Yeast cell cytochromes

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

Figure 12.2 Copper chaperone function, (a) Copper homeostasis in Enterococcus hirae is affected by the proteins encoded by the cop operon. CopA, Cu1+-import ATPase CopB, Cu1+-export ATPase CopY, Cu1+-responsive repressor copZ, chaperone for Cu1+ delivery to CopY. (b) The CTR family of proteins transports copper into yeast cells. Atxlp delivers copper to the CPx-type ATPases located in the post Golgi apparatus for the maturation of Fet3p. (c) Coxl7p delivers copper to the mitochondrial intermembrane space for incorporation into cytochrome c oxidase (CCO). (d) hCTR, a human homologue of CTR, mediates copper-ion uptake into human cells. CCS delivers copper to cytoplasmic Cu/Zn superoxide dismutase (SOD1). Abbreviations IMM, inner mitochondrial membrane OMM, outer mitochondrial membrane PM, plasma membrane PGV, post Golgi vessel. Reprinted from Harrison et al., 2000. Copyright (2000), with permission from Elsevier Science.

The third class of haemoproteins, with hexa-coordinate low-spin iron, are the cytochromes. First discovered by McMunn in 1884, they were rediscovered in 1925 by David Keilin. Using a hand spectroscope he observed the characteristic absorption (Soret) bands of the three cytochromes a, b and c in respiring yeast cells, which disappeared upon oxygenation. 

A wide range of mammalian proteins have been expressed in S. pombe. In a successful example, the human lipocortin I comprised 50% of the total soluble proteins in yeast cells and showed high activity, indicating that the post-translational modifications were mammalian-like [104]. Membrane proteins including cytochrome P450 were expressed at ten times the levels of those in other yeast systems [105]. Also, GPCRs have been expressed in S. pombe, where the human dopamine D2 receptor was correctly inserted into the yeast cell membrane and demonstrated expression levels three times those of S. cerevisiae [106]. 

Following a stimulation by the yeast cell wall extract zymosan, phagocytosing human leukocytes show an enhanced oxygen consumption, the cyanide-insensitive respiratory burst , after a lag period of 30-40 s. The production of superoxide anions, measured by the reduction of Fe(ni)-cytochrome c, followed the same... 

Using mutant proteins as well as a variety of redox pairs and electron-transfer distances the validity of the Marcus equation with respect to the thermodynamic driving force and distance dependence has been verified.153 This is even true for cytochrome c mutants functioning in living yeast cells.146... 

Cytochrome oxidase also serves as a proton pump, so that the process of electron transfer is associated with the vectorial transfer of protons across the membrane, and thus contributes to the establishment of the proton gradient which is used to drive the synthesis of ATP. Cytochrome oxidase is located in the inner mitochondrial membrane of animal, plant and yeast cells (the eukaryotes) and in the cell membrane of prokaryotes. The arrangement is represented schematically in Figure 58. The complexity of cytochrome oxidase and the problems associated with its solubilization from the membrane have presented great obstacles to the elucidation of the structure and mechanism of the enzyme, but its importance has resulted in an enormous literature, which has been reviewed frequently.1296 1299... 

A feature of some pterocarpan phytoalexins (e.g., pisatin and glyceollin of pea and soybean, respectively) is their hydroxylation at position 6a, a reaction catalyzed by a microsomal cytochrome P450 monooxygenase.49 50 A cDNA encoding this enzyme was recently characterized from elicited soybean cell cultures.51 The microsomal protein, expressed in yeast cells, catalyzed the stereoselective hydroxylation of (6a/ , lla/ )-3,9-dihydroxypterocarpan to its 6a-hydroxy derivative. It was also demonstrated that the enzyme expression is regulated at the transcriptional level.51... 

Fig. 1. Schematic overview of copper trafficking and homeostasis inside the yeast cell. The actions of Mad and Ace 1, copper-dependent metalloregulatory transcription factors, control the production of copper import [copper transporter (Ctr) and reductase (Fre)] and detoxification/sequestration [metallothionein (MT)] machineries, respectively. Three chaperone-mediated delivery pathways are shown. Atxl shuttles Cu(I) to the secretory pathway P-type ATPase Ccc2 (right). CCS delivers Cu(I) to the cytoplasmic enzyme copper-zinc superoxide dismutase (SOD) (left). Coxl7 shuttles Cu(I) to cytochrome c oxidase (CCO) in the mitochondria (bottom). Mitochondrial proteins Scol and Sco2 may also play a role in copper delivery to the CuA and CuB sites of CCO. Copper metabolism and iron metabolism are linked through the actions of Fet3, a copper-containing ferroxidase required to bring iron into the cell (lower right) (see text).

Sakaki, T., Shibata, M., Yabusaki, Y., Murakami, H., and Ohkawa, H., 1990, Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae, DNA Cell Biol. 9 6039614. 

While the in vitro studies on assembly have provided relatively little information, in vivo data can give us some suggestions on possible pathway of assembly. For a long time it has been known that in rho yeast cells, where cytochrome b is not produced, cytochrome c, is still accumulated in the inner membrane. This agrees with the plasmid studies of overproduction. On the other hand the cytoplasmically synthesized subunits of cytochrome c oxidase accumulate in much lower quantities in the absence of subunits I, II and III, which are mitochondrial products. It is unlikely that this diminished accumulation is due to substantially reduced gene expression. This may indicate that certain subunits are stabilized by their counterparts. 

Fig. 12.6. The onset of synthesis of various mitochondrial polypeptides upon transferring anaerobically grown yeast cells to aerobic conditions. Yeast cells were grown overnight under anaerobic conditions. At time zero they were transferred to aerobic conditions, and at the indicated time periods samples of cells were removed and lysed in the presence of NaOH and mercaptoethanol. Samples containing about 50 /ig of protein were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel. The proteins were electrotransferred to nitrocellulose sheets and decorated with specific antibodies and l-labelled protein A. Paper pieces corresponding to the labelled protein spots were cut out from the immune blot and counted in a y counter. The amount of counts obtained in the samples of 8 h aerobic conditions was taken as 100%. The antibodies used were directed against the following polypeptides porin of the mitochondrial outer membrane (29 k) /8 subunit of the proton-ATPase (iS-F,) subunit IV of cytochrome c oxidase (OxIV) and subunit V of cytochrome c oxidase (OxV).

Calcium chromate has been shown to induce cytoplasmic petite mutations in mitochondria of Saccharomyces cerevisiae K Calcium chromate also dramatically depressed the content of the mitochondrial gene products cytochrome aa3 and cytochrome b, in whole yeast cells. Chromate ( 8 nM) was readily taken up by rat thymocytes and after 30 min 9% of the Cr was found in the mitochondria although 62% was found in the nuclei . Isolated rat thymus mitochondria and nuclei readily took up CrOj . After one hour incubation of Erlich ascites tumor cells with CrOj (380 /nuclear fraction and 12% was in the mitochondrial-microsomal fraction. Levels of chromium in rat liver mitochondria reached a plateau six hours after i.v. injection of chromate (0.02 mg/kg) and remained at that level through 5 days. Liver nuclear chromium levels in the same animals, although similar to mitochondrial levels at 6 h, reached a maximum at 12 h and steadily decreased after that time. Therefore the nuclear chromium levels were lower than the mitochondrial chromium levels at later times (24-120 h) after injection. The subcellular distribution of chromium in the liver of rats injected i.v. with chromate (0.56 mg/kg) was also found to be time dependent in another study. The distribution of chromium in rat liver mitochondria increased from 5% at 15 min to 21% at 72 h and also increased in the nuclear fraction from 22% at 15 min to 52% at 72 h. Incubation of isolated rat liver mitochondria with chromate (0.3-16.6 electron transport chain of the mitochondrial iner membrane. 

Fig. I. Absorption spectra of whole yeast cells of a wild-type strain (O, left) and a cytochrome c-deficient mutant (c i, right). The pellet of cells was examined, in the reduced state, at the temperature of liquid nitrogen. The absorption peak of cytochrome c occurs at 550 mu at room temperature, and at 547 mfx at the temperature of liquid nitrogen. The vertical bars above the continuous tracing represent absorption bands as seen through a low-dispersion spectroscope. From Slonimski el al. [33].

Fig. 4. Induction of cytochrome oxidase activity (as k second" per gram of protein per milliliter) in anaerobically grown, stationary-phase yeast cells. From Chen and Chara-lampous [99].

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