Struktur und Funktions-Analyse der ScPex13p SH3-Domäne und ihre Interaktionen mit ScPex5p und ScPex14p

Title and Contents 1\. Zusammenfassung 6 2\. Abstract 7 3\. Introduction 8 4\. Materials and Methods 22 5\. Results 43 6\. Discussion 87 7\. Reference 97The docking complex of the peroxisomal protein import machinery consists of at least Pex13p and Pex14p. The C-terminal SH3 domain of Pex13p interacts with the non-PXXP-ligand of Pex5p and the type II SH3-ligand of Pex14p. The ScPex13p (Saccharomyces cerevisiae) SH3 domain was heterologously overexpressed in E. coli and purified in large scale for NMR studies. The solution structure of the Pex13p SH3 domain comprising 62 amino acids from residues 309 to 370 was determined by NMR spectroscopy. This NMR structure was compared with the computer model illustrated in this work. The structure consists of five ?-strand regions and three loops. One of these loops, the n-Src loop, is longer than that in other SH3 domains but appears in all the known Pex13p SH3 domains of different species. The novel binding pocket for Pex5p which binds an ?-helical non-PXXP-peptide and the binding pocket for Pex14p harboring a type II of PXXP motif were identified by the NMR studies. Chemical shift assays revealed both binding pockets for the conventional and non-conventional ligands to be distinct. Pex5p- and Pex14p-peptides can bind simultaneously to the SH3 domain; an access of Pex5p dissociates the Pex14p bound on the SH3 domain. The binding sites of the Pex13p SH3 domain in Pex5p and Pex14p were also located by combination of two hybrid assays and peptide scanning. The binding site in Pex14p was mapped to a classical PXXP motif (type II), which locates between amino acids 86 and 92 in Pex14p; the binding site in Pex5p was mapped to a non classical peptide comprising amino acids 202-215. The interactions among three proteins, Pex5p, Pex13p (SH3 domain) and Pex14p were characterized by NMR studies and surface plasmone resonance studies. These studies indicate that the Pex13p is involved in the dynamics of the peroxisomal protein import process.Pex13p stellt zusammen mit Pex14p eine Komponente des peroxisomalen Docking- Komplexes dar. Die C-terminale SH3-Domäne von Pex13p interagiert sowohl mit Pex14p als auch mit Pex5p, dem Rezeptor für Proteine mit einem peroxisomalen Targetingsignal-Typ I (PTS1). Die SH3-Domäne von ScPex13p (Saccharomyces cerevisiae) wurde erfolgreich in E. coli überexprimiert und in großen Mengen für NMR-Studien aufgereinigt. Die 3D-Struktur der SH3-Domäne von Pex13p, die 62 Aminosäuren von 309 bis 370 des Proteins umfasst, wurde mittels NMR- Spektroskopie ermittelt. Diese NMR-Struktur und das Computer-Modell wurden miteinander verglichen. Die NMR-Strukur umfasst fünf ?-Faltblatt-Regionen und drei Schleifen. Eine der drei Schleifen, die n-Src-Schleife, ist länger als diejenige der anderen SH3-Domänen. Ein Sequenz-Vergleich ergab, dass die längere n-Src-Schleife in allen bekannten SH3-Domänen von Pex13p vorkommt. Die neuartigen Bindungstaschen der SH3-Domäne des Pex13p für Pex5p und Pex14p wurden charakterisiert. Die beiden Bindungstaschen sind räumlich voneinander getrennt und die Proteine Pex5p und Pex14p können gleichzeitig an die SH3-Domäne des Pex13p binden. Pex14p kann jedoch von der SH3-Domäne des Pex13p durch einen Überschuss an Pex5p abgelöst werden. Die Bindungsmotive in Pex5p und Pex14p für die SH3-Domäne des Pex13p wurden mittels NMR-Studien und Peptid-Bibliotheken identifiziert. In Pex14p stellt diese ein klassisches PXXP-Bindungsmotiv (AS 86-92) dar und entspricht somit einem Typ-II- Bindungsmotiv der SH3-Domäne. In ScPex5p wurde ein neuartiges Motiv, das sich von AS 202-215 erstreckt, identifiziert. Die wechselseitigen Interaktionen zwischen den drei Proteinen Pex5p, Pex13p (SH3- Domäne) und Pex14p wurden durch in vitro Studien charakterisiert. Diese Studien unterstützen die Bedeutung von Pex13p an der Dynamik des peroxisomalen Protein-Imports

Identification and Characterization of the Human Orthologue of Yeast Pex14p

Pex14p is a central component of the peroxisomal protein import machinery, which has been suggested to provide the point of convergence for PTS1- and PTS2-dependent protein import in yeast cells. Here we describe the identification of a human peroxisome-associated protein (HsPex14p) which shows significant similarity to the yeast Pex14p. HsPex14p is a carbonate-resistant peroxisomal membrane protein with its C terminus exposed to the cytosol. The N terminus of the protein is not accessible to exogenously added antibodies or protease and thus might protrude into the peroxisomal lumen. HsPex14p overexpression leads to the decoration of tubular structures and mislocalization of peroxisomal catalase to the cytosol. HsPex14p binds the cytosolic receptor for the peroxisomal targeting signal 1 (PTS1), a result consistent with a function as a membrane receptor in peroxisomal protein import. Homo-oligomerization of HsPex14p or interaction of the protein with the PTS2-receptor or HsPex13p was not observed. This distinguishes the human Pex14p from its counterpart in yeast cells and thus supports recent data suggesting that not all aspects of peroxisomal protein import are conserved between yeasts and humans. The role of HsPex14p in mammalian peroxisome biogenesis makes HsPEX14 a candidate PBD gene for being responsible for an unrecognized complementation group of human peroxisome biogenesis disorders

Atmospheric methane oxidation is affected by grassland type and grazing and negatively correlated to total soil respiration in arid and semiarid grasslands in Inner Mongolia

Methane (CH 4) is an important trace greenhouse gas and atmospheric CH 4 uptake by high-affinity methanotrophs in grassland soil accounts for an important proportion of the terrestrial CH 4 sink. However, our understanding of the comprehensive effects of grassland type and grazing treatment on active soil methanotrophs and atmospheric CH 4 uptake is still under debate. This study investigates the impact of grazing on CH 4 oxidation rate and active atmospheric CH 4 oxidizing methanotroph communities in two arid and semiarid grassland ecosystems (meadow and desert) by detecting transcripts of methane monooxygenase (pmoA) genes. Atmospheric CH 4 oxidation rates differed according to grassland type and grazing treatment. The highest activity was found in desert grasslands with moderate grazing and the lowest activity in meadow grasslands with exclosures. The differences in activities were linked with changes in abundance, composition and co-occurrence network patterns of active methanotrophs and CO 2 production rate. Redundancy, correlation and random forest analyses indicated that pmoA transcripts, available phosphorus (AP), NO 3 −-N, and CO 2 production rate were the most important factors predicting active methanotroph community composition and atmospheric CH 4 oxidation activity in these grassland ecosystems. A glucose amendment incubation experiment showed that addition of glucose increased heterotrophic microbial respiration and inhibited atmospheric CH 4 oxidation. This study provides evidence that CO 2 production rate is an important factor associated with atmospheric CH 4 oxidation activity in arid and semiarid grassland ecosystems and suggests that interactions between methanotrophs and other heterotrophs influence methanotroph activity in grassland ecosystems. </p