Open Access

Bo Zhang

• Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
• Die eukaryotische Zelle ist in Kompartimente unterteilt, diese nennt man Organellen. Zu den Organellen gehören das Cytoplasma, Bestandteile des Endomembransystems (ER, GolgiApparat, Plasmamembran), Mitochondrien, Chloroplasten und Peroxisomen. Im Gegensatz zu tierischen Zellen, enthalten Pflanzenzellen zwei Energie generierende Organelle: Chloroplasten und Mitochondrien. Beide dieser Organellen stammen von einem Gramnegativen prokaryotischen Vorläuferorganismus ab. Ein starkes Argument für die Gültigkeit dieser Endosymbiontentheorie ist das Vorhandensein von β-Fass Proteinen in der Membran von Mitochondrien und Chloroplasten. Diese Proteine bestehen aus parallelen, alternierenden β-Faltblättern, welche sich zu einer zylindrischen Struktur falten und Metaboliten oder ungefalteten Proteinen als Membrandurchgang dienen. Im Laufe der Evolution wurden viele Gene, die ursprünglich im Vorläuferorganismus lokalisiert waren, in den Nukleus überführt. Aktuelle Schätzungen gehen davon aus, dass ca. 4.500 protein-codierende Gene vom Cyoanobakterienvorläufer des Chloroplasten in das nukleäre Genom der Wirtszelle transferiert wurden. Während dieser Gentransfer eine zentralisierte Kontrolle der Genexpression ermöglichte, zog er auch die Entwicklung von Zielführungs- und Importmachanismen für nukleär codierte Gene nach sich...