statements)

References 33 publications

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“…In all other nucleoside monophosphate kinases, the carboxyl-terminal amino acids form part of an amphipathic ␣-helix, which makes a hydrophobic contact to the purine ring of ATP and stabilizes the protein like a clamp (Diederichs and Schulz, 1991). Their mutation in the adenylate kinase from E. coli (Yoneya et al, 1990) or their partial deletion in Aky2p from yeast (Magdolen et al, 1992) lead to inactive proteins exhibiting increased susceptibility to proteolytic degradation. This explains why Pak3p is inactive also.…”

Section: Discussionmentioning

confidence: 99%

“…20 adk1-1 ts -complementing plasmids were isolated from nine different experiments yielding at least 12 independent frameshift mutants. Site-specific in vitro mutagenesis was performed as described previously (Kunkel et al, 1987;Magdolen et al, 1992).…”

Section: Methodsmentioning

confidence: 99%

“…As a consequence, this allele lacks two hydrophobic residues at positions 235 and 239 (family numbering, cf. Magdolen et al (1992)), which are strictly conserved in all other known adenylate kinases. In the cases of Aky2p from yeast (Magdolen et al, 1992) and AK1 from chicken (Yoneya et al, 1990), it has been shown that deletions of the carboxyl terminus or conversion of amino acid 239 into a hydrophilic residue led to drastically diminished protein stability and/or enzymatic activity.…”

Section: Gain Of Biological Activity By Frameshift Mutations-com-mentioning

confidence: 99%

“…Magdolen et al (1992)), which are strictly conserved in all other known adenylate kinases. In the cases of Aky2p from yeast (Magdolen et al, 1992) and AK1 from chicken (Yoneya et al, 1990), it has been shown that deletions of the carboxyl terminus or conversion of amino acid 239 into a hydrophilic residue led to drastically diminished protein stability and/or enzymatic activity. The suspicion that this AK3 allele, PAK3, might have been shortened by a frameshift mutation that has resulted in an inactive protein was corroborated by the fact that the introduction of a ϩ1 frameshift shortly 5Ј-adjacent to the stop codon would extend the reading frame by 27 base pairs.…”

Section: Gain Of Biological Activity By Frameshift Mutations-com-mentioning

confidence: 99%

“…At least eight independent isolates of mutants containing mutation type 2, three of type 3, and one of type 4 were obtained. Although all single base pair insertions 3Ј of position 492 (corresponding to Glu at position 196 in the protein family numbering, Magdolen et al (1992)) potentially lead to proteins that are 9 amino acids longer at the carboxyl terminus than Pak3p, only four nucleotide positions lying within three consecutive triplets were tolerated and led to an active protein product. All alleles capable of complementation contained amphiphilic amino acid residues at positions 235 and 239.…”

Section: Gain Of Biological Activity By Frameshift Mutations-com-mentioning

confidence: 99%

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Strain-dependent Occurrence of Functional GTP:AMP Phosphotransferase (AK3) in Saccharomyces cerevisiae

Schricker

Magdolen²,

Strobel³

et al. 1995

Journal of Biological Chemistry

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The gene for yeast GTP:AMP phosphotransferase (PAK3) was found to encode a nonfunctional protein in 10 laboratory strains and one brewers' strain. The protein product showed high similarity to vertebrate AK3 and was located exclusively in the mitochondrial matrix. The deduced amino acid sequence revealed a protein that was shorter at the carboxyl terminus than all other known adenylate kinases. Introduction of a ؉1 frameshift into the 3-terminal region of the gene extended homology of the deduced amino acid sequence to other members of the adenylate kinase family including vertebrate AK3. Frameshift mutations obtained after in vitro and in vivo mutagenesis were capable of complementing the adk1 temperature-conditional deficiency in Escherichia coli, indicating that the frameshift led to the expression of a protein that could phosphorylate AMP. Some yeasts, however, including strain D273-10B, two wine yeasts, and two more distantly related yeast genera, harbored an active allele, named AKY3, which contained a ؉1 frameshift close to the carboxyl terminus as compared with the laboratory strains. The encoded protein exhibited GTP:AMP and ITP:AMP phosphotransferase activities but did not accept ATP as phosphate donor. Although single copy in the haploid genome, disruption of the AKY3 allele displayed no phenotype, excluding the possibility that laboratory and brewers' strains had collected second site suppressors. It must be concluded that yeast mitochondria can completely dispense with GTP:AMP phosphotransferase activity.Adenylate kinases constitute a family of highly conserved soluble proteins that catalyzes the interconversion of nucleoside phosphates (Noda, 1973) and, thus, fulfills an essential function in maintaining the energy charge in cells (Atkinson, 1977). In mammals three types of isozymes exist. They can be divided into short and long isoforms of 21 and 25 kDa molecular mass, respectively. The short form enzyme, AK, 1 resides in the cytoplasm. Based on differences in primary structure, substrate utilization, and subcellular location, two distinct subgroups of the 25-kDa form can be discriminated, called AK2 and AK3 (for a review, see Schulz (1987)). AK2 is located mainly in the mitochondrial intermembrane space and uses ATP⅐Mg 2ϩ as donor of the high energy phosphate, while AK3, which occurs in the mitochondrial matrix, uses GTP⅐Mg 2ϩ and ITP⅐Mg 2ϩ (Tomasselli et al., 1979a(Tomasselli et al., , 1986). The latter is thought to play a role in the interconversion of non-ATP nucleoside triphosphates, ITP, and GTP (Tomasselli et al., 1979b), generated by substrate chain phosphorylation through succinic thiokinase in the tricarboxylic acid cycle.By contrast, procaryotes were shown to contain only a single member of the adenylate kinase family, a long form isozyme (Brune et al., 1985). In yeast, the major isoform of adenylate kinases, Aky2p, is a protein of 24 kDa molecular mass displaying highest homology to mammalian AK2 isozymes. As in bacteria, this protein was considered to be the only adenylate kinase in ...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Gain Of Biological Activity By Frameshift Mutations-com-mentioning

confidence: 99%

Section: Gain Of Biological Activity By Frameshift Mutations-com-mentioning

confidence: 99%

Section: Gain Of Biological Activity By Frameshift Mutations-com-mentioning

confidence: 99%

See 3 more Smart Citations

Strain-dependent Occurrence of Functional GTP:AMP Phosphotransferase (AK3) in Saccharomyces cerevisiae

Schricker

Magdolen²,

Strobel³

et al. 1995

Journal of Biological Chemistry

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The structure of bovine mitochondrial adenylate kinase: Comparison with isoenzymes in other compartments

Schlauderer

Schulz

1996

Protein Science

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In vertebrates, there are different adenylate kinases in the compartments cytosol, mitochondrial intermembrane space, and mitochondrial matrix. Here, we report the spatial structure of the intermembrane species established in two crystal forms by X-ray diffraction analyses at 1.92 and 2.1 A resolution. In both structures, the enzyme is unligated, and thus in an "open" conformation. The enzyme was prepared from bovine liver, containing at least five variants arisen from posttranscriptional and posttranslational modifications. It could only be crystallized after removing some of these variants. A comparison with the known structures of the adenylate kinases from cytosol and mitochondrial matrix reveals structural differences that should play a role in protein targeting because none of these enzymes contains a cleavable signal peptide. A further comparison with adenylate kinases from Grampositive bacteria showed that the structural Zn2+ ion of these species is replaced by a strictly conserved assembly of hydrogen bonded residues.

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Mitochondrial Import of Cytochrome C

Dumont

1993

Protein Synthesis and Targeting in Yeast

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In vivo import of yeast adenylate kinase into mitochondria affected by site‐directed mutagenesis

Cited by 25 publications

References 33 publications

Strain-dependent Occurrence of Functional GTP:AMP Phosphotransferase (AK3) in Saccharomyces cerevisiae

Strain-dependent Occurrence of Functional GTP:AMP Phosphotransferase (AK3) in Saccharomyces cerevisiae

The structure of bovine mitochondrial adenylate kinase: Comparison with isoenzymes in other compartments

Mitochondrial Import of Cytochrome C

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