Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae

Journal of Biological Chemistry

1995

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114

We have confirmed by spectral analysis that cytochrome oxidase is not present in strains of the yeast Saccharomyces cerevisiae having a primary deficiency in cytochrome c, and we have demonstrated by immunological procedures that such strains lack the mitochondrially encoded subunits I, II, and III of cytochrome oxidase. Furthermore, pulse-chase experiments demonstrated that subunit II is rapidly degraded in vivo. This degradation can be at least partially suppressed by disruption of the nuclear gene YME1, which encodes a putative ATP-Zn 2؉ -dependent protease. We suggest that the cytochrome oxidase subunits are not properly assembled in the absence of cytochrome c, and that Yme1 and possibly other proteases degrade the unassembled mitochondrial-encoded subunits of cytochrome oxidase.

Section: Resultsmentioning

confidence: 99%

“…Low Temperature Spectrophotometric Recordings of Intact CellsTotal amounts of cytochromes a⅐a 3 , b, c, and c 1 were quantitatively estimated by absorbance recordings of intact cells at Ϫ196°C, with an Aviv Model 14 spectrophotometer as described previously (32).…”

Section: Genetic Nomenclature and Yeast Strains-the Symbols Cyc1mentioning

confidence: 99%

Degradation of Cytochrome Oxidase Subunits in Mutants of Yeast Lacking Cytochrome c and Suppression of the Degradation by Mutation of yme1

Pearce

Journal of Biological Chemistry

1995

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114

“…The strains were grown on 10 g/l yeast extract, 20 g/l peptone and 20 g/l xylose at 30 • C for 3 days. The absorption spectra were recorded as previously described (Hickey et al, 1991).…”

Section: Shake-flask Fermentationmentioning

confidence: 99%

SHAM‐sensitive alternative respiration in the xylose‐metabolizing yeast Pichia stipitis

Shi

Cruz

et al. 2002

Yeast

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SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood. We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption. The resulting sto1-deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum. Levels of cytochromes b, c, c 1 and a·a 3 did not change in the sto1-mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool. Interestingly, the sto1-deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose. Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis. We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism. Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source. We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P. stipitis. Published in

“…Total amounts of cytochrome c were determined by spectroscopic examination of intact cells at Ϫ196ЊC (44) and by comparing the intensities of the c ␣ -bands at 547 nm to the c ␣ -bands of strains having known amounts of cytochrome c. A more accurate determination of cytochrome c content in intact cells was made by low-temperature (Ϫ196ЊC) spectrum recording with a modified Cary 14 spectrophotometer (17).…”

Section: Genetic Nomenclature and Yeast Strains Cyc1mentioning

confidence: 99%

Initiation of Translation Can Occur Only in a Restricted Region of the CYC1 mRNA of Saccharomyces cerevisiae

Yun

Molecular and Cellular Biology

1995

The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a ''sensitive'' region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions ؊24 to ؉37 but not in the remaining 3 distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5 cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.Mutationally-altered forms of the CYC1 gene, which encodes iso-1-cytochrome c in the yeast Saccharomyces cerevisiae have been used to elucidate the major features of translation. The deficiencies of iso-1-cytochrome c in certain cyc1 mutants and the corresponding deduced DNA sequences of cyc1 mutations indicated that AUG was the only codon capable of initiating translation at appreciable levels (48, 49), a finding confirmed in yeast cells with other systems (7, 9). Also, early studies indicated that translation of CYC1 mRNA initiated at the most upstream AUG codon (45-47) and that translation could initiate at a relocated AUG initiator codon within a 37-nucleotide region around the normal initiation site (45-49). These results and the results with deletion mutants (2) suggested the lack of any additional sequence requirements for initiation of translation. However, nucleotides preceding the AUG initiator codon at positions Ϫ1 and Ϫ3 slightly influenced initiation of translation, and the introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpi...