The distribution of melanopsin (OPN4) protein in the human brain.

Until now, melanopsin (OPN4) - a specialized photopigment being responsive especially to blue light wavelengths - has not been found in the human brain at protein level outside the retina. More specifically, OPN4 has only been found in about 2% of retinal ganglion cells (i.e. in intrinsically photosensitive retinal ganglion cells), and in a subtype of retinal cone-cells. Given that Allen Institute for Brain Science has described a wide distribution of OPN4 mRNA in two human brains, we aimed to investigate whether OPN4 is present in the human brain also at protein level. Western blotting and immunohistochemistry, as well as immunoelectron microscopy, were used to analyse the existence and distribution of OPN4 protein in 18 investigated areas of the human brain in samples obtained in forensic autopsies from 10 male subjects (54 ± 3.5 years). OPN4 protein expression was found in all subjects, and, furthermore, in 5 out of 10 subjects in all investigated brain areas localized in membranous compartments and cytoplasmic vesicles of neurons. To our opinion, the wide distribution of OPN4 in central areas of the human brain evokes a question whether ambient light has important straight targets in the human brain outside the retinohypothalamic tract (RHT). Further studies are, however, needed to investigate the putative physiological phototransductive actions of inborn OPN4 protein outside the RHT in the human brain.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

Cholesterol is catabolized to bile acids by peroxisomal ß-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this ß-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice.

To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2(-/-) female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2(-/-) mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2(-/-)mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal ß-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2(-/-) mice. The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.

Lung cancer-associated myofibroblasts reveal distinctive ultrastructure and function.

BACKGROUND: Cancer-associated stromal cells interact with carcinoma cells and thus participate in tumor growth. Our aim was to characterize the ultrastructure and contractile properties of stromal cells in collagen gel cultured from lung cancer of various histological types and from tumor-free lung. METHODS: Cells cultured from lung cancer (13 adenocarcinomas, six squamous cell carcinomas, one adenosquamous carcinoma, and one pleomorphic carcinoma) and tumor-free lung were analyzed by transmission electron microscopy and three-dimensional collagen gel contraction assays. The expression of α-smooth muscle actin (α-SMA), a recognized myofibroblast marker, was examined by immunoelectron microscopy from individual cells and by Western blotting from the whole cultured cell population. RESULTS: According to their ultrastructure, the cell lines were composed of fibroblastic and myofibroblastic cells. In electron microscopy, cells of lung cancer exhibited more myofibroblastic features displaying higher amounts of actin belts (p = 0.057) and α-SMA labeling (p = 0.010) than cells from tumor-free lung. Myofibroblasts cultured from lung cancer of smokers expressed less α-SMA labeling (p = 0.013) than counterparts from nonsmokers. Western blotting revealed that cancer-associated fibroblasts expressed more α-SMA (p = 0.006) than cells from tumor-free lung, whereas cells from tumor-free central lung of smokers showed less α-SMA (p = 0.039) than counterparts from nonsmokers. Cells cultured from cancer contracted more in collagen gel than those from tumor-free lung. The contractile capacity in collagen gel correlated with the frequency of extracellular component of fibronexus by transmission electron microscopy. CONCLUSIONS: Lung cancer-associated myofibroblasts are different both ultrastructurally and functionally when compared with cells cultured from tumor-free lung. Smoking altered myofibroblastic phenotype, regardless of their origin.

Transmembrane prostatic acid phosphatase (TMPAP) interacts with snapin and deficient mice develop prostate adenocarcinoma.

The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/-)) with C57BL/6J background. The PAP(-/-) mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

Myofibroblast expression in airways and alveoli is affected by smoking and COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by structural changes in alveoli and airways. Our aim was to analyse the numbers of alpha-smooth muscle actin (α-SMA) positive cells, as a marker of myofibroblasts, in different lung compartments in non-smokers and smokers with normal lung function or COPD. METHODS: α-SMA, tenascin-C (Tn-C) and EDA-fibronectin in alveolar level and airways were assayed by immunohistochemistry and quantified by image analysis. Immunohistochemical findings were correlated with clinical data. α-SMA protein was also analysed by Western blotting from fibroblastic cells cultured from peripheral lung of non-smokers, smokers without COPD and smokers with COPD. RESULTS: In many cases, the endings of the detached alveolar walls were widened, the structures of which were named as widened alveolar tips. Widened alveolar tips contained α-SMA positive cells, which were obviously myofibroblasts. There were less alveolar tips containing positive cells for α-SMA in alveoli and α-SMA positive cells in bronchioles in smokers and in COPD compared to non-smokers. The quantity of α-SMA positive cells was increased in bronchi in COPD. Tn-C was elevated in bronchi in COPD and smokers' lung. The α-SMA protein level was 1.43-fold higher in stromal cells cultured from non-smokers than in those of smokers. CONCLUSIONS: Myofibroblasts are localized variably in normal and diseased lung. This indicates that they have roles in both regeneration of lung and pathogenesis of COPD. The widened alveolar tips, these newly characterized histological structures, seemed to be the source of myofibroblasts at the alveolar level.

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.

Myocardial overexpression of Mecr, a gene of mitochondrial FAS II leads to cardiac dysfunction in mouse.

It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy.

Pxmp2 is a channel-forming protein in Mammalian peroxisomal membrane.

BACKGROUND: Peroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma. METHODS/PRINCIPAL FINDINGS: In this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2(-/-) mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. CONCLUSIONS/SIGNIFICANCE: Pxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of "bulky" metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters.

Channel-forming activities of peroxisomal membrane proteins from the yeast Saccharomyces cerevisiae.

Highly-purified peroxisomes from the yeast Saccharomyces cerevisiae grown on oleic acid were investigated for the presence of channel (pore)-forming proteins in the membrane of these organelles. Solubilized membrane proteins were reconstituted in planar lipid bilayers and their pore-forming activity was studied by means of multiple-channel monitoring or single-channel analysis. Two abundant pore-forming activities were detected with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl, respectively. The high-conductance pore (0.6 nS in 1.0 m KCl) is slightly selective to cations (P(K+)/P(Cl-) approximately 1.3) and showed an unusual flickering at elevated (> +/-40 mV) holding potentials directed upward relative to the open state of the channel. The data obtained for the properties of the low-conductance pore (0.2 nS in 1.0 m KCl) support the notion that the high-conductance channel represents a cluster of two low-conductance pores. The results lead to conclusion that the yeast peroxisomes contain membrane pore-forming proteins that may aid the transfer of small solutes between the peroxisomal lumen and cytoplasm.

UK114, a YjgF/Yer057p/UK114 family protein highly conserved from bacteria to mammals, is localized in rat liver peroxisomes.

Mammalian UK114 belongs to a highly conserved family of proteins with unknown functions. Although it is believed that UK114 is a cytosolic or mitochondrial protein there is no detailed study of its intracellular localization. Using analytical subcellular fractionation, electron microscopic colloidal gold technique, and two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with mass spectrometric analysis we show here that a large portion of UK114 is present in rat liver peroxisomes. The peroxisomal UK114 is a soluble matrix protein and it is not inducible by the peroxisomal proliferator clofibrate. The data predict involvement of UK114 in peroxisomal metabolism.

Localization of a portion of the liver isoform of fatty-acid-binding protein (L-FABP) to peroxisomes.

The liver isoform of fatty-acid-binding protein (L-FABP) facilitates the cellular uptake, transport and metabolism of fatty acids and is also involved in the regulation of gene expressions and cell differentiation. Consistent with these functions, L-FABP is predominantly present in the cytoplasm and to a lesser extent in the nucleus; however, a significant portion of this protein has also been detected in fractions containing different organelles. More recent observations, notably on L-FABP-deficient mice, indicated a possible direct involvement of L-FABP in the peroxisomal oxidation of long-chain fatty acids. In order to clarify the links between L-FABP and peroxisomal lipid metabolism, we reinvestigated the subcellular distribution of the protein. Analytical subcellular fractionation by a method preserving the intactness of isolated peroxisomes, two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with MS analysis, and immunoelectron microscopy of liver sections demonstrate the presence of L-FABP in the matrix of peroxisomes as a soluble protein. Peroxisomal L-FABP was highly inducible by clofibrate. The induction of L-FABP was accompanied by a marked increase in the binding capacity of peroxisomal matrix proteins for oleic acid and cis-parinaric acid. The peroxisomal beta-oxidation of palmitoyl-CoA and acyl-CoA thioesterase activity were stimulated by L-FABP, indicating that the protein modulates the function of peroxisomal lipid-metabolizing enzymes. The possible role of intraperoxisomal L-FABP in lipid metabolism is discussed.

The rat liver peroxisomal membrane forms a permeability barrier for cofactors but not for small metabolites in vitro.

The functional role of the peroxisomal membrane as a permeability barrier to metabolites has been a matter of controversy for more than four decades. The initial conception, claiming free permeability of the membrane to small solute molecules, has recently been challenged by several observations suggesting that the peroxisomal membrane forms a closed compartment. We have characterized in vitro the permeability of rat liver peroxisomal membrane. Our results indicate that the membrane allows free access into peroxisomes for small hydrophilic molecules, such as substrates for peroxisomal enzymes (glycolate, urate), but not to more bulky cofactors (NAD/H, NADP/H, CoA). Although access for cofactors is not prevented completely by the membrane, the membrane barrier severely restricts their rate of entry into peroxisomes. The data lead to conclusion that, in vivo, peroxisomes may possess their own pool of cofactors, while they share a common pool of small metabolites with the cytoplasm. The results also indicate that molecular size plays an important role in in vivo distinction between cofactors and metabolic intermediates.

Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology.

Among the recently recognized aspects of mitochondrial functions, in yeast as well as humans, is their ability to synthesize fatty acids in a malonyl-CoA dependent manner. We describe here the identification of the 3-hydroxyacyl-ACP dehydratase involved in mitochondrial fatty acid synthesis. A colony-colour-sectoring screen was applied in Saccharomyces cerevisiae in a search for mutants that, when grown on a non-fermentable carbon source, were unable to lose a plasmid that carried a chimeric construct coding for mitochondrially localized bacterial analogue. Our mutants, which are respiratory deficient, lack cytochromes and display abnormal mitochondrial morphology, were found to have a lesion in the yeast YHR067w/RMD12 gene. The Yhr067p is predicted to be a member of the thioesterase/thioester dehydratase-isomerase superfamily enzymes. Hydratase 2 activity in mitochondrial extracts from cells overexpressing YHR067w was increased. These overexpressing cells also display a striking mitochondrial enlargement phenotype. We conclude that YHR067w encodes a novel mitochondrial 3-hydroxyacyl-thioester dehydratase 2 and suggest renaming it HTD2. The mitochondrial phenotypes of the null and overexpression mutants suggest a crucial role of YHR067w in maintenance of mitochondrial respiratory competence and morphology in yeast.

The behavior of peroxisomes in vitro: mammalian peroxisomes are osmotically sensitive particles.

It has been known for a long time that mammalian peroxisomes are extremely fragile in vitro. Changes in the morphological appearance and leakage of proteins from purified particles demonstrate that peroxisomes are damaged during isolation. However, some properties of purified peroxisomes, e.g., the latency of catalase, imply that their membranes are not disrupted. In the current study, we tried to ascertain the mechanism of this unusual behavior of peroxisomes in vitro. Biochemical and morphological examination of isolated peroxisomes subjected to sonication or to freezing and thawing showed that the membrane of the particles seals after disruption, restoring permeability properties. Transient damage of the membrane leads to the formation of peroxisomal "ghosts" containing nucleoid but nearly devoid of matrix proteins. The rate of leakage of matrix proteins from broken particles depended inversely on their molecular size. The effect of polyethylene glycols on peroxisomal integrity indicated that these particles are osmotically sensitive. Peroxisomes suffered an osmotic lysis during isolation that was resistant to commonly used low-molecular-mass osmoprotectors, e.g., sucrose. Damage to peroxisomes was partially prevented by applying more "bulky" osmoprotectors, e.g., polyethylene glycol 1500. A method was developed for the isolation of highly purified and nearly intact peroxisomes from rat liver by using polyethylene glycol 1500 as an osmoprotector.

Cell-specific regulation of gamma-glutamylcysteine synthetase in human interstitial lung diseases.

The pathogenesis of interstitial lung diseases (ILDs) is known to be associated with reactive oxygen and nitrogen metabolites and increased oxidant stress. One of the major antioxidants in human lung is glutathione (GSH) and enzymes linked to its synthesis. The rate-limiting enzyme of GSH synthesis is gamma-glutamylcysteine synthetase (gamma-GCS) containing catalytically active heavy (gamma-GCSh) and regulatory light (gamma-GCSl) subunits. It can be hypothesized that gamma-GCS is the major determinant in explaining reduced GSH levels in fibrotic lung disorders. We investigated the regulation of gamma-GCS by transforming growth factor beta(1) (TGF-beta(1)) and tumor necrosis factor alpha (TNF-alpha) in human lung cells and its expression and distribution in fibrotic (biopsy-proven idiopathic pulmonary fibrosis, for instance, usual interstitial pneumonia, UIP, n = 15), inflammatory, and granulomatous diseases of human lung parenchyma (desquamative interstitial pneumonia, n = 10; ILD associated with collagen diseases, n = 10; sarcoidosis, n = 19 and allergic alveolitis, n = 8). In human lung alveolar epithelial cells, gamma-GCSh was decreased by TGF-beta(1), whereas TNF-alpha caused a transient enzyme induction. In normal lung, gamma-GCS was mainly localized to the bronchiolar epithelium. In UIP, the highest immunoreactivities were observed in the airway epithelium and metaplastic alveolar epithelium, but fibroblastic foci were negative. In sarcoidosis, the highest reactivities were detected in the epithelium, alveolar macrophages and pulmonary granulomas. gamma-GCS was ultrastructurally localized to the cytoplasm of regenerating type II pneumocytes and macrophages. In conclusion, gamma-GCS is widely expressed in sarcoidosis and regenerating epithelium but is low in the fibrotic areas of usual interstitial pneumonia, probably because of enzyme down-regulation.

A mouse model for alpha-methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis and intolerance to dietary methyl-branched lipids.

alpha-Methylacyl-CoA racemase (Amacr) deficiency in humans leads to sensory motor neuronal and liver abnormalities. The disorder is recessively inherited and caused by mutations in the AMACR gene, which encodes Amacr, an enzyme presumed to be essential for bile acid synthesis and to participate in the degradation of methyl-branched fatty acids. To generate a model to study the pathophysiology in Amacr deficiency we inactivated the mouse Amacr gene. As per human Amacr deficiency, the Amacr(-/-) mice showed accumulation (44-fold) of C27 bile acid precursors and decreased (over 50%) primary (C24) bile acids in bile, serum and liver, however the Amacr(-/-) mice were clinically symptomless. Real-time quantitative PCR analysis showed that, among other responses, the level of mRNA for peroxisomal multifunctional enzyme type 1 (pMFE-1) was increased 3-fold in Amacr(-/-) mice. This enzyme can be placed, together with CYP3A11 and CYP46A1, to make an Amacr-independent pathway for the generation of C24 bile acids. Exposure of Amacr(-/-) mice to a diet supplemented with phytol, a source for branched-chain fatty acids, triggered the development of a disease state with liver manifestations, redefining the physiological significance of Amacr. Amacr is indispensable for the detoxification of dietary methyl-branched lipids and, although it contributes normally to bile acid synthesis from cholesterol, the putative pMFE-1-mediated cholesterol degradation can provide for generation of bile acids, allowing survival without Amacr. Based upon our mouse model, we propose elimination of phytol from the diet of patients suffering from Amacr deficiency.

Characterization of 2-enoyl thioester reductase from mammals. An ortholog of YBR026p/MRF1'p of the yeast mitochondrial fatty acid synthesis type II.

A data base search with YBR026c/MRF1', which encodes trans-2-enoyl thioester reductase of the intramitochondrial fatty acid synthesis (FAS) type II in yeast (Torkko, J. M., Koivuranta, K. T., Miinalainen, I. J., Yagi, A. I., Schmitz, W., Kastaniotis, A. J., Airenne, T. T., Gurvitz, A., and Hiltunen, K. J. (2001) Mol. Cell. Biol. 21, 6243-6253), revealed the clone AA393871 (HsNrbf-1, nuclear receptor binding factor 1) in human EST data bank. Expression of HsNrbf-1, tagged C-terminally with green fluorescent protein, in HeLa cells, resulted in a punctated fluorescence signal, superimposable with the MitoTracker Red dye. Wild-type polypeptide was immunoisolated from the extract of bovine heart mitochondria. Recombinant HsNrbf-1p reduces trans-2-enoyl-CoA to acyl-CoA with chain length from C6 to C16 in an NADPH-dependent manner with preference to medium chain length substrate. Furthermore, expression of HsNRBF-1 in the ybr026cDelta yeast strain restored mitochondrial respiratory function allowing growth on glycerol. These findings provide evidence that Nrbf-1ps act as a mitochondrial 2-enoyl thioester reductase, and mammalian cells may possess bacterial type fatty acid synthetase (FAS type II) in mitochondria, in addition to FAS type I in the cytoplasm.

Structure-function analysis of enoyl thioester reductase involved in mitochondrial maintenance.

Candida tropicalis enoyl thioester reductase Etr1p and the Saccharomyces cerevisiae homologue Mrf1p catalyse the NADPH-dependent reduction of trans-2-enoyl thioesters in mitochondrial fatty acid synthesis (FAS). Unlike prokaryotic enoyl thioester reductases (ETRs), which belong to the short-chain dehydrogenases/reductases (SDR), Etr1p and Mrf1p represent structurally distinguishable ETRs that belong to the medium-chain dehydrogenases/reductases (MDR) superfamily, indicating independent origin of two separate classes of ETRs. The crystal structures of Etr1p, the Etr1p-NADPH complex and the Etr1Y79Np mutant were refined to 1.70A, 2.25A and 2.60A resolution, respectively. The native fold of Etr1p was maintained in Etr1Y79Np, but the mutant had only 0.1% of Etr1p catalytic activity remaining and failed to rescue the respiratory deficient phenotype of the mrf1Delta strain. Mutagenesis of Tyr73 in Mrf1p, corresponding to Tyr79 in Etr1p, produced similar results. Our data indicate that the mitochondrial reductase activity is indispensable for respiratory function in yeast, emphasizing the significance of Mrf1p (Etr1p) and mitochondrial FAS for the integrity of the respiratory competent organelle.