Yeast Mitochondrial Inner Membrane Research Articles

Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria

Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome Complex of Yeast Mitochondria

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc 1 complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc 1 complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc 1 complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain.

Bcs1p can rescue a large and productive cytochrome bc1 complex assembly intermediate in the inner membrane of yeast mitochondria

The yeast cytochrome bc 1 complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc 1 complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc 1 assembly and the formation of a functionally inactive bc 1 core structure of about 500-kDa. This immature bc 1 core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc 1 core structure leading to the formation of the functional homodimeric bc 1 complex. Following Bcs1p expression, the mature bc 1 complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc 1 complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc 1 complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc 1 core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc 1 complex and gives new insights into the molecular mechanisms involved in the last steps of bc 1 assembly.

Induction of permeability of the inner membrane of yeast mitochondria

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a nonspecific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca2+-dependent pores, neither the classical Ca2+/P(i)-dependent, cyclosporin A-sensitive pore even under de-energization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca2+ concentrations (in the presence of the Ca2+ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca2+ uptake and is differently regulated compared to the mPTP of animal mitochondria.

Aquaporin-8-facilitated mitochondrial ammonia transport

Aquaporin-8 (AQP8) is a membrane channel permeable to water and ammonia. As AQP8 is expressed in the inner mitochondrial membrane of several mammalian tissues, we studied the effect of the AQP8 expression on the mitochondrial transport of ammonia. Recombinant rat AQP8 was expressed in the yeast Saccharomyces cerevisiae. The presence of AQP8 in the inner membrane of yeast mitochondria was demonstrated by subcellular fractionation and immunoblotting analysis. The ammonia transport was determined in isolated mitochondria by stopped flow light scattering using formamide as ammonia analog. We found that the presence of AQP8 increased by threefold mitochondrial formamide transport. AQP8-facilitated mitochondrial formamide transport in rat native tissue was confirmed in liver (a mitochondrial AQP8-expressing tissue) vs. brain (a mitochondrial AQP8 non-expressing tissue). Comparative studies indicated that the AQP8-mediated mitochondrial movement of formamide was markedly higher than that of water. Together, our data suggest that ammonia diffusional transport is a major function for mitochondrial AQP8.

Use of the addressing sequence of yeast D-lactate dehydrogenase for insertion of CYP11A1p into the inner membrane of yeast mitochondria

Mammalian cytochrome P450scc (CYP11A1p) is a pseudointegral protein of the inner membrane of mitochondria with the active center exposed in the matrix. Upon import of the CYP11A1p precursor into yeast mitochondria, only a minor part was incorporated into the inner mitochondrial membrane and acquired catalytic activity (Kovaleva, I. E., Novikova, L. A., Nazarov, P. A., Grivennikov, S. I., and Luzikov, V. N. (2003) Eur. J. Biochem., 270, 222-229). The present work is an attempt to increase the efficiency of this process by substitution of the inherent N-terminal presequence of CYP11A1p by the addressing signal of D-lactate dehydrogenase (D-LD) of the yeast Saccharomyces cerevisiae. D-LD is known to be inserted into the inner membrane of mitochondria through its transmembrane domain located close to the N-terminus of the polypeptide chain in such a way that the protein globule is exposed in the intermembrane space. The hybrid protein D-LD(1-72)-mCYP11A1p synthesized in yeast cells was imported into yeast mitochondria, underwent processing, and was inserted into the inner membrane on the side of the intermembrane space. In the presence of adrenodoxin and adrenodoxin reductase, the hybrid protein exhibited cholesterol side-chain cleavage activity. Thus, CYP11A1p insertion into the inner membrane of mitochondria mediated by the D-LD topogenic signal resulted in the catalytically active mCYP11A1p domain in the hybrid protein.

Molecular Dissection of the Protein Import Complexes of the Inner Membrane of Yeast Mitochondria

Molecular Dissection of the Protein Import Complexes of the Inner Membrane of Yeast Mitochondria

Role of the ABC transporter Mdl1 in peptide export from mitochondria.

ATP-binding cassette (ABC) adenosine triphosphatases actively transport a wide variety of compounds across biological membranes. Here, the ABC protein Mdl1 was identified as an intracellular peptide transporter localized in the inner membrane of yeast mitochondria. Mdl1 was required for mitochondrial export of peptides with molecular masses of approximately 2100 to 600 daltons generated by proteolysis of inner-membrane proteins by the m-AAA protease in the mitochondrial matrix. Proteolysis by the i-AAA protease in the intermembrane space led to the release of similar-sized peptides independent of Mdl1. Thus, two pathways of peptide efflux from mitochondria exist that may allow communication between mitochondria and their cellular environment.

Yeast and Rat Coq3 and Escherichia coli UbiG Polypeptides Catalyze Both O-Methyltransferase Steps in Coenzyme Q Biosynthesis

Ubiquinone (coenzyme Q or Q) is a lipid that functions in the electron transport chain in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. Q-deficient mutants of Saccharomyces cerevisiae harbor defects in one of eight COQ genes (coq1-coq8) and are unable to grow on nonfermentable carbon sources. The biosynthesis of Q involves two separate O-methylation steps. In yeast, the first O-methylation utilizes 3, 4-dihydroxy-5-hexaprenylbenzoic acid as a substrate and is thought to be catalyzed by Coq3p, a 32.7-kDa protein that is 40% identical to the Escherichia coli O-methyltransferase, UbiG. In this study, farnesylated analogs corresponding to the second O-methylation step, demethyl-Q(3) and Q(3), have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro. Both yeast and rat Coq3p recognize the demethyl-Q(3) precursor as a substrate. In addition, E. coli UbiGp was purified and found to catalyze both O-methylation steps. Futhermore, antibodies to yeast Coq3p were used to determine that the Coq3 polypeptide is peripherally associated with the matrix-side of the inner membrane of yeast mitochondria. The results indicate that one O-methyltransferase catalyzes both steps in Q biosynthesis in eukaryotes and prokaryotes and that Q biosynthesis is carried out within the matrix compartment of yeast mitochondria.

Characterization of Recombinant Adrenodoxin Reductase Homologue (Arh1p) from Yeast: IMPLICATION IN IN VITRO CYTOCHROME P45011β MONOOXYGENASE SYSTEM

The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely adrenodoxin reductase and adrenodoxin. Adrenodoxin reductase transfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian adrenodoxin reductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome c reduction, and, more importantly, in vitro 11beta-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent Km values were determined to be 0.5, 0.6, and 0.1 microM for NADPH, NADH, and bovine adrenodoxin, respectively. These values differ slightly from those of mammalian adrenodoxin reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.

Characterization of the yeast mitochondria unselective channel: a counterpart to the mammalian permeability transition pore?

Large and unselective permeabilities through the inner membrane of yeast mitochondria have been observed for more than 20 years, but the characterization of these permeabilities, leading to hypothesize the existence of a large-conductance unselective channel in yeast inner mitochondrial membrane, was done only recently by several groups. This channel has been tentatively identified as a yeast counterpart to the mammalian permeability transition pore, the crucial role of which is now well-documented in physiopathological phenomena, such as Ca2+ homeostasis, ischemic damages, or programmed cell death. The aim of this review is to make a point on the known characteristics of this yeast mitochondrial unselective channel (YMUC) and to analyze whether or not it can be considered as a "yeast permeability transition pore."

Targeting and translocation of the phosphate carrier/p32 to the inner membrane of yeast mitochondria.

We analyzed the submitochondrial location and biogenesis pathway of the phosphate carrier (PiC), also termed p32, of Saccharomyces cerevisiae mitochondria, PiC/p32 was found to behave as an integral membrane protein that cofractionated with the ADP/ATP carrier of the inner membrane. Import of the precursor of PiC/p32 required a membrane potential across the inner membrane, supporting its localization to the inner membrane. This makes it unlikely that the major function of PiC/p32 is that of an import receptor on the surface of the mitochondrial outer membrane. Furthermore, we found that both receptors MOM72 and MOM19 were involved in the import pathway of the precursor of PiC/p32 with MOM72 being responsible for the bulk of import. Yeast PiC/p32 is thus not only structurally homologous to the ADP/ATP carrier, but has a similar targeting mechanism and submitochondrial location, supporting its classification as a member of the inner membrane carrier family.

Localization of the NADH Kinase in the Inner Membrane of Yeast Mitochondria

The bulk of NADH kinase of Saccharomyces cerevisiae was recovered in the mitochondrial fraction prepared from spheroplasts. Most of the NADH kinase was localized in the inner membrane fraction, which was separated from other mitochondrial components by the combined swelling, shrinking, and sonication procedure. Treatment of mitoplasts with antiserum against the NADH kinase caused inactivation of the enzyme. On the contrary, no influence was observed upon the same treatment of intact mitochondria. p-Chloromercuribenzoate and eosin-5-maleimide inactivated the enzyme without affecting the matrix ATPase. The NADH kinase was enzymatically iodinated in mitoplasts, but not in the intact mitochondria. These results support the conclusion that NADH kinase is localized and functions at the intermembrane space side of the mitochondrial inner membrane. It is evident that the NADH kinase is encoded by nuclear gene(s) because it is synthesized in the presence of chloramphenicol or acriflavine, and a significant amount of the enzyme was detected in mitochondrial DNA-deficient mutants.

Structure of cytochromec oxidase from baker's yeast—a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochromec binding site

Cytochrome c oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K). In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtration. Their amino acid composition as well as their amino- and carbosy-terminal amino acid residues have been determined. Sequence determinations of subunits IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids. The binding site of yeast cytochrome oxidase for cytochrome c was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochrome c from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochrome c and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochrome c and from subunit III of cytochrome oxidase.