Trehalose‐6‐phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose‐6‐phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose‐6‐phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose‐6‐phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.
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.
The single nuclear gene encoding the 17-kDa subunit VI of yeast ubiquinol: cytochrome c oxidoreductase has been inactivated by one-step gene disruption. Disruption was verified by Southern blot analysis of nuclear DNA and immunoblotting. Cells lacking the 17-kDa protein are still capable of growth on glycerol and they contain all other subunits of complex 111 at wild-type levels, implying that the 17-kDa subunit is not essential for either assembly of complex 111, or its function. In vitro, electron transport activity of complex I11 of mutant cells is about 40% of the wild-type complex, but for the total respiratory chain no significant difference in activity was measured between mutant and wild type. The energy-transducing capacity of the complex is not reduced in the absence of the 17-kDa protein. In a relatively high proportion of the transformants, disruption of the 17-kDa gene was accompanied by the appearance of a second mutation causing a petite phenotype. In these cells which lack cytochrome b, the presence of the 17-kDa protein (after complementation) results in stabilization of cytochrome c l .The respiratory chain enzyme ubiquinol : cytochrome c oxidoreductase in yeast is thought to consist of 8 -10 different subunits [l, 21. The enzyme catalyzes electron transfer from ubiquinol to cytochrome c. Coupled to this process is the translocation of protons over the mitochondria1 inner membrane, resulting in an electrochemical gradient which is used by the FoF1-ATPase to generate ATP.Three subunits, cytochrome b, cytochrome c1 and the Rieske Fe-S protein, with molecular masses of 42, 29 and 24 kDa respectively, contain a prosthetic group and play an essential role in electron transfer. The remaining subunits are the two core proteins, I and 11, of 44 and 40 kDa respectively, three smaller polypeptides of 17, 14 and 11 kDa and possibly two proteins of 8 and 5.5 kDa [l, 21. Until recently, nothing was known about a possible function of these proteins in electron transfer, proton translocation and/or assembly of the complex. Since then, however, genes coding for most of these subunits have been cloned [3 -51 and mutants, in which the gene coding for each has been inactivated, have been constructed [5-71. Analysis of these mutants shows that the 11-kDa, 14-kDa and 44-kDa proteins are essential: their absence results in lack of assembly and proteolytic degradation of several other subunits [5, 61. Absence of the 40-kDa core I1 protein also destabilizes the remaining complex but in this mutant approximately 5% of the wild-type complex 111 activity remains, indicating that this protein is not essential for electron transfer [7].In the present study we report on the construction and characterization of a mutant lacking the 17-kDa subunit VI.
The nucleotide sequence of a 2.5 x 103-base segment of yeast nuclear DNA, containing the structural gene for the 40-kDa subunit I1 of the ubiquinol: cytochrome-c oxidoreductase, has been determined. The region contains only one single reading frame of length sufficient to encode a protein of the size of subunit 11. The mature protein is predicted to have a length of 352 amino acids, with a molecular mass of 38714 Da. It is predominantly hydrophilic, with an overall polarity of 45%. Comparison of the sequence of the reading frame with that derived from direct sequence analysis of the N terminus of the mature 40-kDa protein shows that subunit I1 is synthesized as a longer precursor and shows that the extension is N-terminal. The presequence is 16 amino acids long and it contains a number of positively charged residues and lacks acidic ones. It is also rich in neutral, polar amino acids. S1 nuclease protection analysis of DNA . RNA hybrids identifies two major and one minor transcript of the gene, whose 5' termini map approximately 55, 65 and 75 nucleotides upstream of the initiation codon. Sequences 5' of these termini lack obvious homology to the regulatory sequences of other imported mitochondrial proteins, whose synthesis is controlled by oxygen and by catabolite repression. A mutant lacking a functional subunit I1 gene has been constructed by a one-step gene-disruption procedure. This mutant grows only slowly on glycerol and still displays a low level of QH,: cytochrome-c oxidoreductase activity (approx. 5% of that of wild type). The implications of this finding for the possible role of subunit I1 in the complex are discussed.QH, : cytochrome-c oxidoreductase is a proton-translocating enzyme of the respiratory chain in mitochondria. In yeast it consists of eight to ten subunits [l -31, of which only one, apocytochrome b, is mitochondrially encoded [4, 51. The remaining subunits are encoded in the nucleus. The dual genetic origin of the enzyme poses questions concering the coordinate synthesis, import and assembly of individual subunits. To address these, molecular information about the genes and the subunits is required. This is so far available for subunits I11 (cytochrome cl), IV (cytochrome b) and VI-VIII [6-91 (Maarse, unpublished results). We describe here an analysis of the nuclear gene for subunit 11. Comparison of the nucleotide sequence with a partial amino acid sequence of the N terminus of the mature protein reveals an amino-terminal extension which displays several features common to the presequences of other imported mitochondrial proteins. The remaining protein sequence is predominantly hydrophilic, consistent with previous suggestions that the bulk of the subunit protrudes from the mitochondrial inner membrane into the matrix space MATERIALS AND METHODS Strains and mediaSaccharomyces cerevisiae strains DL1 (a, his3, leu2, ura3) and HR2 (a, his3,leu2, trpl) were used for most experiments. Transformation was carried out as described by Klebe et al. [Ill and transformants were selected on minima...
The single nuclear gene encoding the 14‐kDa subunit VII of yeast ubiquinol:cytochrome c oxidoreductase has been inactivated by one‐step gene disruption, as verified by Southern blot analysis and immunoblotting. The resulting mutant has no ubiquinol:cytochrome c oxidoreductase activity and is respiratory‐deficient. Immunoblotting shows that cells lacking the 14‐kDa protein, also have lowered steady‐state levels of other subunits of complex III, the nuclear‐encoded 11‐kDa subunit VIII, the Rieske Fe‐S protein and the mitochondrially encoded cytochrome b can be detected spectrally. The steady‐state levels of the transcripts from genes encoding these proteins are not reduced, implying that the mutation exerts its pleiotropic effects at a posttranscriptional level. The residual amounts of subunits of complex III are recovered in the mutant mitochondria, suggesting that import is unaffected. The results strongly suggest that the 14‐kDa protein plays an essential role in the biosynthesis of the complex, most probably at the level of assembly. Field‐inversion gel electrophoresis was used to separate chromosomes of HR2 wild type and the (14‐kDa‐protein)° mutant, after which the gene encoding the 14‐kDa protein was located on chromosome IV by Southern blot analysis.
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