Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V.

Abstract

In Saccharomyces cerevisiae, subunit V of the inner mitochondrial membrane protein complex cytochrome c oxidase is encoded by two Cytochrome c oxidase is a key enzyme in the regulation of cellular energy production in eucaryotes (18). As a member of the electron transport chain of the inner mitochondrial membrane, this enzyme catalyzes the reduction of oxygen in a reaction that is essentially irreversible (57, 59). Since this is the only irreversible reaction in the mitochondrial electron transport chain, it is equivalent to the committed step in a metabolic pathway. As such it is an important control point in the regulation of electron flow through the chain (18). At present, it is unclear how eucaryotic cells alter their cytochrome c oxidase activity levels in response to changes in energy demand. Is it by changing (increasing or decreasing) the number of assembled holoenzyme molecules in the inner membrane? Is it by modulating the activity of individual enzyme molecules? Or is it by the assembly of isologous forms of subunits, which have different effects on catalysis, into holoenzyme molecules (4,12,23,24,26)?To study these and related questions, we are examining various aspects of the biogenesis of cytochrome c oxidase in Saccharomyces cerevisiae. This enzyme consists of nine different polypeptide subunits (46); subunits I, II, and III are encoded by mitochondrial DNA, whereas subunits IV, V, VI, VII, Vlla, and VIII are encoded by nuclear DNA. The structural genes encoding all but one of these subunits (subunit VII) have been identified and characterized (6, 11-13, 20, 25, 30, 33, 42, 51, 53, 60, 61). Subunits I, II, and III as well as subunits IV, VI, and VIIa are each encoded by a unique gene. In contrast, subunit V is encoded by a small gene family composed of two nonidentical genes, COXSa and COXSb (12).Previously, we isolated and partially characterized both COXS genes (12, 13) and showed that the protein product of either can assemble into holocytochrome c oxidase. Subsequently, two other laboratories also isolated and sequenced * Corresponding author. (25,51). However, because only partial nucleotide sequences for COXSa and COXSb have been available until now the relation between COXSa and COX5b and this COXS gene has been unclear. In studies described here we demonstrate that COXSa and this recently described COXS gene are identical. We also demonstrate that COXSa and COXSb exist in single copy and are markedly different. They encode proteins that differ in 55 of 154 amino acids, their 5'-and 3'-flanking regions lack homology, and one of them, COXSb, possesses an intron that interrupts its initiation codon. In the accompanying paper (54) we describe the effects of null mutations in COX5a and COXSb on in vivo cytochrome c oxidase activity, cell respiration, and growth on nonfermentable carbon sources. We also demonstrate that these two genes encode interchangeable mature protein subunits that are expressed at markedly different levels. MATERIALS AND METHODSPlasmids, strains, and growth media. The...

Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

“…This information combined with the N-terminal amino acid sequence of the mature N. crassa subunit V, known from previous work (60), indicates that N. crassa subunit V protein is 144 amino acids long. By comparison, the corresponding S. cerevisiae subunits Va and Vb (14) and mammalian subunit IV proteins (2) are 133, 134, and 147 amino acids long, respectively. All of these proteins lack cysteine.…”

“…In many Saccharomyces strains, there are two distinct cox5 genes (14,56). The S. cerevisiae genes COX5a and COXSb encode two proteins which are 66% identical.…”

“…Since genes for cytochrome oxidase reside in both nuclear and mitochondrial compartments, analysis of the nuclear genes for cytochrome oxidase subunits is useful for studying the coordinate regulation of mRNA and protein syntheses (6,23,28,32), interactions between the nuclear and mitochondrial genetic systems, and the import of proteins into mitochondria (15). Recently, DNA sequences for yeast and mammalian nuclearly coded cytochrome oxidase subunits have been reported (2,14,23,54,62, and references therein). In N. crassa, which, unlike Saccharomyces cerevisiae, is an obligate aerobe, only the genes for the mitochondrially coded cytochrome oxidase subunits have been isolated and sequenced (10).…”

A Single Precursor Protein for Ferrochelatase-I fromArabidopsis Is Imported in Vitro into Both Chloroplasts and Mitochondria

“…In a reciprocal experiment, Huang et al (6) demonstrated that the yeast cytochrome oxidase subunit Va (COXVa) transit peptide directed chloramphenicol acetyltransferase to both chloroplasts and mitochondria in transgenic tobacco. However, both systems used artificial fusion proteins in heterologous systems, and both presequences used were atypical: that of the Chlamydomonas SSU has the potential to form an amphiphilic ␣-helix more typical of mitochondrial transit peptides (7), whereas the yeast COXVa presequence was predicted to form an antiparallel ␤-sheet (8). Further, since fungi do not possess chloroplasts, there is no need for their mitochondria to have mechanisms to discriminate between precursor proteins, so these data do not necessarily prove mistargeting of proteins between chloroplasts and mitochondria in vivo.…”

Expression of the AAC2 Gene Encoding the Major Mitochondrial ADP/ATP Carrier in Saccharomyces cerevisiae is Controlled at the Transcriptional Level by Oxygen, Heme and HAP2 Factor

“…In the yeast S. cerevisiae there are several mitochondrial enzymes encoded by gene pairs which are differentially regulated by oxygen. These include genes encoding respiratory chain components cytochrome c and subunit V of cytochrome-c oxidase [19,20,[25][26][27], as well as two genes coding for the rate-limiting enzyme in sterol biosynthesis, 3-hydroxy-3-methylglutarylCoA reductase [28]. In contrast to AAC3 which encodes a minor 'hypoxic' isoform of ADP/ATP carrier and it is negatively regulated by oxygen, heme and ROXl factor [9], the AAC2 gene encodes the major ADP/ATP carrier which functions in the oxidative phosphorylation.…”