Loss of NAD(H) from swollen yeast mitochondria
© Bradshaw and Pfeiffer; licensee BioMed Central Ltd. 2006
Received: 30 September 2005
Accepted: 24 January 2006
Published: 24 January 2006
The mitochondrial electron transport chain oxidizes matrix space NADH as part of the process of oxidative phosphorylation. Mitochondria contain shuttles for the transport of cytoplasmic NADH reducing equivalents into the mitochondrial matrix. Therefore for a long time it was believed that NAD(H) itself was not transported into mitochondria. However evidence has been obtained for the transport of NAD(H) into and out of plant and mammalian mitochondria. Since Saccharomyces cerevisiae mitochondria can directly oxidize cytoplasmic NADH, it remained questionable if mitochondrial NAD(H) transport occurs in this organism.
NAD(H) was lost more extensively from the matrix space of swollen than normal, condensed isolated yeast mitochondria from Saccharomyces cerevisiae. The loss of NAD(H) in swollen organelles caused a greatly decreased respiratory rate when ethanol or other matrix space NAD-linked substrates were oxidized. Adding NAD back to the medium, even in the presence of a membrane-impermeant NADH dehydrogenase inhibitor, restored the respiratory rate of swollen mitochondria oxidizing ethanol, suggesting that NAD is transported into the matrix space. NAD addition did not restore the decreased respiratory rate of swollen mitochondria oxidizing the combination of malate, glutamate, and pyruvate. Therefore the loss of matrix space metabolites is not entirely specific for NAD(H). However, during NAD(H) loss the mitochondrial levels of most other nucleotides were maintained. Either hypotonic swelling or colloid-osmotic swelling due to opening of the yeast mitochondrial unspecific channel (YMUC) in a mannitol medium resulted in decreased NAD-linked respiration. However, the loss of NAD(H) from the matrix space was not mediated by the YMUC, because YMUC inhibitors did not prevent decreased NAD-linked respiration during swelling and YMUC opening without swelling did not cause decreased NAD-linked respiration.
Loss of endogenous NAD(H) from isolated yeast mitochondria is greatly stimulated by matrix space expansion. NAD(H) loss greatly limits NAD-linked respiration in swollen mitochondria without decreasing the NAD-linked respiratory rate in normal, condensed organelles. NAD addition can totally restore the decreased respiration in swollen mitochondria. In live yeast cells mitochondrial swelling has been observed prior to mitochondrial degradation and cell death. Therefore mitochondrial swelling may stimulate NAD(H) transport to regulate metabolism during these conditions.
A difference in the mitochondrial respiratory chain of yeast and mammals
When lacking functional mitochondria the yeast Saccharomyces cerevisiae can grow in media containing a fermentable carbon source. Yeast can do this because of the ability to synthesize sufficient ATP by glycolysis and re-oxidize NADH reduced during glycolysis by converting pyruvate to ethanol. Yeast therefore provide a very useful genetic system to study mitochondrial bioenergetics. Electron transport in yeast mitochondria occurs, for the most part, similarly as it does in mammalian mitochondria. However, there is a striking difference. Yeast mitochondria do not contain a multi-subunit complex I NADH dehydrogenase capable of utilizing matrix space NADH to pump protons out of the matrix space as is present in mammalian mitochondria [1, 2]. Instead, yeast utilize two single polypeptide NADH dehydrogenases, one facing the matrix space and the other facing the intermembrane space. Yeast NADH dehydrogenases do not pump protons, but still pass electrons from NADH to ubiquinone similarly as mammalian complex I.
Nucleotide transporters in mitochondria
Several yeast inner mitochondrial membrane proteins able to transport nucleotides have been identified. They are all members of the mitochondrial carrier family of transport proteins. Three are isoforms of the adenine nucleotide translocator (ANT). The ANT exports ATP synthesized in the matrix space for ADP in the cytosol. A guanine nucleotide transporter brings GTP into the matrix space in exchange for GDP . A mitochondrial carrier for pyrimidine nucleotides has also been identified . And finally, a flavin adenine dinucleotide (FAD) transporter is responsible for maintaining a proper balance of matrix space FAD to flavin mononucleotide (FMN) . Mammalian mitochondrial carriers for deoxynucleotides  and adenine nucleotides [7, 8] have also been identified. Since mitochondria have shuttles that transport NAD(H) equivalents across the inner membrane and yeast mitochondria have NADH dehydrogenases facing both directions across the inner membrane, it is highly unlikely that rapid mitochondrial NAD(H) transport occurs during normal physiological conditions. However a slow rate of NAD transport across the mitochondrial inner membrane likely exists to replenish levels diluted by mitochondrial division. In this regard NAD transport activity has been identified in plant [9, 10] and mammalian mitochondria .
The yeast mitochondrial inner membrane contains an unspecific channel
The yeast mitochondrial inner membrane contains a permeability transition pore opened by ATP or respiratory substrates in the absence of phosphate . This pore is alternatively called the yeast mitochondrial unspecific channel (YMUC) . When mitochondria isolated from laboratory strains of yeast were suspended in an isoosmotic mannitol medium, YMUC opening caused colloid-osmotic mitochondrial swelling . Mitochondria isolated from an industrial yeast strain, Yeast Foam, did not swell when the YMUC was opened in a mannitol medium . However, they did swell after YMUC opening in a medium containing KCl. These results as well as other strain-dependent behaviors have caused conflicting results when characterizing yeast mitochondrial ion transport.
When yeast mitochondria were suspended in a KCl medium in the presence of the K+ ionophore valinomycin, a swelling and decreased rate of ethanol respiration was observed . The decreased respiration was hypothesized to be due to a loss of a component from the matrix space. The component postulated to be lost was either the ethanol dehydrogenase, Zn2+ a cofactor for ethanol dehydrogenase, or NAD. The present study was performed to further investigate the mechanism of the decreased respiratory rate of yeast mitochondria oxidizing ethanol.
A decreased rate of matrix space NAD-linked respiration occurs in the absence of phosphate
Colloid-osmotic swelling has been shown to occur due to opening of the YMUC when oxidizing ethanol in the absence of phosphate. Therefore, light scattering of the mitochondrial suspension was monitored under identical conditions to follow mitochondrial volume changes (Fig. 1B). Oxidizing any of these respiratory substrates caused mitochondrial swelling. The rate of swelling was slightly greater for mitochondria oxidizing the given concentrations of NADH or ethanol as compared to the given concentrations of succinate or MGP. This swelling was blocked by phosphate. Therefore the decreased NAD-linked respiratory rate is either due to YMUC opening or mitochondrial swelling.
The decreased respiratory rate is independent of YMUC opening
In the presence of phosphate, the mitochondria had a slower linear rate of respiration on NAD-linked substrates in the sucrose medium than in the mannitol medium (compare Fig. 1A to Fig. 2A). This may be related to the decreased initial absorbance (increased apparent initial matrix space volume) when mitochondria were suspended in the sucrose medium. However after spontaneously contracting for 10 minutes a volume similar to the initial absorbance of mitochondria suspended in a mannitol medium was obtained.
Mg2+ (size ~24 Daltons) was not extensively released from the mitochondrial matrix after ethanol-induced YMUC opening like it was after ATP-induced YMUC opening (see additional file 1: Figure S1). Therefore the respiration-induced YMUC does not transport solutes as large as previously reported (~1,000 Daltons) .
Mitochondrial swelling without YMUC opening causes decreased respiration
NAD addition restores the respiratory rate of swollen mitochondria
Mitochondrial contraction after swelling does not restore the respiratory rate
Hypotonically swelling mitochondria for 4.5 minutes and then recontracting them by the addition of osmotic support (sorbitol) before ethanol addition did not result in a decreased respiratory rate once ethanol was added (Fig. 6). Therefore, not only is swelling needed for the decreased respiratory rate, but also respiration must be occurring during the time of swelling for the decreased respiratory rate to occur. So the loss of NAD from the matrix space of swollen mitochondria appears to be a respiration-dependent process.
NAD(H) is released from swollen mitochondria
A decreased rate of ethanol respiration occurs when mitochondria are suspended in a KCl medium in the presence of valinomycin presumably due to a loss of matrix space components . Our data indicate that NAD(H) is the component lost and it is lost from swollen mitochondria independently of the opening of the YMUC. Since NAD addition can restore the respiratory rate in the presence of a membrane impermeant NADH dehydrogenase inhibitor, external NAD appears to enter the matrix space in swollen mitochondria. The metabolite loss is not completely specific for NAD since NAD addition does not restore MGP respiration in swollen mitochondria. But the loss has some degree of specificity because many nucleotide species are not lost upon mitochondrial swelling (see Fig. 8).
Significance of a mitochondrial NAD(H) transport activity
We believe that a transport of NAD(H) across the inner mitochondrial membrane is the most probable explanation for our data, although other interpretations are possible. A mitochondrial NAD transport activity may be needed to restore matrix space NAD levels diluted by mitochondrial division. Our data indicate that the swelling-induced NAD transport activity may be minimally active in normal, condensed mitochondria to fulfill this role, because a substantial amount of NAD(H) is released from normal, condensed mitochondria, even if NAD-linked respiration is not decreased in these unswollen organelles.
Stimulation of an NAD(H) transport activity in the inner membrane upon mitochondrial swelling would be of physiological relevance since the equilibration of NAD(H) between mitochondria and the cytoplasm may allosterically inhibit or activate certain enzymes. In this regard the SIRT family of NAD-dependent histone deacetylases regulates aging, the cell cycle, and apoptosis . SIR2, a yeast homolog controls the replicative lifespan of yeast. Therefore the cytoplasmic level of NAD(H) may be very important to many aspects of cell growth.
The equilibration of NAD(H) between the cytoplasm and the mitochondrion may also be important in coordinating energy metabolism with nutrient availability. Under conditions of glucose excess for example, the YMUC may open because the cytoplasmic phosphate concentration drops from 4 mM to 1–2 mM [21, 22] causing mitochondrial swelling . The majority of cellular NAD(H) resides in mitochondria . Therefore, an equilibration of NAD(H) between compartments would likely increase the cytoplasmic concentration. This loss of mitochondrial NAD(H) and other tricarboxylic acid cycle intermediates or cofactors would slow the tricarboxylic acid cycle so NAD would be reduced at a slower rate. This would be beneficial to slow respiration and conserve energy when the YMUC is open and dissipating the proton gradient.
The addition of glucose to yeast growing on a nonfermentable carbon source results in the swelling of a portion of the mitochondria . During this change in carbon source the cell switches from a mostly aerobic metabolism to a highly glycolytic metabolism. It has been estimated that during this change in metabolism mitochondria decrease from 12 % of the cell volume to 3% of the cell volume . A switch to a fermentable substrate is also accompanied by a release of lytic enzymes from the vacuole  followed by a release of mitochondrial proteins into the cytoplasm, which are then either degraded or imported back into remaining mitochondria . An increase in cytoplasmic NAD due to its mitochondrial release may stimulate glycolysis or act as a cytoplasmic signaling molecule during such conditions.
The structure of yeast mitochondria during swelling and subsequent contraction has been studied using electron tomography . After swelling and contraction most of the mitochondrial cristae reform, but contain no openings to the intermembrane space. Therefore swelling may cause irreversible structural damage to mitochondria resulting in cytoplasmic ATP depletion and cell death.
It is possible that the irreversible change in membrane structure during mitochondrial swelling may also play a role in the stimulation of NAD(H) transport activity.
Yeast mitochondrial swelling during programmed cell death
Yeast mitochondria have been shown to swell during programmed cell death . Mammalian mitochondria swell under conditions that induce necrotic cell death as a result of permeability transition pore opening . Since cytochrome c is dispensable for cell death induced by expression of the proapoptotic Bax protein in yeast, it is uncertain as to what function mitochondrial swelling would serve in yeast cell death . It is possible that other yeast proteins from the mitochondrial intermembrane space are involved in this process. In either case, NAD(H) may be released from the matrix space and alter cellular metabolism to influence yeast cell death.
Solute selectivity of the respiration-induced YMUC
It has previously been reported that the respiration-induced YMUC is able to transport solutes up to 1000 Daltons using a solute exclusion method with different molecular weights of polyethylene glycol (PEG) . However there is also evidence that PEG molecules of molecular weight less than 1000 Daltons are membrane permeable , which would question the suitability of this method for the size determination of small pores. Consistent with this previous observation, yeast mitochondria spontaneously swell when they are suspended in a medium containing 400 or 600 molecular weight PEG indicating that these PEG molecules may be membrane permeant. YMUC opening by ethanol addition does not increase the rate of swelling (see additional file 1: Figure S3) suggesting PEG-400 and PEG-600 are too large to be transported by YMUC.
Mitochondrial nucleotides have also been shown to be released from the YMUC . However, repeating these mitochondrial nucleotide level measurements using a shorter incubation period this time (10 minutes) revealed no large-scale efflux of nucleotides. It is possible that mitochondrial phospholipid hydrolysis from the large yeast mitochondrial phospholipase A activity  and long incubation time previously employed led to a loss of inner membrane integrity and nucleotide efflux. Since Mg2+, sucrose, PEG-400, PEG-600 and other small molecules (EDTA and raffinose, data not shown) are not permeable through the respiration-induced YMUC, nucleotides that are of a slightly larger size are almost certainly not permeable. Therefore the nucleotides that are lost from the mitochondria during YMUC opening and swelling appear to be lost independently of YMUC opening. The ability to maintain a linear rate of NAD-linked respiration after YMUC opening in a sucrose medium is further evidence that YMUC opening by itself does not allow loss of NAD from the matrix space.
The Ndt1 (YIL006W) and Ndt2 (YEL006W) genes have recently been identified as encoding yeast mitochondrial NAD transporters . Both proteins are excellent candidates to mediate a mitochondrial swelling stimulated transport of NAD. Reconstituted Ndt1 transported NAD rapidly when in exchange for another nucleotide or slowly by a uniport mechanism. It was suggested that NAD transport into the matrix space in yeast cells occurs most frequently in exchange for AMP or GMP. The Ndt1 Ndt2 double mutant had a slow growth phenotype when growing on nonfermentable carbon sources but still maintained a low level of mitochondrial NAD. This suggests that another transporter aids Ndt1 and Ndt2 in maintaining basal matrix space NAD levels.
NAD(H) loss from isolated yeast mitochondria is stimulated upon times of matrix space expansion. Added NAD is able to restore the decreased respiratory rate of swollen mitochondria by entering the matrix space, being reduced and donating electrons to the internal NADH dehydrogenase. Therefore NAD appears to be transported across the inner membrane in swollen organelles. Since yeast mitochondria contain NADH dehydrogenases facing both the matrix space and intermembrane space, and the matrix space is normally in a condensed state, a mitochondrial transport of NAD(H) is only likely to occur at a minimal rate under normal conditions. This may be enough to maintain matrix space NAD(H) levels throughout mitochondrial divisions. NAD(H) transport activity would also be stimulated when mitochondria swell prior to their degradation. Mitochondrial swelling is known to occur during switches from nonfermentable to fermentable carbon sources as well as prior to yeast cell death. The redistribution of cellular NAD(H) during these times may facilitate the metabolic changes needed for these processes to occur.
Yeast growth and mitochondrial isolation
The yeast strains W303-1A and Yeast Foam were grown aerobically at 30°C in a medium containing 2% lactate, 1% yeast extract, 2% peptone, 0.05% dextrose, and 0.01% adenine at pH 5.0. Yeast cells were harvested during logarithmic growth phase (A600 = 1.8–2.2). Mitochondria were isolated from spheroplasts as previously described , except 0.6 M sucrose was used in the homogenization buffer instead of 0.6 M mannitol. The isolated yeast mitochondria were suspended in 0.6 M mannitol, 20 mM HEPES (TEA+), and 0.1 mM EGTA, pH 6.8. Protein concentration was determined by a mini-biuret method using BSA as the standard. Briefly, 25 μl of mitochondrial suspension was added together with 75 μl of 10 % Na+ deoxycholate. Then 900 μl of biuret reagent was added. The mixture was incubated for 15 minutes, and the absorbence read at 540 nm.
Mitochondrial light scattering, respiration, and membrane potential determination
Solute permeability was monitored by light scattering using an SLM-Aminco DW-2C spectrophotometer in split beam mode at A540 using mitochondria suspended at 1 mg protein /ml. Respiration was monitored with a Clark oxygen electrode (Yellow Springs) suspending the mitochondria at a concentration of 1 mg/ml at 25°C. The mitochondrial membrane potential was monitored by absorbance of the dye safranine O as described in .
A decavanadate solution was made from a 0.1 M stock solution of Na+ orthovanadate adjusted to pH 6.0 to obtain an orange color typical of this polymeric species and then further diluted .
Determination of nucleotides
Total NAD plus NADH fluorescence measurements were performed as described in . High performance liquid chromatography analysis of the yeast mitochondrial nucleotides was performed after an alkaline extraction of the mitochondrial suspension, using a C18 reversed phase column on a Beckman model 110A instrument. Nucleotide retention times were monitored using a UV detector at A280 .
We would like to thank Dr. Dennis W. Jung for helpful discussions. We would also like to thank Dr. Stephon Manon for the gift of the Yeast Foam strain. This research was supported by an NIH grant to DP (GM071396).
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