酿酒酵母胞质内NADH的代谢调控

酿酒酵母(Saccharomyces cerevisiae)的生长过程有大量的胞内NADH产生。有氧途径中,胞外的NADH脱氢酶、三磷酸甘油穿梭酶系是线粒体内NADH氧化的最主要机制。该文主要讨论以下三个方面的内容:不同生理环境下促成线粒体胞内NADH氧化的各主要机制的作用;借助电子传递链开启NADH从胞质脱氢酶到线粒体的通道,各代谢动力学的有序进行;各种酶形成超分子复合物,尤其是起关键调控作用的酶形成具相似生理功能的高整合性功能酶。     

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel,VDAC

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found inNeurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 µg/ml) increases the voltage dependence of VDAC channels over 2–3-fold. At higher protein concentrations (50–100 µg/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifierin vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.     

Perspectives on the mitochondrial multiple conductance channel

A multiple conductance channel (MCC) with a peak conductance of over 1 nS is recorded from mitoplasts (mitochondria with the inner membrane exposed) using patch-clamp techniques. MCC shares many general characteristics with other intracellular megachannels, many of which are weakly selective, voltage-dependent, and calcium sensitive. A role in protein import is suggested by the transient blockade of MCC by peptides responsible for targeting mitochondrial precursor proteins. MCC is compared with the peptide-sensitive channel of the outer membrane because of similarities in targeting peptide blockade. The pharmacology and regulation of MCC by physiological effectors are reviewed and compared with the properties of the pore hypothesized to be responsible for the mitochondrial inner membrane permeability transition.     

线粒体ATP敏感钾通道与钙激活钾通道激活对正常与缺血脑线粒体通透性转变的影响

目的：明确线粒体ATP敏感钾通道与钙激活钾通道对正常和缺血脑线粒体渗透性转变的作用。方法：实验采用分光光度法,在分离的线粒体上分别观察两种线粒体钾通道激动剂对正常与缺血脑线粒体肿胀的影响。结果：在正常脑线粒体,diazoxide与NSl619能有效抑制由钙诱导的线粒体氏20下降,但其效应可被atractyloside所阻断。与正常相比,缺血损伤后的脑线粒体在钙离子诱导下线粒体A520下降较快,diazoxide与NS1619仍可抑制由钙诱导的线粒体A520下降,其作用同样为atractykxside所阻断。结论：线粒体ATP敏感钾通道与钙激活钾通道激活在离体条件均具有保护脑线粒体的作用,其作用可能是通过影响线粒体通透性转变而实现。     

A novel NADH kinase is the mitochondrial source of NADPH in Saccharomyces cerevisiae

Mitochondria require NADPH for anti-oxidant protection and for specific biosynthetic pathways. However, the sources of mitochondrial NADPH and the mechanisms of maintaining mitochondrial redox balance are not well understood. We show here that in Saccharomyces cerevisiae, mitochondrial NADPH is largely provided by the product of the POS5 gene. We identified POS5 in a S.cerevisiae genetic screen for hyperoxia-sensitive mutants, or cells that cannot survive in 100% oxygen. POS5 encodes a protein that is homologous to NAD(+) and NADH kinases, and we show here that recombinant Pos5p has NADH kinase activity. Pos5p is localized to the mitochondrial matrix of yeast and appears to be important for several NADPH-requiring processes in the mitochondria, including resistance to a broad range of oxidative stress conditions, arginine biosynthesis and mitochondrial iron homeostasis. Pos5p represents the first member of the NAD(H) kinase family that has been identified as an important anti-oxidant factor and key source of the cellular reductant NADPH.     

cAMP-dependent phosphoprotein changes in the yeast Saccharomyces cerevisiae

Abstract cAMP-dependent phosphoprotein changes were determined using 1-dimensional SDS-gel electrophoresis in a cAMP-requiring yeast mutant ( Saccharomyces cerevisiae AM18). During cAMP starvation, the yeast cells accumulated 3 ³²P-labeled bands with M _r/ 72000, 54000, and 37000. The M _r/ 72000 protein was the most prominent phosphorylated protein. After the readdition of cAMP, these phosphoproteins lost their ³²P-label while phosphoproteins with M _r/ 76000, 65000, 56000 and 30000 were accumulated. Similar phosphoprotein changes were also detected in cdc35 at the nonpermissive temperature, but not in wildtype (A363A) or cdc7 strains of S. cerevisiae .     

Linearity, saturation and blocking in a large multiionic channel: divalent cation modulation of the OmpF porin conductance

Measurement of unitary conductance is a fundamental step in the characterization of a protein ion channel permeabilizing a membrane. We study here the effect of salts of divalent cations on the OmpF channel conductance with a particular emphasis in dissecting the role of the electrolyte itself, the role of the counterion accumulation induced by the protein channel charges and other effects not found in salts of monovalent cations. We show that current saturation and blocking are not exclusive properties of narrow (single-file) ion channels but may be observed in large, multiionic channels like bacterial porins. Single-channel conductance measurements performed over a wide range of salt concentrations (up to 3 M) combined with continuum electrodiffusion calculations demonstrate that current saturation cannot be simply ascribed to ion interaction with protein channel residues.     

Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures

Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.     

Role of mitochondrial calcium transport in the control of substrate oxidation

This paper reviews the model of the control of mitochondrial substrate oxidation by Ca²⁺ ions. The mechanism is the activation by Ca²⁺ of four mitochondrial dehydrogenases, viz: glycerol 3-phosphate dehydrogenase, the pyruvate dehydrogenase multienzyme complex (PDH), NAD-linked isocitrate dehydrogenase (NAD-IDH) and 2-oxoglutarate dehydrogenase (OGDH). This results in the increase, or near-maintenance, of mitochondrial NADH/NAD ratios in the activated state, depending upon the tissue and the degree of "downstream" activation by Ca²⁺, likely at the level of the F₁F₀ ATP-ase. Higher values of the redox span of the respiratory chain allow for greatly increased fluxes through oxidative phosphorylation with a minimal drop in protonmotive force and phosphorylation potential. As PDH, NAD-IDH and OGDH are all located within the inner mitochondrial membrane, it is changes in matrix free Ca²⁺ ( [Ca²⁺]m ) which act as a signal to these activities. In this article, we review recent work in which ([Ca²⁺]m) is measured in cells and tissues, using different techniques, with special emphasis on the question of the degree of damping of ([Ca²⁺]m) relative to changes in cytosol free Ca²⁺ in cells with rapid transients in cytosol Ca²⁺, e.g. cardiac myocytes. Further, we put forward the point of view that the failure of mitochondrial energy transduction to keep pace with cellular energy needs in some forms of heart failure may involve a failure of ([Ca²⁺]m) to be raised adequately to allow the activation of the dehydrogenases. We present new data to show that this is so in cardiac myocytes isolated from animals suffering from chronic, atreptozocin-induced diabetes. This raises the possibility of therapy based upon partial inhibition of mitochondrial Ca²⁺ efflux pathways, thereby raising ([Ca²⁺]m) at a given, time-average value of cytosol free Ca²⁺.     

Apoptosis induction by the natural product cancer chemopreventive agent deguelin is mediated through the inhibition of mitochondrial bioenergetics

Deguelin exhibits chemopreventive properties in animal carcinogenesis models. The mechanism underpinning the chemopreventive effects of deguelin has not been fully elucidated. However, it has been suggested that this agent reduces ornithine decarboxylase activity, and perhaps the activity of other signaling intermediates associated with tumorigenesis, by inhibiting mitochondrial bioenergetics. We sought to determine if deguelin could trigger apoptosis by inhibiting mitochondrial bioenergetics. Therefore, we compared and contrasted the effects of deguelin on cells from two human cutaneous squamous cell carcinoma cell lines (parental cells) and their respiration-deficient clones lacking mitochondrial DNA (rho0). While deguelin promoted marked apoptosis in the parental cells in a dose- and time-dependent manner, it failed to do so in the rho0 clones. Furthermore, short-term exposure to deguelin diminished oxygen consumption by the parental cells and promoted mitochondrial permeability transition as evidenced by the dissipation of mitochondrial inner transmembrane potential, reactive oxygen species production, cardiolipin peroxidation, caspase activation, and mitochondrial swelling. Mitochondrial permeability transition was not observed in the rho0 clones exposed to deguelin. These results demonstrate that deguelin induces apoptosis in skin cancer cells by inhibiting mitochondrial bioenergetics and provide a novel mechanism for the putative anticancer activity of this agent.     

The migration of mitochondrial DNA fragments to the nucleus affects the chronological aging process of Saccharomyces cerevisiae

Migration of fragmented mitochondrial DNA (mtDNA) to the nucleus has been shown to occur in multiple species including yeast, plants, and mammals. Several human diseases, including Pallister–Hall syndrome and mucolipidosis, can be initiated by mtDNA insertion mutagenesis of nuclear DNA. In yeast, we demonstrated that the rate of mtDNA fragments translocating to the nucleus increases during chronological aging. The yeast chronological lifespan (CLS) is determined by the survival of nondividing cell populations. Whereas yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, strains with decreased mtDNA transfer rates to the nucleus exhibited an extended CLS. Although one of the most popular theories of aging is the free radical theory, migration of mtDNA fragments to the nucleus may also contribute to the chronological aging process by possibly increasing nuclear genomic instability in cells with advanced age.     

Protein overexport in a Saccharomyces cerevisiae mutant depends on mitochondrial genome integrity and function

The wild-type yeast Saccharomyces cerevisiae (S. cerevisiae) is able to export less than 1 percent of the protein to be secreted. The reasons for retention of most of the secretory proteins on the cell surface of S. cerevisiae are unknown. Recently, temperature-sensitive (ts) mutants of S. cerevisiae showing an oversecretion phenotype were isolated. In order to study the influence of the mitochondrial genome status on protein export in yeast cells, we have isolated several types of respiratory impaired mitochondrial mutants of either the parental S. cerevisiae strain or their derivative ts protein-overexporting mutants. In this paper we demonstrate by quantitative analyses of exported proteins and by SDS-PAGE analysis that protein overexport in ts mutants requires mitochondrial genome integrity and function.     

Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state

Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (E(GSH)) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with E(GSH)-specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of E(GSH) in the IMS, thus explaining a steady-state E(GSH) in the IMS which is similar to the cytosol. Moreover, we show that the local E(GSH) contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells.     

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.     

Regulation of the mitochondrial outer membrane channel,VDAC

The channel-forming protein, VDAC, located in the mitochondrial outer membrane, is probably responsible for the high permeability of the outer membrane to small molecules. The ability to regulate this channelin vitro raises the possibility that VDAC may perform a regulatory rolein vivo. VDAC exists in multiple, quasi-degenerate conformations with different permeability properties. Therefore a modest input of energy can change VDAC's conformation. The ability to use a membrane potential to convert VDAC from a high (open) to a low (closed) conducting form indicates the presence of a sensor in the protein that allows it to respond to the electric field. Titration and modification experiments point to a polyvalent, positively charged sensor. Soluble, polyvalent anions such as dextran sulfate and Konig's polyanion seem to be able to interact with the sensor to induce channel closure. Thus there are multiple ways of applying a force on the sensor so as to induce a conformational change in VDAC. Perhaps cells use one or more of these methods.     

Plasma membrane electron transport in Saccharomyces cerevisiae depends on the presence of mitochondrial respiratory subunits

Most investigations into plasma membrane electron transport (PMET) in Saccharomyces cerevisiae have focused on the inducible ferric reductase responsible for iron uptake under iron/copper-limiting conditions. In this paper, we describe a PMET system, distinct from ferric reductase, which reduces the cell-impermeable water-soluble tetrazolium dye, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-1), under normal iron/copper conditions. WST-1/1-methoxy-phenazine methosulphate reduction was unaffected by anoxia and relatively insensitive to diphenyleneiodonium. Dye reduction was increased when intracellular NADH levels were high, which, in S. cerevisiae , required deletion of numerous genes associated with NADH recycling. Genome-wide screening of all viable nuclear gene-deletion mutants of S. cerevisiae revealed that, although mitochondrial electron transport per se was not required, the presence of several nuclear and mitochondrially encoded subunits of respiratory complexes III and IV was mandatory for PMET. This suggests some form of interaction between components of mitochondrial and plasma membrane electron transport. In support of this, mitochondrial tubular networks in S. cerevisiae were shown to be located in close proximity to the plasma membrane using confocal microscopy.     

Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae

We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.     

In Saccharomyces cerevisiae,cations control the fate of the energy derived from oxidative metabolism through the opening and closing of the yeast mitochondrial unselective channel

The yeast mitochondrial unspecific channel (YMUC) sensitivity to inorganic (Ca²⁺ or Mg²⁺) or organic (hexyl or octyl-guanidine) cations was measured. The rate of oxygen consumption in State 3 and State 4, the transmembrane potential (), mitochondrial swelling, and the polyethylene-glycol mediated recontraction were used to follow opening of the YMUC. Addition of 0.4 mM PO₄ did not close the YMUC, although it did enhance the sensitivity to Ca²⁺ (I₅₀ decreased from 50 to 0.3 mM) and Mg²⁺ (I₅₀ decreased from 5 to 0.83 mM Mg²⁺). The Ca²⁺ concentration needed to close the YMUC was higher than the concentrations usually observed in the cell. Nonetheless, Mg²⁺, Ca²⁺, and PO₄ exhibited additive effects. These cations did not inhibit contraction of preswollen mitochondria, suggesting that the YMUC/cation interaction was labile. Octyl-guanidine (OG-I₅₀ 7.5 M) was the only cation which inhibited mitochondrial recontraction, probably as a result of membrane binding stabilization through its hydrophobic tail. The PO₄-dependent, Ca²⁺/Mg²⁺-mediated closure of the YMUC may be a means to control the proportion of oxidative energy producing ATP or being lost as heat.