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The majority of mitochondrial phosphatidylethanolamine (PtdEtn), a phospholipid essential for aerobic growth of yeast cells, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p) in the inner mitochondrial membrane (IMM). To identify components that become essential when the level of mitochondrial PtdEtn is decreased, we screened for mutants that are synthetically lethal with a temperature-sensitive (ts) allele of PSD1. This screen unveiled mutations in PHB1 and PHB2 encoding the two subunits of the prohibitin complex, which is located to the IMM and required for the stability of mitochondrially encoded proteins. Deletion of PHB1 and PHB2 resulted in an increase of mitochondrial PtdEtn at 30 degrees C. On glucose media, phb1Delta psd1Delta and phb2Delta psd1Delta double mutants were rescued only for a limited number of generations by exogenous ethanolamine, indicating that a decrease of the PtdEtn level is detrimental for prohibitin mutants. Similar to phb mutants, deletion of PSD1 destabilizes polypeptides encoded by the mitochondrial genome. In a phb1Delta phb2Delta psd1(ts) strain the destabilizing effect is dramatically enhanced. In addition, the mitochondrial genome is lost in this triple mutant, and nuclear-encoded proteins of the IMM are assembled at a very low rate. At the nonpermissive temperature mitochondria of phb1Delta phb2Delta psd1(ts) were fragmented and aggregated. In conclusion, destabilizing effects triggered by low levels of mitochondrial PtdEtn seem to account for synthetic lethality of psd1Delta with phb mutants.

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Several cellular oxidoreductases are involved in the metabolism of quinone compounds by catalyzing oneor two electron reductions of quinones. The cytotoxic effects of quinones are commonly thought to arise from their one-electron reduction, for example, by cytochrome P450 reductases, resulting in oxidative cycling of deleterious oxygen species.In contrast to other cellular reductases, NAD(P)H:quinone acceptor oxidoreductases (QRs) catalyze strict two-electron reductions and thereby afford protection against cytotoxic and neoplastic effects of electrophilic quinones [1]. For example, it has been shown that QR-null mice are more susceptible to quinone toxicity than their wild-type counterparts [2]. Furthermore, QRs are found in many solid tumors at increased levels and therefore can be used to target the tumor cells through bioreductive activation of quinone-based pro-drugs such as mitomycins [3].Cytosolic mammalian QRs have been crystallized from human (quinone reductase type 1 and 2) [4,5], rat [6] and mouse [4]. All of them are homodimers, containing FAD as a cofactor and exhibiting a flavodoxin-like fold that is characterized by twisted, parallel b-strands at the centre flanked by helices on both sides. The FAD cofactor is bound to the enzyme in the same manner, with the isoalloxazine ring system of the molecule accessible to the solvent and the ribityl phosphate and AMP moiety buried in the protein. NAD(P)H:quinone acceptor oxidoreductases are flavoenzymes expressed in the cytoplasm of many tissues and afford protection against the cytotoxic effects of electrophilic quinones by catalyzing a strict two-electron reduction. Such enzymes have been reported from several mammalian sources, e.g. human, mouse and rat, and from plant species. Here, we report identification of Lot6p (YLR011wp), the first soluble quinone reductase from the unicellular model organism Saccharomyces cerevisiae. Localization studies using an antibody raised against Lot6p as well as microscopic inspection of Lot6p-GFP demonstrated accumulation of the enzyme in the cytosol of yeast cells. Despite sharing only 23% similarity to type 1 human quinone reductase, Lot6p possesses biochemical properties that are similar to its human counterpart. The enzyme catalyzes a two-electron reduction of a series of natural and artificial quinone substrates at the expense of either NADH or NADPH. The kinetic mechanism follows a ping-pong bi-bi reaction scheme, with K M values of 1.6-11 lm for various quinones. Dicoumarol and Cibacron Marine, two well-known inhibitors of the quinone reductase family, bind to Lot6p and inhibit its activity. In vivo experiments demonstrate that the enzymatic activity of Lot6p is consistent with the phenotype of both Dlot6 and Lot6p overexpressing strains, suggesting that Lot6p may play a role in managing oxidative stress in yeast.Abbreviations duroquinone, 2,3,5,6-tetramethyl-1,4-benzoquinone; GFP, green fluorescent protein; Ni-NTA, nickel-nitrilotriacetic acid agarose; QR, quinone reductase.

The majority of phosphatidylethanolamine, an essential component of yeast mitochondria, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p), a component of the inner mitochondrial membrane. Here, we report that deletion of OXA1 encoding an inner mitochondrial membrane protein translocase markedly affects the mitochondrial phosphatidylethanolamine level. In an oxa1Δ mutant, cellular and mitochondrial levels of phosphatidylethanolamine were lowered similar to a mutant with PSD1 deleted, and the rate of phosphatidylethanolamine synthesis by decarboxylation of phosphatidylserine in vivo and in vitro was decreased. This was due to a lower PSD1 transcription rate in the oxa1Δ mutant compared with wild‐type and compromised assembly of Psd1p into the inner mitochondrial membrane. Lack of Mba1p, another component involved in the assembly of mitochondrial proteins into the inner mitochondrial membrane, did not affect the amount of phosphatidylethanolamine or the assembly of Psd1p. Deletion of the inner membrane protease Yme1p enhanced Psd1p stability suggesting that Yme1p contributed substantially to the proteolytic turnover of Psd1p in wild‐type. In summary, our results demonstrate a link between the mitochondrial protein import machinery, assembly and stability of Psd1p, and phosphatidylethanolamine homeostasis in yeast mitochondria.