The Outer Chloroplast Envelope Protein OEP16-1 for Plastid Import of NADPH:Protochlorophyllide Oxidoreductase A in Arabidopsis thaliana

The outer plastid envelope protein OEP16-1 was previously identified as an amino acid-selective channel protein and translocation pore for NADPH:protochlorophyllide oxidoreductase A (PORA). Reverse genetic approaches used to dissect these mutually not exclusive functions of OEP16-1 in planta have led to descriptions of different phenotypes resulting from the presence of several mutant lines in the SALK_024018 seed stock. In addition to the T-DNA insertion in the AtOEP16-1 gene, lines were purified that contain two additional T-DNA insertions and as yet unidentified point mutations. In a first attempt to resolve the genetic basis of four different lines in the SALK_024018 seed stock, we used genetic transformation with the OEP16-1 cDNA and segregation analyses after crossing out presumed point mutations. We show that AtOEP16-1 is involved in PORA precursor import and by virtue of this activity confers photoprotection onto etiolated seedlings during greenin

Untersuchungen zur Funktion der Peroxine Pex17p und Pex13p im peroxisomalen Matrixproteinimport der Hefe Saccharomyces cerevisiae\textit {Saccharomyces cerevisiae}

Peroxisomen sind in Eukaryonten ubiquitär vorhandene Organellen, deren Proteine posttranslational importiert werden. Die vorliegende Arbeit beschäftigt sich neben der Kartierung von Pex14p-Bindestellen in Pex17p mit der Isolierung und Charakterisierung von Pex13p- und Pex17p-haltigen Membranproteinkomplexen aus der Hefe $\textit {S. cerevisiae}$. Beide Peroxine sind Komponenten des Importomers, der peroxisomalen Importmaschinerie für Matrixproteine. Mit Pex17p- und Pex13p-TEV-ProteinA wurden jeweils 10 Peroxine als Interaktionspartner koisoliert. Mittels BN- und 2D-PAGE wurden zwei stabile $\it Core$-Komplexe (Pex14p/Pex17p und Pex2p/Pex10p/Pex12p) identifiziert. Mit diesen sind die Peroxine Pex5p und Pex13p schwach assoziiert. Weitere Analysen der Pex13p- und Pex17p-haltigen Membranproteinkomplexe erfolgten in verschiedenen $\it pex$-Deletionsmutanten. Desweiteren wurde die Bedeutung der C-terminalen Pex14p-Bindestelle in Pex17p und der SH3-Domäne von Pex13p für die Assemblierung der Komplexe untersucht

A substrate-independent, 14:3:3 protein-mediated plastid import pathway of NADPH : protochlorophyllide oxidoreductase (POR) A

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A substrate-independent, 14:3:3 protein-mediated plastid import pathway of NADPH:protochlorophyllide oxidoreductase A

Plastids are semiautonomous organelles that contain only limited coding information in their own DNA. Because most of their genome was transferred to the nucleus after their endosymbiotic origin, plastids must import the major part of their protein constituents from the cytosol. The exact role of cytosolic targeting factors in the regulation of plastid protein import has not been determined. Here, we report that the nucleus-encoded NADPH:protochlorophyllide (Pchlide) oxidoreductase A plastid precursor (pPORA) can use two different plastid import pathways that differ by the requirements for cytosolic 14:3:3 proteins and Hsp70. pPORA synthesized in a wheat germ lysate segregated into different precursor fractions. While import of free pPORA and only Hsp70-complexed pPORA was Pchlide-dependent and involved the previously identified Pchlide-dependent translocon, 14:3:3 protein- and Hsp70-complexed pPORA was transported into Pchlide-free chloroplasts through the Toc75-containing standard translocon at the outer chloroplast membrane/translocon at the inner chloroplast membrane machinery. A 14:3:3 protein binding site was identified in the mature region of the 35S-pPORA, which governed 14:3:3 protein- and Hsp70-mediated, Pchlide-independent plastid import. Collectively, our results reveal that the import of pPORA into the plastids is tightly regulated and involves different cytosolic targeting factors and plastid envelope translocon complexes

Targeting of nucleus-encoded proteins to chloroplasts in plants.

Most chloroplast proteins are encoded in the nucleus and synthesized on free, cytosolic ribosomes in precursor form. Each precursor has an amino-terminal extension called a transit peptide, which directs the protein through a post-translational targeting pathway and is removed upon arrival inside the organelle. This 'protein import' process is mediated by the coordinate action of two multiprotein complexes, one in each of the envelope membranes: the TOC and TIC (Translocon at the Outer/ Inner envelope membrane of Chloroplasts) machines. Many components of these complexes have been identified biochemically in pea; these include transit peptide receptors, channel proteins, and molecular chaperones. Intriguingly, the Arabidopsis genome encodes multiple, homologous genes for receptor components of the TOC complex. Careful analysis indicated that the different receptor isoforms operate in different import pathways with distinct precursor recognition specificities. These 'substrate-specific' import pathways might play a role in the differentiation of different plastid types, and/or act to prevent deleterious competition effects between abundant and nonabundant precursors. Until recently, all proteins destined for internal chloroplast compartments were thought to possess a cleavable transit peptide, and to engage the TOC/TIC machinery. New studies using proteomics and other approaches have revealed that this is far from true. Remarkably, a significant number of chloroplast proteins are transported via a pathway that involves the endoplasmic reticulum and Golgi apparatus. Other recent reports have elucidated an intriguing array of protein targeting routes leading to the envelope membranes themselves