Studies on the assembly of complex II in yeast mitochondria using chimeric human/yeast genes for the iron-sulfur protein subunit

Saghbini, Michael; Pl, Broomfield; Ie, Scheffler

doi:10.1021/bi00167a021

Cited by 15 publications

(8 citation statements)

References 17 publications

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“…in the absence of the Fp subunit the Ip subunit does not become stably associated with the inner mitochondrial membrane but is instead rapidly degraded upon import into the mitochondria (26). Related studies with mutated Ip subunits so far have failed to yield evidence that the Ip and Fp subunits can assemble without first becoming membrane associated (27). Because simple models of stepwise assembly of complex II have no experimental support, coordinated import and assembly of all four peptides seems to be required for biogenesis of complex II.…”

Section: Discussionmentioning

confidence: 99%

“…Related studies with mutated Ip subunits so far have failed to yield evidence that the Ip and Fp subunits can assemble without first becoming membrane associated (27). Because simple models of stepwise assembly of complex II have no experimental support, coordinated import and assembly of all four peptides seems to be required for biogenesis of complex II.…”

Section: A Chinese Hamster Mutant Cell Line With a Defect In C Ii-3 2mentioning

confidence: 99%

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A Chinese Hamster Mutant Cell Line with a Defect in the Integral Membrane Protein CII-3 of Complex II of the Mitochondrial Electron Transport Chain

Oostveen

Meijer

et al. 1995

Journal of Biological Chemistry

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In this study, a respiration-deficient Chinese hamster cell line with a defect in succinate dehydrogenase activity is shown to result from a single base change in a codon in the coding sequence for the membrane anchor protein C II-3 (also referred to as QPs-1). A premature translation stop results in the truncation of 33 amino acids from the C terminus. Bovine cDNA encoding this peptide complements the mutation. There is about 82% identity between these two mammalian proteins. The gene for C II-3 was mapped on human chromosome 1, and because it is also found on minichromosomes characterized by our laboratory, we can localize it on the short arm within 1-2 megabases from the centromere.A series of respiration-deficient Chinese hamster cell mutants isolated by our laboratory can grow normally in tissue culture as long as an adequate supply of glucose is available for glycolysis (1-3). Depletion of glucose leads to rapid cessation of growth and cell death. The mutants were grouped into seven complementation groups by somatic cell fusions (1), and one, represented by mutant CCL16-B9, was characterized to be almost completely deficient in succinate dehydrogenase activity (SDH) 1 (4,5). This enzyme, which links the reactions of the Krebs cycle to the electron transport chain, is part of a complex of four polypeptides (complex II) in the inner mitochondrial membrane. The active site for the substrate is on the flavoprotein (Fp, 70 kDa), while the iron-sulfur protein (Ip, 27 kDa) is believed to link it to two small integral membrane proteins (C II-3 and C II-4 , 15 and 7-9 kDa, respectively). A b-type heme group is associated with the membrane proteins. Electrons from the oxidation of the substrate in the mitochondrial matrix pass from the Fp via three non-heme iron-sulfur centers in the Ip , , ) to the integral membrane proteins and from there to ubiquinone. The function of the heme group associated with the membrane proteins is not completely clear (see Refs. 6 -9 for reviews).All four peptides are encoded by nuclear genes in eukaryotic organisms. Thus, the precursor polypeptides are synthesized in the cytosol and subsequently or concurrently imported into mitochondria. Following processing to their mature forms, the biogenesis of a functional complex II requires covalent attachment of flavin to the largest subunit (Fp) (10), formation of the three non-heme iron-sulfur clusters in the Ip subunit, and assembly of the heme with the membrane anchor proteins. Although this complex is the smallest and least intricate of the electron transport complexes in the inner mitochondrial membrane, much remains to be learned about the mechanism or pathway of its assembly.With the Fp-Ip complex dissociated from the membrane by chaotropic ions, SDH activity can be assayed with artificial electron acceptors such as tetrazolium ions, phenazine methosulfate and dichlorophenol-indophenol, or ferricyanide. Based on the absence of activity in the mutant CCL16-B9, we hypothesized that the defective protein was either the Ip or the Fp subun...

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Section: Discussionmentioning

confidence: 99%

Section: A Chinese Hamster Mutant Cell Line With a Defect In C Ii-3 2mentioning

confidence: 99%

A Chinese Hamster Mutant Cell Line with a Defect in the Integral Membrane Protein CII-3 of Complex II of the Mitochondrial Electron Transport Chain

Oostveen

Meijer

et al. 1995

Journal of Biological Chemistry

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“…3) and a majority of Cα atoms superimpose. It explains why chimeras of human and yeast Ips will confer activity on Ip‐deficient yeast [27].…”

Section: Iron–sulfur Subunitmentioning

confidence: 99%

Progress in understanding structure–function relationships in respiratory chain complex II

Ackrell¹

2000

FEBS Letters

126

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Complex II (succinate:quinone oxidoreductase) of aerobic respiratory chains oxidizes succinate to fumarate and passes the electrons directly into the quinone pool. It serves as the only direct link between activity in the citric acid cycle and electron transport in the membrane. Finer details of these reactions and interactions are but poorly understood. However, complex II has extremely similar structural and catalytic properties to quinol :fumarate oxidoreductases of anaerobic organisms, for which X-ray structures have recently become available. These offer new insights into structure^function relationships of this class of flavoenzymes, including evidence favoring protein movement during catalysis.z 2000 Federation of European Biochemical Societies.

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“…pGC40 was created by replacing the StuI-XbaI fragment of pGCAA9 with the 298-bp BalI-NheI fragment of the HIS3 coding sequence. The construction of the chimeric human/yeast Ip genes has been described elsewhere (Saghbini et al, 1994).…”

Section: Plasmid Constructionsmentioning

confidence: 99%

“…The resulting transcript was regulated normally in YPD and YPG (pGC40.2 in Figure 4). In addition, a series of chimeric genes were constructed by swapping coding segments of the yeast Ip with the corresponding human cDNA sequences (Saghbini et al, 1994). In every case the open reading frame is preserved and some of these chimeric proteins are functionally active.…”

Section: The Coding Sequence Of the Ip Gene Is Notmentioning

confidence: 99%

Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role.

Cereghino

Atencio

Saghbini

et al. 1995

MBoC

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We have demonstrated previously that glucose repression of mitochondrial biogenesis in Saccharomyces cerevisiae involves the control of the turnover of mRNAs for the iron protein (Ip) and flavoprotein (Fp) subunits of succinate dehydrogenase (SDH). Their half-lives are >60 min in the presence of a nonfermentable carbon source (YPG medium) and <5 min in glucose (YPD medium). This is a rare example in yeast in which the half-life of an mRNA can be controlled by manipulating external conditions. In our current studies, a series of Ip transcripts with internal deletions as well as chimeric transcripts with heterologous sequences (internally or at the ends) have been examined, and we established that the 5'-untranslated region (5' UTR) of the Ip mRNA contains a major determinant controlling its differential turnover in YPG and YPD. Furthermore, the 5' exonuclease encoded by the XRN1 gene is required for the rapid degradation of the Ip and Fp mRNAs upon the addition of glucose. In the presence of cycloheximide the nucleolytic degradation of the Ip mRNA can be slowed down by stalled ribosomes to allow the identification of intermediates. Such intermediates have lost their 5' ends but still retain their 3' UTRs. If protein synthesis is inhibited at an early initiation step by the use of a prtl mutation (affecting the initiation factor eIF3), the Ip and Fp mRNAs are very rapidly degraded even in YPG. Significantly, the arrest of translation by the introduction of a stable hairpin loop just upstream of the initiation codon does not alter the differential stability of the transcript in YPG and YPD.These observations suggest that a signaling pathway exists in which the external carbon source can control the turnover of mRNAs of specific mitochondrial proteins. Factors must be present that control either the activity or more likely the access of a nuclease to the select mRNAs. As a result, we propose that a competition between initiation of translation and nuclease action at the 5' end of the transcript determines the half-life of the Ip mRNA.

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Studies on the assembly of complex II in yeast mitochondria using chimeric human/yeast genes for the iron-sulfur protein subunit

Cited by 15 publications

References 17 publications

A Chinese Hamster Mutant Cell Line with a Defect in the Integral Membrane Protein CII-3 of Complex II of the Mitochondrial Electron Transport Chain

A Chinese Hamster Mutant Cell Line with a Defect in the Integral Membrane Protein CII-3 of Complex II of the Mitochondrial Electron Transport Chain

Progress in understanding structure–function relationships in respiratory chain complex II

Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role.

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