Energy conversion coupled to cyanide-resistant respiration in the yeasts Pichia membranifaciens and Debaryomyces hansenii

Abstract

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (ΔΨ_m), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.

Cyanide-resistant respiration, Energy conversion, Pichia membranifaciens, Debaryomyces hansenii

1 Introduction

In mitochondria of all known eukaryotes, cytochrome c oxidase acts as the terminal oxidase for the electron transfer chain, reducing oxygen to water. In higher plants, some protozoa and fungi, in addition a second oxidase, branching from the core pathway at the ubiquinone pool level, also catalyses the four-electron reduction of oxygen to water. This alternative oxidase (AOX) is insensitive to cytochrome pathway inhibitors, such as antimycin A or cyanide, but is specifically inhibited by salicylhydroxamic acid (SHAM) and propylgallate, and confers a cyanide-resistant respiration (CRR) to these organisms [1].

It has been generally accepted that in plants electron transfer through the alternative oxidase is not coupled to proton translocation, so that two of the three sites of energy conservation are bypassed and the free energy is released as heat [2–4]. The remaining coupling site, complex I (reduced nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase), couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the inner mitochondrial membrane, allowing some energy conversion [5]. In yeasts, no attention has been paid to energy associated with the alternative pathway, probably because most studies on energy conversion have used Saccharomyces cerevisiae, a cyanide-sensitive yeast in which the complex I is absent [6,7].

In a previous work [8] we have reported that occurrence of CRR is very frequent among yeasts. We have proposed that, as an alternative to cytochrome respiration, yeasts have developed two strategic catabolic pathways: either aerobic fermentation in the so-called Crabtree-positive yeasts, or CRR in non-fermentative and in Crabtree-negative yeasts (capable of fermentation but not under aerobic conditions). Only a relatively small number of yeasts is capable of aerobic fermentation and only in these CRR has not been found. CRR being such a frequent pathway among yeasts, it is important to identify its contribution to energy conversion. In the present work, a non-fermentative yeast, Pichia membranifaciens, and a Crabtree-negative yeast, Debaryomyces hansenii, in which the emergence of CRR has been reported [8], were used as models to study the contribution of the cyanide-resistant respiration to the overall energy production.

2 Materials and methods

2.1 Chemicals

Antimycin A, rotenone and salicylhydroxamic acid (SHAM) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rhodamine 123 was purchased from Molecular Probes (Leiden, The Netherlands). Zymolyase 100T (Arthrobacter luteus) was purchased from Seikagaku Corporation (Amsterdam, The Netherlands).

2.2 Microorganisms and growth conditions

P. membranifaciens PYCC 5017 and D. hansenii PYCC 2968 were kindly provided by the Portuguese Yeast Culture Collection (PYCC, SA Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825 Monte da Caparica, Portugal).

Yeast cells were grown in a mineral medium with vitamins [9] and 2% (w/v) glucose at 28°C, in an orbital shaker at 150 rpm, unless otherwise stated.

2.3 Determination of growth parameters

Exponentially growing populations were used as inocula to identical growth media, supplemented with 10 mg l⁻¹ antimycin A and/or 3.2 mM SHAM (previously dissolved in dimethyl sulfoxide (DMSO)), in 500-ml flasks, magnetically stirred in waterbaths at 25°C. Absorbance was measured at 640 nm (A) in a Milton Roy Company (Rochester, NY, USA) Spectronic 20D and used to calculate specific growth rates.

2.4 Determination of adenosine triphosphate (ATP) content

CRR was induced by lowering the pH in exponentially growing cultures of P. membranifaciens to 2.2, or by resuspending D. hansenii growing in early exponential phase in 50 mM potassium phosphate buffer, pH 6.0. After a 4-h incubation at 28°C in an orbital shaker (150 rpm) for P. membranifaciens, or with aeration in a waterbath for D. hansenii, cells were harvested by a 5-min centrifugation (18 000×g) at 4°C and washed twice with ice-cold water. Three-grams (wet weight) pellets were resuspended in 100 ml of 50 mM potassium phosphate buffer, pH 6.0. After incubation with respiratory chain inhibitors for 45 min, 1 ml of cell suspension was mixed with the same volume of ethanol at 80°C for 2 min and kept on ice [10]. After dilution with 2 ml of demineralised water, the cell extract (100 μl) was incubated with 40 mM HEPES, 15 mM MgSO₄ buffer (100 μl) for 5 min, and 100 μl of this mixture was added to 100 μl of luciferin/luciferase reagent (ATP bioluminescent assay kit; Sigma). Relative light units (RLU) were determined in a BIOCOUNTER M 2500 photometer from Lumac.

2.5 Confocal and epifluorescence microscopy analysis

P. membranifaciens cells in exponential phase were centrifuged, washed twice with water and resuspended in sterile commercial water (Paracélsia), pH 6.0 (3 g wet weight per 100 ml). Cells were stained with 200 nM rhodamine 123 (Rh123) for confocal microscopy analysis, as described by Ludovico et al. [11]. Samples of stained cell suspensions (20 μl) were placed between a slide and a coverslip, after mixing with an equal volume of antifading reagent (Vectashield mounting medium for fluorescence H-1000, from Vector Laboratories, Burlingame, CA, USA) and analysed by confocal microscopy. The analysis was performed on a confocal microscope (Bio-Rad, Hercules, CA, USA).

Cells of D. hansenii in the early exponential phase (A₆₄₀∼0.4) were incubated for 4 h with 10 mg l⁻¹ antimycin A, at 28°C in an orbital shaker at 150 rpm. They were harvested by centrifugation for 6 min at 18 000×g, and washed twice with ice-cold water. As described above, cell pellets (3 g wet weight) were resuspended in water (100 ml). After a 10-min incubation with 6.4 mM SHAM, cells were stained with 1 μM Rh123, mounted on a slide as above and analysed by epifluorescence microscopy (EFM). The EFM analysis was performed on a Dialux 20 epifluorescence microscope (Zeiss, Wetzlar, Germany) equipped with a 50-W mercury vapour lamp.

2.6 Flow-cytometric analysis

P. membranifaciens cells in the stationary phase were centrifuged and washed twice with water. The pellets were resuspended in water (3 g wet weight per 100 ml) and respiratory inhibitors were added. Cells were stained with 50 nM Rh123 essentially as described by Ludovico et al. [11]. After a 25-min incubation with Rh123 at room temperature, in the dark, cellular fluorescence was measured with a flow cytometer.

Flow-cytometric measurements (FCM) were performed on a EPICS XL-MCL (Beckman-Coulter Corporation, Hialeah, FL, USA) flow cytometer, equipped with an argon-ion laser emitting a 488-nm beam at 15 mW. The green fluorescence was collected through a 488-nm blocking filter, a 550-nm long-pass dichroic and a 525-nm band-pass. About 20 000 cells per sample were analysed. An acquisition protocol was defined to measure forward scatter (FS log), side scatter (SS log) and green fluorescence (FL1 log) on a four-decades logarithmic scale. Green fluorescence (FL1 log) was gated in a scattergram of SS log×FS log in order to include in the fluorescence measurements the sub-population with the highest frequency and homogeneity. The data were analysed with the Multigraph software included in the system-II acquisition software for the EPICS XL/XL-MCL version 1.0.

2.7 Preparation of yeast mitochondria

Mitochondria were prepared essentially as described by Faye et al. [12]. Cells were harvested by centrifugation (18 000×g) for 5 min at 4°C, and washed once with 1.2 M sorbitol, after which 10 g (wet weight) were resuspended in 30 ml digestion buffer and incubated in an orbital shaking waterbath at 30°C. To prepare the digestion buffer 30 ml of 2 M sorbitol were mixed with 3 ml of 1 M potassium phosphate buffer, pH 7.5, 0.1 ml of 0.5 M ethylenediamine tetraacetic acid (EDTA), 0.5 ml mercaptoethanol, 16 ml of water and 2 units ml⁻¹ of zymolyase 100T. When the digestion was complete, 150 ml of a 1.2 M sorbitol solution was added to the suspension, which was centrifuged at 3000×g for 10 min (4°C). The pellet was then washed twice with 150 ml of 1.2 M sorbitol. The protoplasts were resuspended in a 0.5 M sorbitol, 20 mM Tris, pH 7.5, 1 mM EDTA solution (30–50 ml per 10 g of starting cells), and blended for 15–30 s. The homogenate was centrifuged twice at 1000×g for 5–10 min and the supernatant was collected and centrifuged (18 000×g) for 15 min (4°C). The mitochondria (pellet) were washed twice with 0.5 M sorbitol (15 000×g, 10–15 min) and suspended in 0.5 M sorbitol buffer.

For the assay of complex I, the mitochondria suspension was strongly sonicated for 1 min, disturbing the membrane integrity and allowing the access of external NADH to the internal face of the inner mitochondrial membrane.

2.8 Measurement of oxygen consumption

Oxygen consumption was measured with a Clark type electrode (Diamond General chemical microsensor, Ann Arbor, MI, USA). Of either a previously aerated cell suspension or a mitochondrial suspension 3 ml was incubated in a small reactor at 28°C. Substrates and inhibitors were added as indicated.

3 Results and discussion

3.1 Growth parameters in the presence of inhibitors

As a first approach, to check how important CRR is for yeast performance, we evaluated growth in the presence of inhibitors of respiration. The growth of P. membranifaciens on 0.5% (w/v) glucose was not affected by antimycin A and/or SHAM. This result was unexpected since the inhibitors had been effective in short incubation periods and the yeast is not able to ferment sugars at all. Probably the discrepancy can be explained by the ability of the yeast to degrade or to extrude the inhibitors by pumping mechanisms, after a long incubation period during the growth experiment.

In the case of D. hansenii, growth was evaluated on 0.5% (w/v) ethanol since fermentation of glucose can occur at least to some extent. The yeast was able to grow on ethanol in the presence of 3.2 mM SHAM with a slight reduction of 13% of the specific growth rate (μ_g), and also in the presence of 10 mg l⁻¹ antimycin A, although in this case the μ_g was reduced by 80%. However, when both inhibitors were present simultaneously growth stopped. Although nothing can be inferred from the results with P. membranifaciens, the behaviour of D. hansenii suggests that this yeast was able to obtain some energy when the cyanide-resistant respiration was the only pathway available. Similar results were observed by Shi et al. [13] in a Pichia stipitis mutant (PsCYC1) in which the cytochrome c gene had been disrupted, blocking the electron flow through the cytochrome c oxidase and forcing the cells to depend on the alternative respiratory pathway. This mutant was also able to grow on ethanol, indicating that CRR could produce some energy to support growth.

3.2 ATP content

The effect of different respiratory inhibitors on the ATP content of cells carrying both the cytochrome and the cyanide-resistant pathways, was explored as a clue to the ability of the CRR to contribute to energy conversion.

In preliminary experiments we established the conditions under which CRR was particularly active in the yeasts studied. CRR was induced in P. membranifaciens by lowering the pH of the culture to 2.2 and in D. hansenii by resuspending the cells in phosphate buffer, pH 6.0. After a 4-h incubation period under these conditions, oxygen consumption was cyanide-resistant in both yeasts (data not shown) and the ATP content suffered a decrease to 63% in P. membranifaciens and 50% in D. hansenii (Fig. 1). A similar behaviour has been described for Yarrowia lipolytica in which stationary cells (cyanide-resistant) have half the content in ATP of the exponential cells (cyanide-sensitive) [14]. It is interesting to notice that the ATP content of exponentially growing Y. lipolytica cells was 7 μmol (g dry weight)⁻¹[14], much lower than the values of 22 and 12 μmol (g dry weight)⁻¹ we report for exponentially growing P. membranifaciens and D. hansenii cells, respectively.

1

ATP contents in P. membranifaciens (A) and in D. hansenii (B) cells. CS represents the cyanide-sensitive cells (exponentially growing cultures) and CR represents cells with cyanide-resistant respiration, which was induced by either incubating the exponential culture at a pH 2.2 for 4 h in P. membranifaciens, or by resuspending the cells in phosphate buffer, pH 6.0, for 4 h, in D. hansenii. Incubation with respiratory chain inhibitors is represented by subscripts: A: 10 mg l⁻¹ antimycin A, S: 3.2 mM salicylhydroxamic acid (SHAM), AS: 10 mg l⁻¹ antimycin A plus 3.2 mM SHAM, ASD: 10 mg l⁻¹ antimycin A plus 3.2 mM SHAM plus 50 mM 2-deoxy-glucose. The ATP determinations were carried out in a minimum of duplicates and reported as mean values. Error bars represent standard deviations.

The presence of either antimycin A or SHAM did not affect the ATP content in cyanide-resistant D. hansenii cells, whereas in P. membranifaciens a decrease to 38 or 29%, respectively, was observed. The addition of both inhibitors together resulted in a marked decrease in the ATP content in both yeasts, but not to its complete depletion. This was achieved only upon addition of 2-deoxy-glucose, used as a control. This sugar is phosphorylated with ATP and is accumulated in this form leading to energy starvation [15]. The detection in cells incubated with antimycin A of rather high levels of ATP which were not found in cells incubated with antimycin A plus SHAM strongly indicated that formation of ATP occurs due to activity of cyanide-resistant respiration. This result was particularly evident in D. hansenii. The ATP levels were almost identical in cells without inhibitors and in cells in which either cytochrome or the alternative pathway was blocked, suggesting that both were efficient for energy production. When both pathways were blocked together almost total ATP depletion occurred.

Joseph-Horne et al. [16] have referred that there is evidence in some filamentous fungi that the complex I/AOX pathway can drive ATP synthesis, without any observable loss of cell viability. In Gaeumannomyces graminis, for example, inhibition of AOX decreased the rate of ATP synthesis, regardless of whether electron flow could still occur through the bc₁ complex [17]. Such regulation lends support to a role for fungal AOX in mitochondrial ATP production [16].

3.3 Mitochondrial membrane potential

Electrical potential differences exist across the inner mitochondrial membrane, the mitochondrial matrix being negative with respect to the cytosol. This potential is dissipated when energy metabolism is inhibited. When lipophilic cations, like some cationic dyes, are allowed to equilibrate in cells with functional mitochondria, the membrane potential differences generate gradients of cation concentration across mitochondrial and cytoplastic membranes, the concentration being highest inside the mitochondria [18].

The assessment of the mitochondrial membrane potential (ΔΨ_m) in cyanide-resistant cells, under conditions inhibitory to the cytochrome pathway, was used to provide information on the CRR contribution to the overall energy production. In the last 20 years, Rh123, a fluorescent lipophilic dye with a delocalised positive charge, has been widely used in mammalian cells to assess ΔΨ_m (see for example, [19–23]). More recently, a Rh123 staining protocol was optimised to assess ΔΨ_m in yeast populations [11]. The specific distribution of Rh123 in mitochondria of P. membranifaciens was confirmed by confocal microscopy (Fig. 2). The ΔΨ_m of stationary-phase cells of this yeast (cyanide-resistant) in the presence of respiratory inhibitors was further analysed by flow cytometry (Fig. 3), as described in Section 2. Antimycin A and SHAM each induced a decrease in the mean fluorescence intensity (MFI), indicating a decrease of ΔΨ_m (by 30 and 44%, respectively). The simultaneous presence of both inhibitors resulted in a greater decrease of MFI (62%) indicating a lower ΔΨ_m value. These results suggest, again, a contribution of the CRR to energy production in P. membranifaciens.

2

Photomicrograph obtained by confocal microscopy of P. membranifaciens cells stained with 200 nM rhodamine 123 (Rh123).

3

Mean fluorescence intensity (MFI) of stationary cells of P. membranifaciens (cyanide-resistant) stained with Rh123, determined by flow-cytometric analysis. Cells were stained with 50 nM Rh123 and respiratory inhibitors were added. Addition of inhibitors to the control cells (CR) is represented by a subscript: A: 10 mg l⁻¹ antimycin A, S: 3.2 mM salicylhydroxamic acid (SHAM), AS: 10 mg l⁻¹ antimycin A plus 3.2 mM SHAM. The MFI determinations were carried out in a minimum of triplicates and reported as mean values. Error bars represent standard deviations.

The analysis by flow cytometry was not possible with D. hansenii. Actually both the scattergrams (forward scatter versus side scatter) and the green fluorescence histograms displayed high coefficients of variation pointing to a very heterogeneous cell population. Yet it was possible to monitor, by epifluorescence microscopy analysis, the Rh123 distribution in D. hansenii cells and to demonstrate that the CRR generates a ΔΨ_m in this species (Fig. 4). The effects of presence or absence of SHAM on ΔΨ_m-dependent accumulation of Rh123 inside the mitochondria in antimycin A-treated cells of D. hansenii, in which the alternative pathway is the only respiratory pathway operational [8], were compared. While in the absence of SHAM cells accumulated Rh123 inside the mitochondria (Fig. 4A), its presence completely prevented Rh123 accumulation since no fluorescence was observed (Fig. 4B). This is a clear indication that CRR is responsible for ΔΨ_m formation and the consequent accumulation of Rh123. There are also reports on the rapid collapse of mitochondrial ΔΨ_m caused by the inhibition of AOX in isolated mitochondria from several filamentous fungi [16].

4

Photomicrographs obtained by epifluorescence microscopy (EFM) of D. hansenii cells after 4-h incubation with antimycin A, where the cyanide-resistant respiration is the only pathway available, stained with 1 μM Rh123. A: Control cells. B: Cells incubated with 6.4 mM SHAM. Above are the pictures of the same cells in phase contrast microscopy.

An interesting observation was made when oxygen consumption of P. membranifaciens cultures, with CRR induced by incubation at pH 2.2, was measured using 50 mM glucose as a substrate. The oxygen uptake rate (8.0 mmol O₂ (g dry weight)⁻¹ h⁻¹), was stimulated by 16% upon addition of antimycin A. Addition of other cytochrome pathway inhibitors promoted a similar behaviour: 33% for potassium cyanide and 27% for sodium azide. In the case of D. hansenii, the addition of antimycin A induced a stimulation of the oxygen consumption by 20%.

Studies performed in Y. lipolytica cells possessing CRR have also revealed a stimulation of respiration by cyanide, accompanied by a marked decrease in ATP and increases in ADP and AMP [14]. Since activity of the yeast alternative oxidase is stimulated by AMP, Medentsev et al. [24] have interpreted the increase in respiration rate in presence of cyanide as due to activation of the alternative oxidase by an increase in AMP concentration. But this activation is very fast. We suggest that the increase in oxygen consumption by the alternative pathway may correspond to a response of the mitochondria when only this pathway yielding a relatively low ΔΨ_m is functional.

3.4 Presence of complex I

As it is generally assumed that not any phosphorylating site is directly associated with the alternative oxidase, the results presented above can only be explained by the presence of complex I, an NADH dehydrogenase of the inner mitochondrial membrane in which proton translocation occurs. Thus, the search for this complex in P. membranifaciens and in D. hansenii was mandatory.

It is well established that the mitochondrial inner membrane is not permeable to NADH. However, yeast mitochondria are able to oxidise externally added NADH through a mechanism, not coupled to proton translocation, involving other NADH dehydrogenases that can be located at the outer or at the inner membrane of the mitochondria [6]. In this experiment we measured the oxygen consumption in previously sonicated mitochondria, in order to allow NADH (2 mM) to enter the matrix and to be oxidised by the putative complex I. In mitochondria isolated from P. membranifaciens cells, a decrease of 67% in the oxygen consumption rate was observed after the addition of 100 μM rotenone. Since rotenone specifically inhibits complex I and not the alternative NADH dehydrogenases [6], we conclude that the observed inhibition was due to the presence of complex I in this yeast and that the remaining oxygen consumption was probably due to the alternative NADH dehydrogenases. This result confirms the suspicion of the existence of complex I in this yeast, referred to by Kitano et al. [25] who found positive signals of the presence of the enzyme subunit genes (ND genes) in P. membranifaciens by Southern hybridisation with probes from Hansenula wingei ND1, ND2 and ND3 genes.

In D. hansenii, rotenone inhibited the mitochondrial oxygen consumption by 55%. The partial sequence of the genome of this yeast, recently published, has revealed several subunits of the NADH dehydrogenase complex I [26], confirming the presence of this complex.

The same criterion to detect the presence of complex I has been used by Büschges et al. [27] who have found similar inhibitory effects on mitochondria from several obligately aerobic yeasts.

3.5 Cyanide-resistant respiration and complex I in yeasts

In a previous work [8] we have reported that CRR is very common among yeasts. However, it is not found in Crabtree-positive yeasts (capable of aerobic fermentation). This suggests that in yeasts two strategies have developed for the fine adjustment of the amount of energy that can be obtained from the glycolytic flux. A maximum energy production would be produced from the cytochrome pathway, and this amount can be reduced by diverting carbon flux either to fermentation (in Crabtree-positive yeasts) or to the alternative oxidase (in non-fermentative and in Crabtree-negative yeasts).

In the present work we present evidence that in P. membranifaciens and in D. hansenii energy is produced while electrons flow through the alternative pathway. In these yeasts we also demonstrate the presence of complex I, which is absent in S. cerevisiae[7,27]. Following our previous reasoning and taking into account that complex I should be present in order to have energy produced in the alternative pathway, we were prompted to go through the literature and to look for a correlation between the simultaneous occurrence of CRR and complex I in different yeast species. It is important to notice that Nosek and Fukuhara [7] state the possible occurrence of mitochondrial NADH dehydrogenase genes in several yeasts in which a mitochondrial ND gene from Candida parapsilosis was used as a hybridisation probe. The authors indicate that there are reasons to suppose that even the group of species showing low levels of hybridisation signals do contain the ND genes.

The outcome of our literature reconnaissance is shown in Table 1. In almost all the yeasts in which CRR has been described (12 out of 13), complex I was also present. Pichia anomala is the only exception. It would be interesting to evaluate more carefully the existence of complex I in this yeast, since in this case the presence of CRR is very well documented [35]. In almost all the yeasts in which CRR is absent (six out of eight), complex I was also absent. The exceptions are Cryptococcus albidus, a non-fermentative yeast, and Pichia angusta, a Crabtree-negative yeast. Both should display CRR, according to our previous reasoning, but we were not able to detect it [8]. We intend to study more strains of these species in order to elucidate this discrepancy. It appears that whenever the alternative pathway is present, complex I is also present so that energy can be produced from this pathway. It should be emphasised that, like CRR, complex I is not found in yeasts capable of aerobic fermentation. In Crabtree-positive yeasts, fermentation provides the alternative way to obtain energy. The diversion of the metabolic flux through CRR or through fermentation, both relatively poor energy-yielding pathways, provides yeasts with mechanisms for the fine adjustment of available energy under different physiological conditions.

1

Occurrence of complex I and CRR in yeasts

Yeast	Cyanide-resistant respiration	Refs.a	Complex I	Refs.b
Candida albicans	+	[28]	+	[29,7]
C. parapsilosis	+	[30]	+	[7]
Candida sake	+	[8]	+	[7]
Candida shehatae	+	[31]	+	[7]
Candida tropicalis	+	[32]	+	[33,7]
D. hansenii PYCC 2968	+	[8]	+	[7] c
Debaryomyces occidentalis (Schwanniomyces castellii)	+	[34]	+	[34]
P. anomala (Hansenula anomala)	+	[35]	−	[7]
Pichia jadinii (Candida utilis)	+	[36]	+	[6,7]
P. membranifaciens PYCC 5017	+	[8]	+	[7] c
Pichia pastoris	+	d	+	[7]
P. stipitis	+	[37]	+	[37]
Y. lipolytica (Candida lipolytica)	+	[38]	+	[39]
C. albidus	−	[8]	+	[27]
Dekkera bruxellensis	−	[8]	−	[7]
Kluyveromyces lactis	−	[40]	−	[16,7]
Kluyveromyces marxianus	−	[8]	−	[27,7]
P. angusta (Hansenula polymorpha)	−	[8]	+	[7]
Saccharomyces bayanus (Saccharomyces carlsbergensis)	−	[8]	−	[41]
S. cerevisiae	−	[8]	−	[27,7]
Schizosaccharomyces pombe	−	[8]	−	[7]

The designations are according to Kurtzman and Fell [42]. Yeast names between parentheses indicate synonyms previously used in the literature.

^aReferences relative to CRR.

^bReferences relative to complex I.

^cThe presence of complex I was confirmed in this work.

^dThe presence of CRR was detected in this work according to the method described by Veiga et al. [8].

Acknowledgements

This work was supported by PRAXIS/PCNA/C/BIA/98/96. A.V. received a grant PRAXIS XXI/BD/9086/96. We are very grateful to Cláudio Sunkel (Instituto de Biologia Celular e Molecular da Universidade do Porto) who kindly did the confocal microscopy analysis. The ATP content was determined in the laboratory of José Maria da Fonseca.