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

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 (Delta Psi(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.     

Stress situations induce cyanide-resistant respiration in spoilage yeasts

AIMS: To investigate the conditions that promote the expression of cyanide-resistant respiration (CRR) in the spoilage yeasts Pichia membranifaciens and Debaryomyces hansenii. METHODS AND RESULTS: CRR was detected by sensitivity of oxygen consumption to salicylhydroxamic acid. It was absent in both yeasts in the early exponential phase, but was triggered by several stress situations. Starvation under aerobic conditions, decreasing pH or incubation of the culture in a narrow temperature range below the maximum temperature for growth promoted the emergence of CRR in both yeasts. In D. hansenii, CRR was also induced by 1.5-2 mol l(-1) NaCl. Although the presence of H2O2 and menadione induced CRR, radical scavengers had no effect on the emergence of CRR. Also, the level of reactive oxygen species did not vary with the CRR activity. CONCLUSIONS: Under aerobic conditions, a respiratory pathway alternative to the cytochrome chain is triggered by stress conditions in P. membranifaciens and D. hansenii. SIGNIFICANCE AND IMPACT OF THE STUDY: The relationship between stress situations and CRR must be taken into account in studies on the performance of spoilage yeasts in the food processing environments where several forms of stress are common.     

Cyanide-resistant respiration is frequent, but confined to yeasts incapable of aerobic fermentation

In Pichia membranifaciens, cyanide-resistant respiration (CRR) sensitive to salicylhydroxamic acid emerged after forced aeration of starved cells for 4 h. Surveying a large number of species by this simple methodology, we found that CRR is very frequent among yeasts. Remarkably, considering our results together with previous data in the literature, CRR was present in 24 out of 28 non-fermentative or Crabtree-negative yeasts and absent in 10 out of 12 Crabtree-positive yeasts. We submit that, as alternatives to cytochromic respiration, yeasts developed two strategies: either aerobic fermentation in Crabtree-positive yeasts or CRR in non-fermentative or Crabtree-negative yeasts.     

工业真菌中的交替氧化酶

真菌是现代微生物发酵产业的主力军之一。交替呼吸途径(Alternative respiration pathway,ARP),以其非磷酸化电子传递途径,起到了能量溢流(Energy overflow)的作用,调节细胞能量代谢,平衡碳代谢和电子传递,有利于代谢产物的积累。此外,交替呼吸对真菌的抗逆反应和条件致病菌的生理作用也都具有非常重要的影响。交替氧化酶(Alternative oxidase,AOX)是线粒体中交替呼吸途径的末端氧化酶,广泛存在于高等植物及部分真菌和藻类中。由于交替氧化酶对水杨氧肟酸(Salicylhydroxamic acid,SHAM)敏感而对细胞色素呼吸抑制剂氰化物不敏感,交替氧化酶AOX介导的交替呼吸途径又被称为抗氰呼吸途径(Cyanide-resistant respiration,CRR)。近年来,研究交替呼吸途径和交替氧化酶已成为细胞呼吸代谢领域的热门课题。本文主要对真菌交替呼吸途径和交替氧化酶的结构与其在工业真菌体内功能的最新研究进展作一简要的综述。     

Effect of salicylic acid on the alternative pathway of yellow lupine respiration

The effects of salicylic acid (SA) on the rate of respiration and the activity of cyanide-resistant sensitive to salicylhydroxamic acid oxidation pathway in detached etiolated cotyledons of yellow lupine (Lupinus luteus L.) and mitochondria isolated from these cotyledons were studied. Cotyledon treatment with 1 mM SA for 12 h increased the rate of oxygen uptake predominantly due to the activation of cyanide-resistant respiration (CRR) and alternative pathway of mitochondrial oxidation. It was established that the lupine genome encodes at least two isoforms of alternative oxidase (AO), LuAOX1 and LuAOX2, with the mol wt of about 35 kD. These proteins are always present in the mitochondria of etiolated lupine cotyledons, but their level increased rapidly after cotyledon treatment with SA, probably by increasing the mRNA content of the corresponding genes. SA-induced expression of Aox genes was correlated with the activation of CRR and an increase in the maximal activity (capacity) of AO in both detached yellow lupine cotyledons and mitochondria isolated from them.     

A prokaryotic alternative oxidase present in the bacterium Novosphingobium aromaticivorans

The alternative oxidase (AOX) is a terminal oxidase present in the respiratory chain of all plants as well as some yeasts and trypanosomes, but has not previously been found in a prokaryote. We have identified an AOX homologue in Novosphingobium aromaticivorans, the first AOX found in a prokaryote. We have cloned the gene for the N. aromaticivorans AOX and showed it to have a terminal oxidase activity when heterologously expressed in Escherichia coli. We have also shown that this novel AOX is expressed in N. aromaticivorans cells, and that its expression level is greatly influenced by the oxygen level and carbon source of the growth media.     

The cyanide sensitivity of the residual respiration in Schizosaccharomyces japonicus

Abstract Schizosaccharomyces japonicus , a highly respiratory deficient yeast species, contains two terminal oxidase systems. One is highly sensitive to cyanide like the main terminal oxidase system of respiratory sufficient yeasts. The alternative system is hardly sensitive to cyanide like the usual terminal oxidase system of other respiratory deficient yeasts, and such as that found in respiratory sufficient yeasts besides the sensitive system. The order of magnitude of each system in Sch. japonicus is only a few μ l O₂· (mg dry biomass)⁻¹· h⁻¹, the insensitive system having the lowest activity. As a result the alternative system may pass unnoticed. This situation may be unique among yeasts.     

Alternative respiration of fungus <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis>

Respiratory characteristics of germinating spores, developing mycelium and mitochondria of the fungus Phycomyces blakesleeanus were investigated by means of oxygen Clark-type electrode. The effects of respiratory inhibitors and metabolic compounds on oxygen consumption were tested. It was demonstrated that P. blakesleeanus apart of cyanide-sensitive respiration, CSR, possess alternative respiration, (cyanide-resistant respiration, CRR) which is constitutive and whose capacity decreases during development. Maximum is observed for activated spores where CRR capacity is significantly greater than CSR. After treatment with antimycin A, a third type of respiration insensitive to antimycin A and low concentration of SHAM (sufficient for inhibition of CRR), but sensitive to cyanide and high concentration of SHAM, has been expressed.     

Function of the alternative oxidase: is it still a scavenger?

The alternative oxidase is a respiratory chain protein found in all higher plants, fungi, non-fermentative yeasts and trypanosomes. Its primary structure suggests that it is a new member of the di-iron carboxylate protein family. Recent sequence analysis indicates an evolutionary relationship between primitive members of this protein family and the alternative oxidase, suggesting that its early function was to scavenge di-oxygen. However, modelling of plant growth kinetics suggests a different function.     

Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina

Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long-lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.     

Structure, divergence, and distribution of the CRR centromeric retrotransposon family in rice

The centromeric retrotransposon (CR) family in the grass species is one of few Ty3-gypsy groups of retroelements that preferentially transpose into highly specialized chromosomal domains. It has been demonstrated in both rice and maize that CRR (CR of rice) and CRM (CR of maize) elements are intermingled with centromeric satellite DNA and are highly concentrated within cytologically defined centromeres. We collected all of the CRR elements from rice chromosomes 1, 4, 8, and 10 that have been sequenced to high quality. Phylogenetic analysis revealed that the CRR elements are structurally diverged into four subfamilies, including two autonomous subfamilies (CRR1 and CRR2) and two nonautonomous subfamilies (noaCRR1 and noaCRR2). The CRR1/CRR2 elements contain all characteristic protein domains required for retrotransposition. In contrast, the noaCRR elements have different structures, containing only a gag or gag-pro domain or no open reading frames. The CRR and noaCRR elements share substantial sequence similarity in regions required for DNA replication and for recognition by integrase during retrotransposition. These data, coupled with the presence of young noaCRR elements in the rice genome and similar chromosomal distribution patterns between noaCRR1 and CRR1/CRR2 elements, suggest that the noaCRR elements were likely mobilized through the retrotransposition machinery from the autonomous CRR elements. Mechanisms of the targeting specificity of the CRR elements, as well as their role in centromere function, are discussed.     

EPR studies of the mitochondrial alternative oxidase. Evidence for a diiron carboxylate center

The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (-J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.     

Emerging applications of the methylotrophic yeasts

The use of methylotrophic yeasts for the production of single-cell-protein (SCP), alcohol oxidase and fine chemicals has been proposed. Fermentation technology developed for the growth of these yeasts on methanol at high cell densities has been commercialized. However, it is the production of heterologous recombinant proteins by Pichia pastoris that is emerging as the most significant application of the methylotrophic yeasts.     

Emerging applications of the methylotrophic yeasts

Abstract The use of methylotrophic yeasts for the production of single-cell-protein (SCP), alcohol oxidase and fine chemicals has been proposed. Fermentation technology developed for the growth of these yeasts on methanol at high cell densities has been commercialized. However, it is the production of heterologous recombinant proteins by Pichia pastoris that is emerging as the most significant application of the methylotrophic yeasts.     

Dihydrodipicolinate reductase-like protein, CRR1, is essential for chloroplast NAD(P)H dehydrogenase in Arabidopsis

Chloroplast NAD(P)H dehydrogenase (NDH) is a homolog of the bacterial NADH dehydrogenase NDH-1 and is involved in cyclic electron transport around photosystem I. In higher plants, 14 subunits of the NDH complex have been identified. The subunit that contains the electron donor-binding site or an electron donor to NDH has not been determined. Arabidopsis crr1 (chlororespiratory reduction 1) mutants were isolated by chlorophyll fluorescence imaging on the basis of their lack of NDH activity. CRR1 is homologous to dihydrodipicolinate reductase (DHPR), which functions in a lysine biosynthesis pathway. However, the dihydrodipicolinate-binding motif was not conserved in CRR1, and the crr1 defect was specific to accumulation of the NDH complex, implying that CRR1 is not involved in lysine biosynthesis in Arabidopsis. Similarly to other nuclear-encoded genes for NDH subunits, CRR1 was expressed only in photosynthetic tissue. CRR1 contained a NAD(P)H-binding motif and was a candidate electron donor-binding subunit of the NDH complex. However, CRR1 was detected in the stroma but not in the thylakoid membranes, where the NDH complex is localized. Furthermore, CRR1 was stable in crr2-2 lacking the NDH complex. These results suggest that CRR1 is involved in biogenesis or stabilization of the NDH complex, possibly via the reduction of an unknown substrate.     

A eukaryotic factor required for accumulation of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

The NAD(P)H dehydrogenase (NDH) complex in chloroplasts mediates photosystem I cyclic and chlororespiratory electron transport. Eleven chloroplast genes and three nuclear genes have been identified as encoding Ndh subunits, but the entire subunit composition is still unknown. An Arabidopsis (Arabidopsis thaliana) chlororespiratory reduction (crr3) mutant was isolated based on its lack of transient increase in chlorophyll fluorescence after actinic light illumination; this was due to a specific defect in accumulation of the NDH complex. The CRR3 gene (At2g01590) encodes a novel protein containing a putative plastid-targeting signal and a transmembrane domain. Consistent with the gene structure, CRR3 localized to the membrane fraction of chloroplasts. In addition to the essential function of CRR3 in stabilizing the NDH complex, the NDH complex is also required for the accumulation of CRR3. These results suggest that CRR3 interacts with the NDH complex in the thylakoid membrane. In contrast to other subunits in the chloroplast NDH complex, CRR3 is not conserved in cyanobacteria from which the chloroplast NDH complex is believed to have originated. We propose that CRR3 is a subunit of the NDH complex, which is specific to the chloroplast.     

Multistep assembly of chloroplast NADH dehydrogenase-like subcomplex A requires several nucleus-encoded proteins, including CRR41 and CRR42, in Arabidopsis

Chloroplast NADH dehydrogenase-like complex (NDH) mediates photosystem I cyclic electron transport and chlororespiration in thylakoids. Recently, substantial progress has been made in understanding the structure of NDH, but our knowledge of its assembly has been limited. In this study, a series of interactive proteomic analyses identified several stroma-localized factors required for the assembly of a stroma-protruding arm of NDH (subcomplex A). In addition to further characterization of the previously identified CHLORORESPIRATORY REDUCTION1 (CRR1), CRR6, and CRR7, two novel stromal proteins, CRR41 and CRR42, were discovered. Arabidopsis thaliana mutants lacking these proteins are specifically defective in the accumulation of subcomplex A. A total of 10 mutants lacking subcomplex A, including crr27/cpn60β4, which is specifically defective in the folding of NdhH, and four mutants lacking NdhL-NdhO subunits, were extensively characterized. We propose a model for subcomplex A assembly: CRR41, NdhO, and native NdhH, as well as unknown factors, are first assembled to form an NDH subcomplex A assembly intermediate (NAI500). Subsequently, NdhJ, NdhM, NdhK, and NdhI are incorporated into NAI500 to form NAI400. CRR1, CRR6, and CRR42 are involved in this process. CRR7 is likely to be involved in the final step, in which the fully assembled NAI, including NdhN, is inserted into thylakoids.