The aromatic nature of residue 66 of the 11‐kDa subunit of ubiquinol‐cytochrome <i>c</i> oxidoreductase of the yeast <i>Saccharomyces cerevisiae</i> is important for the assembly of a functional enzyme

Hemrika, Wieger; Lôbo-Hajdu, Gisele; Berden, J.A.; Grivell, Leslie A.

doi:10.1016/0014-5793(94)00346-7

Cited by 14 publications

(12 citation statements)

References 21 publications

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“…We conclude from this that the hydrophobic stretch in this protein (see Fig. 1) anchors the protein firmly in the lipid bilayer, where it may interact with cytochrome b as has previously been suggested [26].…”

Section: Subunit VIII Is An Integral Membrane Proteinsupporting

confidence: 75%

Topological organization of subunits VII and VIII in the ubiquinolcytochrome c oxidoreductase of Saccharomyces cerevisiae

1996

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Based on sensitivity of the protein to proteinase K digestion we now suggest that the N-terminus of subunit VIII is similarly oriented, implying that this subunit does not span the membrane. Despite this, however, subunit VIII cannot be extracted from the membrane even after treatment with 0.1 M Na2CO3 at pH 11.5, showing that the protein is integrally embedded in the membrane. A similar behaviour was displayed by another low molecular weight protein of the complex, subunit VII, which faces the matrix side. A model for the topology of these subunits in the membrane is discussed with respect to the structure of the complex and their involvement in quinone binding.,~?ey words: Membrane topology; Ubiquinol-cytochrome c ~.xidoreductase; Epitope tagging; Integral membrane protein; ( ~accharomyces cerevisiae) tein [8], have shown that at least this part of subunit VIII is exposed to the intermembrane space [9]. In addition, hydrophobicity plots of subunit VIII from S. cerevisiae, K. lactis, S. pombe and the homologous subunit from bovine heart predict the presence of a conserved hydrophobic stretch potentially capable of spanning a lipid bilayer (Fig. 1 and [10]), thus implying that the N-terminus of the protein faces the mitochondrial matrix.In order to verify the accuracy of these predictions, we have tagged the N-terminal half of the protein with two different epitopes (c-myc [11] and HA [12]). As indicated in Fig. I, these epitopes were not placed at the N-terminus of the protein, since the first 25 amino acids may contain a non-cleaved import signal [13]. Using limited protease treatment we have determined the position of the c-myc and the HA epitope with respect to the inner membrane. The outcome of this is combined with carbonate extraction experiments on the entire bca complex in order to further model the topology and subunit arrangement of this large respiratory enzyme.

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Section: Subunit VIII Is An Integral Membrane Proteinsupporting

confidence: 75%

Topological organization of subunits VII and VIII in the ubiquinolcytochrome c oxidoreductase of Saccharomyces cerevisiae

1996

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“…In yeast, mutagenesis of QCR8 indicated an important role of this subunit in the assembly of a functional enzyme and in inhibitor myxothiazol binding, suggesting an impaired Q P site [105, 106]. The crystal structure shows that subunit 7 contributes to both the matrix and TM regions.…”

Section: Structures Of Supernumerary Subunits and Their Functionalmentioning

confidence: 99%

Structural analysis of cytochrome bc1 complexes: Implications to the mechanism of function

Xia¹,

Esser²,

Tang³

et al. 2013

Biochimica et Biophysica Acta (BBA) - Bioenergetics

158

174

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Summary The cytochrome bc1 complex (bc1) is the mid-segment of the cellular respiratory chain of mitochondria and many aerobic prokaryotic organisms; it is also part of the photosynthetic apparatus of non-oxygenic purple bacteria. The bc1 complex catalyzes the reaction of transferring electrons from the low potential substrate ubiquinol to high potential cytochrome c. Concomitantly, bc1 translocates protons across the membrane, contributing to the proton-motive force essential for a variety of cellular activities such as ATP synthesis. Structural investigations of bc1 have been exceedingly successful, yielding atomic resolution structures of bc1 from various organisms and trapped in different reaction intermediates. These structures have confirmed and unified results of decades of experiments and have contributed to our understanding of the mechanism of bc1 functions as well as its inactivation by respiratory inhibitors.

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“…Table 1 List of the different mutants The desired substitution was present in 50-75% of these plasmids depending on the mutant. The mutated QCR8 genes Mutant name and vector Amino acid sequence Reference were ligated as a HindlII-SalI fragment into the single-copy Wild-type/pRS316 66YWYWWT° this study shuttle vectors YCplac33 or pRS316, and the 66SASAA70/ LTN2/YCplac33 66SASAA7° [4] LTN2/YEplac 195 66 SASAA70…”

Section: Production Of the Mutantsmentioning

confidence: 99%

“…assembly and catalytic function of the bcl complex [3,4]. First, mutant 66SASAA7° was restored to nearly 70% of the wild-type the residues 69Vv'~cVKNG 73 were replaced by a cysteine (69C) level in a phenotypic revertaot with the sequence ~FASAA 7°.…”

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confidence: 99%

“…quence [6] was noted: residue 61 is tryptophan instead of Since these pleiotropic effects prevent further analysis of the cysteine [9], increasing the aromatic nature of this domain. role of the 11-kDa subunit in the mechanism of action of the The sequence conservation amongst the protein family that bcl complex, studies were performed in which in vitro-muta-contains the yeast 11-kDa subunit VIII, the bovine 9.5-kDa genised QCR8 genes were transformed to a qcr8 null (DLL80) subunit VII and other homologous proteins, referred to here strain [2][3][4].…”

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The aromatic domain ⁶⁶YWYWW⁷⁰ of subunit VIII of the yeast ubiquinol‐cytochrome c oxidoreductase is important for both assembly and activity of the enzyme

et al. 1996

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The aromatic character of the region 66ywYWW7° ending at 66Y fused to residues 67SCSQAC72 coming from a of the ll-kDa subunit VIII of ubiquinol-cytochrome c oxidostop-oligonucleotide [5]. . This mutant the present study, three other site-directed mutants (66SAYAA7°, showed a reduced enzymic activity and alterations in the bind-66SASAWT° and ~SWYWW 7°) were constructed and analysed, ing of the Qout inhibitor myxothiazol, implying that this reThe data indicate that the presence of one aromatic residue is enough for a substantial level of assembly and that its position gion of the yeast 11-kDa protein contributes to the Qout bindmodulates the level of both assembly and electron transfer ing site. Additionally, the region 66ywYw~V7° in the S. activity. The results also confirm the relevance of this region of cerevisiae subunit VIII was replaced by non-aromatic residues subunit VIII for the formation of the Qout reaction domain, as (66SASAA7°) by site-directed mutagenesis, resulting in the reported by Hemrika et al. [(1993) Eur. J. Biochem. 215, 601-nearly complete absence of assembled complex III [4]. Based 609]. It is further shown that the lowered specific activity of the on the occurrence of a spontaneous second mutation, the mutant described by these authors is not due to the introduction 66FASAAT° mutant, which had a 60-70% recovery of the of a cysteine in the sequence of subunit VIII.wild-type activity, it was suggested that the aromatic nature of residue 66 was important for assembly of a functional en-

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The aromatic nature of residue 66 of the 11‐kDa subunit of ubiquinol‐cytochrome c oxidoreductase of the yeast Saccharomyces cerevisiae is important for the assembly of a functional enzyme

Cited by 14 publications

References 21 publications

Topological organization of subunits VII and VIII in the ubiquinolcytochrome c oxidoreductase of Saccharomyces cerevisiae

Topological organization of subunits VII and VIII in the ubiquinolcytochrome c oxidoreductase of Saccharomyces cerevisiae

Structural analysis of cytochrome bc1 complexes: Implications to the mechanism of function

The aromatic domain ⁶⁶YWYWW⁷⁰ of subunit VIII of the yeast ubiquinol‐cytochrome c oxidoreductase is important for both assembly and activity of the enzyme

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The aromatic nature of residue 66 of the 11‐kDa subunit of ubiquinol‐cytochrome c oxidoreductase of the yeast Saccharomyces cerevisiae is important for the assembly of a functional enzyme

Cited by 14 publications

References 21 publications

Topological organization of subunits VII and VIII in the ubiquinolcytochrome c oxidoreductase of Saccharomyces cerevisiae

Topological organization of subunits VII and VIII in the ubiquinolcytochrome c oxidoreductase of Saccharomyces cerevisiae

Structural analysis of cytochrome bc1 complexes: Implications to the mechanism of function

The aromatic domain 66YWYWW70 of subunit VIII of the yeast ubiquinol‐cytochrome c oxidoreductase is important for both assembly and activity of the enzyme

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The aromatic domain ⁶⁶YWYWW⁷⁰ of subunit VIII of the yeast ubiquinol‐cytochrome c oxidoreductase is important for both assembly and activity of the enzyme