Martine Guérin

Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL.

The characteristics of mitochondria of yeast cells expressing the pro‐apoptotic gene Bax or coexpressing Bax and the anti‐apoptotic gene Bcl‐xL have been investigated in whole cells, isolated mitochondria and permeabilized spheroplasts. It is found that Bax‐induced growth arrest of yeast cells is related to two defects in the respiratory chain: (i) a decrease in the amount of cytochrome c oxidase, the terminal enzyme of the respiratory chain, and (ii) a dramatic increase in the release of cytochrome c to the cytosol. Other components of the inner mitochondrial membrane (bc1 complex and F0F1‐ATPase) are unaffected. Coexpression of Bcl‐xL almost fully prevented the effect of Bax. Surprisingly, these results obtained in yeast parallel similar observations reported in mammalian cells.

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.”

The ‘SUN’ family: yeast SUN4/SCW3 is involved in cell septation

SUN4 is the fourth member of the SUN gene family from S. cerevisiae, whose products display high homology in their 258 amino acid C‐terminal domain. SIM1, UTH1, NCA3 (the founding members) are involved in different cellular processes (DNA replication, ageing, mitochondrial biogenesis) and it is shown herein that SUN4 plays a role in the cell septation process. sun4Δ cells are larger than wild‐type and begin a new cell cycle before they have separated from their mother cell. This phenotype is more pronounced in sun4Δ cells also deleted for UTH1. FACS analysis shows apparent polyploidy which disappears when the cell cycle is arrested by mating factor or nocodazole, indicating that cell septation is delayed without modification of the doubling time. Elutriated sun4Δ uth1Δ daughter cells are born larger, and therefore enter S phase sooner than their wild‐type counterpart. S phase duration, as well as timing of Clb2 degradation, is normal, but cell septation is delayed. Sun4p/Scw3p was recently described as a cell wall protein (Cappellaro et al., 1998 ) and, consistent with this notion, electron micrographs of sun4Δ cells show defects in the final steps of cell wall septation. Our data suggest that Sun4p and Uth1p might contribute to the regulated process of cell wall morphogenesis and septation. Copyright

The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control

In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the −10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C–G‐rich. Replacing a C/G bp with T/A at position −7 partially relieves the supercoiling response while changing the sequence between −5 and +1 (‐CCCCCG‐) for ‐GTTAA‐ abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine‐starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoters sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

In vivo evidence for back and forth oscillations of the transcription elongation complex

We have used a combination of DNA and RNA footprinting experiments to analyze the structural rearrangements experienced by a transcription elongation complex that was halted in vivo by a protein readblock. We show that the complex readblocked within an (ATC/TAG)n sequence is in a dynamic equilibrium between upstream‐ and downstream‐ translocated conformers. By increasing the strength of the putative RNA‐DNA hybrid, the ternary complex is readily trapped in the downstream‐translocated conformation, where the melted DNA region is limited to 8 bp. The shift of the equilibrium towards the downstream location is also achieved by introducing within the 5′ end of the message an RNA sequence that can pair with a segment of the transcript in the vicinity of the halted ternary complex. Our results demonstrate that within certain template DNA sequences, the back and forth oscillations of the ternary complex actually occur in a multipolymerase system and inside the cell. Furthermore, the cis‐acting effect of the upstream RNA sequence underscores an important phenomenon in gene regulation where a transcript may regulate its own elongation.

Conditions allowing different states of ATP- and GDP-induced permeability in mitochondria from different strains of Saccharomyces cerevisiae

The effect of ATP and other nucleotides on the respiration of Saccharomyces cerevisiae mitochondria was investigated. It was observed that ATP induced a stimulation of the respiration rate only in the presence of a salt in mitochondria from the bakers yeast Yeast Foam, whereas an ATP-induced stimulation occurred even in the absence of salt in mitochondria from three different laboratory strains. In both cases, the stimulation was related to a collapse of the transmembrane potential, suggesting the opening of ion- and/or proton-conducting pathways. Not only ATP, but also GTP and CTP, induced these pathways. Moreover, a similar stimulation was obtained with GDP and its analog GDP-beta-S. The fact that, as opposed to NTPs, GDP did not induce any non-specific anion channel, allowed us to use it to demonstrate unambiguously that a proton-conducting pathway was opened through the inner mitochondrial membrane of laboratory strains but not of Yeast Foam. Three additional aspects of this nucleotide-induced permeability were investigated. (i) The proton-conducting pathway was insensitive to Mg2+, whereas the anion-conducting pathway was fully inhibited by 4 mM Mg2-. (ii) The proton-conducting pathway of mitochondria isolated from laboratory strains was opened by the action of nucleotides outside the mitochondrion, since it was fully insensitive to (carboxy)atractyloside, and fully active in mitochondria isolated from op1 and delta anc strains. On the other hand, the cation-conducting pathway of Yeast Foam mitochondria was partly sensitive to (carboxy)atractyloside and insensitive to bongkrekic acid, suggesting a role of the conformational state of ANC in this activity. (iii) Both the proton and cation-conducting pathways were inhibited by very low concentrations of vanadate, under conditions where this oxyanion was polymerized to decavanadate: a competitor to nucleotide-binding sites on some enzymes.

High resolution mapping of E.coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand.

We have used chemical probes and UV light to perform a high resolution mapping of an Escherichia coli transcription elongation complex that was arrested in vivo by a protein readblock at a position distal to the promoter. The in situ probing data provide a precise picture of a constrained ternary complex in which the front edge of the polymerase is located at <6 bp from the catalytic center. Furthermore, our analyses reveal protein contacts with the non‐transcribed strand within the arrested ternary complex. Thus, these results contribute substantially to the emerging view of a flexible transcription elongation complex in which the non‐transcribed strand is an important regulatory element.

K+/H+ exchange in yeast mitochondria: sensitivity to inhibitors, solubilization and reconstitution of the activity in proteoliposomes

The K+/H+ exchange activity of the inner mitochondrial membrane was investigated in the yeast Saccharomyces cerevisiae. Swelling experiments in potassium acetate indicated that the K+/H+ exchange was active without any additional treatment after the mitochondria isolation, such as a Mg2+ depletion. As in mammalian mitochondria, the activity of yeast mitochondria was stimulated by increasing pH and was inhibited by the amphiphilic amines quinine and propranolol and by the carboxyl reagent dicyclohexylcarbodiimide. However, the activity was poorly inhibited by Mg2+ and consequently was only slightly stimulated by the Mg2+/H+ exchanger A23187. On the other hand, Zn2+ was very efficient for inhibiting the exchange and consequently the activity was strongly stimulated by the permeant metal-chelator o-phenanthroline. The [86Rb]Rb+ accumulation in mitochondria and mitoplasts was only partially inhibited by quinine and propranolol suggesting that part of the accumulation monitored under these conditions was due to cation leak through the inner membrane together with adsorption on the membrane. The DCCD-sensitive activity could be reconstituted from mitochondria and from mitoplasts solubilized with Triton X-100; this activity, measured by [86Rb]Rb+ accumulation, was quinine- and propranolol-sensitive. A spectrophotometric method, based on the capacity of negatively charged proteoliposomes to swell, was then developed in order to continuously follow the reconstituted activity.

Investigation of the Yeast Mitochondriai Unselective Channel in intact and permeabilized spheroplasts

The existence of an activity corresponding to the nucleotide‐induced Yeast Mitochondria Unselective Channel (YMUC2) of isolated mitochondria was investigated in permeabilized and intact spheroplasts of the bakers yeast Yeast Foam. In nystatin‐permeabilized spheroplasts, ATP and GDP‐β‐S induced a decavanadate‐sensitive stimulation of the respiration only under conditions equivalent to those previously reported for isolated mitochondria (low phosphate concentration, presence of a salt). On intact spheroplasts, parallel measurements of respiration rate, [ATP]/[ADP] ratio and mitochondrial transmembrane potential allowed to show that the addition of the glucose analog 2‐deoxyglucose decreased the permeability of the inner mitochondrial membrane owing to cellular ATP depletion. This strongly supports the hypothesis that Yeast Mitochondria Unspecific Channel is active in situ and inhibited by cellular [ATP] depletion.

Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.