The mitochondrial K+/H+ exchanger

Mitochondrien sind von einer Doppelmembran umschlossene Organellen, die verschiedene essentielle Zellprozesse kontrollieren, wie etwa ATP-Produktion, Apoptose, Ionenhomöostase, Volumenkontrolle und viele weitere Stoffwechselwege. Um diese Funktionen zu erfüllen, erzeugen Mitochondrien ein Membranpotential (Dy) an der inneren Membran. Aufgrund des Ausstoßes von H+ durch Redoxpumpen der Elektronentransportkette, wird dieses Membranpotential, welches innen negative ist, aufrecht erhalten. In Anwesenheit von hohen Mengen an K+ und anderen zellulären Kationen wie Na+ und Li+, bewirkt das innen negative Dy einen starken Ioneneinstrom. Um die mitochondriale Ionenhomöostase und Osmolarität aufrecht zu erhalten, muß diesem Ionenüberschuss mittels Kationen/Protonen Austauschern entgegengewirkt werden, wie es von Nobelpreisträger Peter Mitchell vor einem halben Jahrhundert vorgeschlagen wurde. Bisherige Daten konnten eindeutig zeigen, dass die Mdm38p/Letm1 Familie - Proteine der inneren Mitochondrienmembran mit einer Transmembranhelix - für den mitochondrialen K+/H+ Austausch essentiell ist. Die Deletion des MDM38 Gens in der Hefe führt zu schwerwiegendem atmungsdefekten Wachstum, mitochondrialer K+ Überladung, fast vollständig fehlender K+/H+ Austausch-Aktivität, Depolarisation der Mitochondrienmembran, sowie starker Schwellung und Fragmentierung des mitochondrialen Netzwerkes. Obwohl eine tragende Funktion von MDM38 in der Kontrolle des mitochondrialen K+/H+ Austausches nicht bezweifelt werden kann, ist die molekulare Identität des eigentlichen Austauschers weiterhin unbekannt. In diesem Forschungsprojekt konzentrierten wir uns auf die Charakterisierung von Proteinen, welche im mitochondrialen Kationen/Protonen Austausch involviert sind, mit dem letztendlichen Ziel der Identifizierung des tatsächlichen mitochondrialen K+/H+ Austauschers. In einer genomweiten Suche nach Überexpressions-Suppressoren der mdm38∆ Mutante identifizierten wir Mrs7p, ein Mdm38p homologes Protein, sowie das nicht verwandte Protein Ydl183cp, als starke Suppressoren des beeinträchtigten mitochondrialen K+/H+ Austausches. Dreifach deletierte Mutanten besaßen keinerlei K+/H+ Austausch-Aktivität und zeigten reduzierte zelluläre Lebensfähigkeit sowie autophagischen Abbau defekter Mitochondrien. Desweiteren sind Mdm38p und beide Suppressoren Komponenten von hochmolekularen Proteinkomplexen. Demzufolge entwickelten wir eine Arbeitshypothese, basierend auf der Idee, dass Mdm38p als ein Hilfsprotein direkt mit dem unbekannten K+/H+ Austauscher interagiert. Durch biochemische Analysen mithilfe von Affinitätschromatographie identifizierten wir mögliche Interaktionspartner von Mdm38p: das essentielle mitochondriale Chaperonprotein Ssc1p und das Ergosterol biosynthetische Enzym Erg5p. Hingegen gelang es uns nicht ein Protein mit mehreren Transmembranhelices zu identifizieren, was einem erwarteten Merkmal eines K+/H+ Austauschers entsprechen würde. Nichtsdestotrotz legen unsere Resultate einen engen Zusammenhang zwischen der Membransterol-Zusammensetzung, mitochondrialer Ionenhomöstase, Membranfusion und zellulärer Lebensfähigkeit nahe. Entgegen unserer ursprünglichen Annahme, sprechen die hier gezeigten Daten stark dafür, dass Mdm38p selbst den gesuchten Austauscher darstellt. Dies wäre der erste bekannte Austauscher mit nur einer Transmembranhelix. In einem zweiten Ansatz führten wir eine genomweite synthetisch-genetische Analyse der mdm38∆ Mutante durch. Dabei identifizierten wir sechs Suppressormutanten, die das atmungsabhängige Wachstum und die mitochondriale Morphologie in der Doppelmutante wiederherstellten. Momentan können wir nur darüber spekulieren wie diese Suppressoren, welche in verschiedenen zellulären Funktionen – von transkriptioneller Regulation bis zu zytoskelettaler Organisation – involviert sind, den Wachstums- und Morphologie-Phänotyp wiederherstellen. Basierend auf diesen Erkenntnissen liegen spannende Experimente vor uns.Mitochondria are double membrane-bound organelles controlling several essential cellular processes like ATP production, apoptosis, ion homeostasis, volume control, and many other metabolic pathways. To fulfill these functions, mitochondria establish an inside negative membrane potential (Dy) across the inner membrane, which is maintained by the ejection of H+ by redox pumps of the electron transport chain. In presence of high abundance of K+ and other cellular cations like Na+ and Li+, the inside negative Dy is a strong driving force for an inwardly directed ion flux. To maintain mitochondrial ion homeostasis and osmolarity, excess cation fluxes have to be counteracted by cation/proton antiporters, as proposed half a century ago by Nobel laureate Peter Mitchell. Solid data showed that the Mdm38p/Letm1 family of single membrane spanning, inner mitochondrial membrane proteins is essential for mitochondrial K+/H+ exchange. Deletion of yeast MDM38 results in a severe respiratory growth defect, mitochondrial K+ overload, almost abolished mitochondrial K+/H+ exchange activities, depolarization of the mitochondrial membrane, drastic mitochondrial swelling, and fragmentation of the mitochondrial reticulum. Though a role of MDM38 in controlling mitochondrial K+/H+ exchange is undoubted, the molecular identity of the exchanger per se still remains elusive. In this research project we focused on the characterization of proteins involved in mitochondrial cation/proton exchange, ultimately aiming at the identification of the actual mitochondrial K+/H+ exchanger. In a genome-wide screen for multicopy suppressors of the mdm38∆ mutant, we identified the Mdm38p homolog Mrs7p and the unrelated Ydl183cp as strong suppressors of impaired mitochondrial K+/H+ exchange. Triple deletion mutants exhibited completely abolished K+/H+ exchange, reduced cellular viability, and autophagic degradation of defective mitochondria. Moreover, both suppressors and Mdm38p were shown to be components of high molecular weight protein complexes. Accordingly, we established a working hypothesis based on the idea of Mdm38p functioning as an auxiliary protein directly interacting with the unknown K+/H+ exchanger. By biochemical analyses based on affinity chromatography, we identified the putative interaction partners of Mdm38p: the essential mitochondrial chaperon Ssc1p and the ergosterol biosynthetic enzyme Erg5p. However, we failed to identify a protein with several transmembrane helices as expected features for the K+/H+ exchanger. Nevertheless, we provide strong evidence for a direct link between membrane sterol composition, mitochondrial ion homeostasis, membrane fusion, and cellular viability. Contrary to our initial consideration, data presented here strongly suggest that Mdm38p itself may represent the exchanger. This would be the first known exchanger containing a single membrane spanning helix. In a second approach, we performed a genome-wide synthetic genetic array analysis of the mdm38∆ mutant, and identified six suppressor mutants restoring respiratory growth and mitochondrial morphology in the double mutant. Currently we can only speculate how these suppressors, with various cellular functions in transcriptional regulation or cytoskeletal organization, may restore the growth and morphology phenotype. Based on these findings there are exciting experiments ahead

Evaluation of the efficacy of mycotoxin modifiers and mycotoxin binders by using an in vitro rumen model as a first screening tool

Ruminal microbiota of cattle are not able to detoxify all mycotoxins. In addition, detoxification can be hampered by adverse ruminal conditions (e.g., low ruminal pH). Hence, in the cattle husbandry, mycotoxin binders and modifiers could be used to prevent animal exposure to mycotoxins. In this study, an in vitro rumen model, including feed matrix, was established as first screening tool to test the efficacy of five products claiming to detoxify mycotoxins. The detoxifiers had different modes of action: (a) binding (three products); (b) enzymatic detoxification of zearalenone (ZEN; one product, ZenA); and (c) bacterial transformation of trichothecenes (one product, BBSH 797). For the mycotoxin binders, the binding to the mycotoxins enniatin B (ENN B), roquefortine C (ROQ-C), mycophenolic acid (MPA), deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) were tested at a dose recommended by the manufacturers. The in vitro model demonstrated that all binders adsorbed ENN B to a certain extent, while only one of the binders also partially adsorbed ROQ-C. The binders did not change the concentrations of the other mycotoxins in the ruminal fluid. The enzyme ZenA detoxified ZEN very quickly and prevented the formation of the more toxic metabolite α-zearalenol (α-ZEL), both at normal (6.8) and low ruminal pH (5.8). The addition of BBSH 797 enhanced detoxification of DON and NIV, both at normal and low ruminal pH. The in vitro rumen model demonstrated that the addition of ZenA seems to be a very promising strategy to prevent estrogenic effects of ZEN contaminated feed, and BBSH 797 is efficient in the detoxification of trichothecenes

Enzymatic Degradation of Zearalenone in the Gastrointestinal Tract of Pigs, Chickens, and Rainbow Trout

The estrogenic mycotoxin zearalenone (ZEN) is a common contaminant of animal feed. Effective strategies for the inactivation of ZEN in feed are required. The ZEN-degrading enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) converts ZEN to hydrolyzed ZEN (HZEN), thereby enabling a strong reduction in estrogenicity. In this study, we investigated the efficacy of ZenA added to feed to degrade ZEN in the gastrointestinal tract of three monogastric animal species, i.e., pigs, chickens, and rainbow trout. For each species, groups of animals received (i) feed contaminated with ZEN (chickens: 400 µg/kg, pigs: 200 µg/kg, rainbow trout: 2000 µg/kg), (ii) feed contaminated with ZEN and supplemented with ZenA, or (iii) uncontaminated feed. To investigate the fate of dietary ZEN in the gastrointestinal tract in the presence and absence of ZenA, concentrations of ZEN and ZEN metabolites were analyzed in digesta of chickens and rainbow trout and in feces of pigs. Upon ZenA administration, concentrations of ZEN were significantly decreased and concentrations of the degradation product HZEN were significantly increased in digesta/feces of each investigated animal species, indicating degradation of ZEN by ZenA in the gastrointestinal tract. Moreover, upon addition of ZenA to the diet, the concentration of the highly estrogenic ZEN metabolite α-ZEL was significantly reduced in feces of pigs. In conclusion, ZenA was effective in degrading ZEN to HZEN in the gastrointestinal tract of chickens, pigs, and rainbow trout, and counteracted formation of α-ZEL in pigs. Therefore, ZenA could find application as a ZEN-degrading feed additive for these animal species

Pun1p is a metal ion-inducible, calcineurin/Crz1p-regulated plasma membrane protein required for cell wall integrity

Under conditions of environmental stress, the plasma membrane is involved in several regulatory processes to promote cell survival, like maintenance of signaling pathways, cell wall organization and intracellular ion homeostasis. PUN1 encodes a plasma membrane protein localizing to the ergosterol-rich membrane compartment occupied also by the arginine permease Can1. We found that the PUN1 (YLR414c) gene is transcriptionally induced upon metal ion stress. Northern blot analysis of the transcriptional regulation of PUN1 showed that the calcium dependent transcription factor Crz1p is required for PUN1 induction upon heavy metal stress. Here we report that mutants deleted for PUN1 exhibit increased metal ion sensitivity and morphological abnormalities. Microscopical and ultrastructural observations revealed a severe cell wall defect of pun1∆ mutants. By using chemical cross-linking, Blue native electrophoresis, and co-immunoprecipitation we found that Pun1p forms homo-oligomeric protein complexes. We propose that Pun1p is a stress-regulated factor required for cell wall integrity, thereby expanding the functional significance of lateral plasma membrane compartments