Dokument: Analyzing the function of a peroxisomal NAD carrier in Arabidopsis and yeast
| Titel: | Analyzing the function of a peroxisomal NAD carrier in Arabidopsis and yeast | |||||||
| Weiterer Titel: | Funktionsanalyse eines peroxisomalen NAD Transporters in Arabidopsis und Hefe | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=34427 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20150601-103426-0 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Schroers, Martin [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Weber, Andreas P. M. [Betreuer/Doktorvater] Prof. Dr. Jahns, Peter [Gutachter] Dr. Linka, Nicole [Betreuer/Doktorvater] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 580 Pflanzen (Botanik) | |||||||
| Beschreibung: | Eukaryotic cells contain different highly specialized cell organelles which are encased by at least one lipid bilayer membrane. These membranes are impermeable for most metabolites without assistance of specific solute transport proteins. These proteins
are also called carriers and enable to maintain a functional metabolic network extending over different organelles inside the cell (Linka and Weber, 2010). This thesis contributes to the knowledge of peroxisomal transport proteins in plants. The main focus of this thesis was to extend the knowledge of the peroxisomal NAD Carrier (PXN) of the model plant Arabidopsis thaliana. The peroxisomal metabolism depends on the import of substrates and cofactors, but our knowledge of the transporters involved is very limited. PXN mutants exhibit a slowed mobilization of storage oils, linking PXN function to the peroxisomal β-oxidation of fatty acids (Bernhardt et al., 2012). In vitro studies showed PXN to be able to transport e.g., NAD, NADH, AMP, and CoA (Agrimi et al., 2012; Bernhardt et al., 2012). In our experiments we could confirm that PXN imports AMP into liposomes preloaded with CoA at a concentration of 10 mM, but also showed that it does not mediate the transport of CoA on lower concentrations (Manuscript 1). Based on these findings CoA import is unlikely to be the in vivo role of PXN. Phenotype suppression studies of yeast mutants impaired in peroxisomal β-oxidation were used to get additional results. We could demonstrate the ability of PXN to suppress the phenotype of mdh3Δ mutants. Mdh3p is part of the malate/oxaloacetate redox shuttle in yeast, which regenerates NAD in the peroxisomal matrix (van Roermund et al., 1995). Yeast cells deficient in Mdh3p function showed a β-oxidation activity of about 30% compared to wild-type cells when grown on oleate as sole carbon source. Expression of PXN in the mdh3Δ background raised this activity significantly to 73% of wild-type levels (Manuscript 1). Thereby we showed PXN to be able to supply peroxisomal β-oxidation with NAD independently of the malate/oxaloacetate shuttle. To identify the counter exchange substrate which is exported from the peroxisome upon NAD import we analyzed an mdh3Δ/npy1Δ double mutant. Npy1p is a peroxisomal NADH pyrophosphatase catalyzing the hydrolysis of NADH to AMP and NMNH (AbdelRaheim et al., 2001). PXN accepts both NADH and AMP as substrates in vitro and loss of Npy1p function results in higher intraperoxisomal NADH levels and less AMP. Expression of PXN in the yeast double mutant did not restore β-oxidation activity, making AMP the most likely counter substrate for NAD uptake in vivo. To verify these findings we targeted PXN to the mitochondria of ndt1Δ/ndt2Δ yeast cells. This mutant lacks the activity of both mitochondrial NAD carriers which import NAD against AMP and is unable to grow on ethanol as sole carbon source (Todisco et al., 2006). Expression of mitochondrial-targeted PXN in the ndt1Δ/ndt2Δ mutant partly suppressed the growth phenotype on ethanol. Thus the import of NAD against AMP is likely to be the in vivo function of PXN. In Manuscript 2 we investigated how the PXN-mediated import of NAD into plant peroxisomes is regulated. Thus we studied if PXN activity is regulated by phosphorylation of the serine residue at position 155 (S155) of the polypeptide chain. This phosphorylation site is located in an elongated hydrophilic loop region which is unique among MCF transporters but conserved in PXN homologues in different higher plant and algae species. Phosphorylation of S155 enhanced the speed of PXN mediated NAD/AMP exchange without altering the proteins affinity to NAD. Manuscript 3 elucidated the involvement of peroxisomal metabolism during the breakdown of small-chain fatty acids which for example arise from the catabolism of branched-chain amino acids (BCAAs). BCAA breakdown is important during different stress conditions, where these amino acids serve as alternative energy source (Araujo et al., 2011). Based on our results peroxisomal metabolism is involved in BCAA degradation as well as in breakdown of exogenously supplied BCAA catabolites like propionate, isobutyrate, and acrylate. PXA1 activity is essential for the import of propionyl-CoA, isobutyryl-CoA, and possibly 2-methylbutyryl-CoA into peroxisomes during BCAA breakdown. Intraperoxisomal ATP provided by PNC1 and PNC2 is mandatory during these processes, while the role of PXN is less distinct. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Biochemie der Pflanzen | |||||||
| Dokument erstellt am: | 01.06.2015 | |||||||
| Dateien geändert am: | 01.06.2015 | |||||||
| Promotionsantrag am: | 09.04.2015 | |||||||
| Datum der Promotion: | 12.05.2015 |
