Podcasts about toc75

In order to sustain their structure and metabolism, chloroplasts and other plastid types must import the majority of their proteins from the cytosol across the envelope membrane. Translocation of these precursor proteins across the double envelope membrane is achieved by two multimeric complexes - the so-called TOC and TIC complexes (Translocon at the Outer envelope of Chloroplast and Translocon at the Inner envelope of Chloroplast, respectively). N-terminal transit peptides essential for import of the precursor proteins are cleaved after their entry into the stroma. It was thus far believed that all of the different cytosolic precursor proteins would enter the chloroplast through the same, jointly acting TOC/TIC machineries. Recent evidence, however, suggests that multiple, regulated import pathways exist in plastids that involve different import machineries. Different combinations of TOC and TIC proteins were shown to establish different import sites in Arabidopsis thaliana with specificity for either photosynthetic proteins (the general import pathway) or non-photosynthetic „housekeeping“ proteins. Moreover, numerous non-canonical import pathways such as the import of Tic32 and AtQORH mediated by the yet unknown novel import pathway and the import via the secretory pathway were shown to exist. Proteomics studies have revealed the presence of a large number of plastid proteins lacking predictable N-terminal transit sequences for import. The import mechanism for the majority of these proteins has not been determined yet. Examples of the transit sequenceless precursor proteins are the chloroplast envelope quinone oxidoreductase homologue, AtQORH and the chloroplast inner envelope protein 32, Tic32. Both proteins are imported into the inner plastid envelope membrane by a non-canonical pathway (Toc159- and Toc75-independent) and without any proteolytic cleavage. In the present study not only the import characteristic of nine tentative ‘non-canonical’ chloroplast precursor proteins but also the new interactions between these precursor proteins and the proteins at the organellar surfaces were analyzed. Moreover, a non-canonical precursor protein without the classical transit peptide, the iron superoxide dismutase (FSD1) could be identified. Biochemical crosslinking experiments revealed that FSD1 interacts with new members of the Toc159 family in pea, namely PsToc132 and PsToc120. Using deletion mutants as well as a peptide scanning approach, regions of the precursor protein, which are involved in receptor binding could be defined. These are distributed across the entire sequence; surprisingly only the extreme N-terminus as well as a C-proximal domain turned out to be essential for targeting and import. En route into the plastid FSD1 engages components of the general import pathway, implying that in spite of the ‘non-canonical’ targeting information and recognition by a specific receptor, this precursor protein follows a similar way across the envelope as the majority of plastid precursor proteins.

The first step of preprotein translocation across the membranes of chloroplasts is facilitated by the Toc translocon. Aim of this work was to elucidate the dynamics and the mechanism of action of this molecular machine. The central, stably associated part of the Toc translocon, the Toc core complex, consists of the pore forming Toc75 and two receptors with GTPase activity, Toc34 and Toc159. The question of Toc159 localization was addressed since controversal results on this topic were reported. In this study, membrane localization of Toc159 was confirmed, which has further implications on the mode of its action. To understand the necessity of multiple isoforms of Toc components as found in Arabidopsis thaliana, expression analysis and tissue-specific localization were conducted. Gathered data suggested the existence of several types of the complex, assembled from different types of subunits. These complexes have different preprotein specificities. Expression analysis provided further arguments for dynamic association of the intermembrane space complex with the Toc core complex. Comparison of gene expression and protein presence of translocon subunits contradicts the function of Tic20 as a general pore for stromal targeted proteins, but not as a protein conducting channel per se. For further analysis of the Toc translocon structure and function, its purification and reconstitution into proteoliposomes was reinvestigated. To this end, a technique for liposome size determination in a single spectrophotometric measurement was developed.

Die Zusammensetzung und Arbeitsweise des Tic Komplexes ist noch ungeklärt. Tic110 ist die einzige von sieben Komponenten, die allgemein akzeptiert ist. Die Funktion und genaue Topologie des Proteins ist aber noch umstritten (Abb.3). Im Rahmen dieser Arbeit wurden verschiedene Experimente zur Klärung der Topologie und Funktion des Proteins durchgeführt. Zum Einen wurde über ein CD-Spektrum eine alpha-helikale Sekundärstruktur für Tic110 gezeigt. Proteasebehandlung sowohl von Vesikeln der inneren Hüllmembran als auch von intakten Chloroplasten lassen vermuten, dass Bereiche von Tic110 in den Intermembranraum zeigen. Auf der anderen Seite weisen Affinitätschromatographieversuche mit dem C-Terminus von Tic110 darauf hin, dass das Protein im Stroma mit HSP93 und HSP70 interagiert. Diese Ergebnisse lassen vermuten, dass ein Teil des C-Terminus in den Intermembranraum ragt und ein anderer Teil ins Stroma. Ob im C-Terminus amphiphile Helices ausgebildet werden können, muss geklärt werden. Mengenmäßig ist Tic110 prominenter in der inneren chloroplastidären Hüllmembran vorhanden als Tic20, der andere „Kandidat“ für die Pore des Tic Komplexes. Im Vergleich zur Menge von Toc75, der Pore der äusseren Hüllmembran, ist Tic110 in ähnlichen Mengen vorhanden. Tic110 ist also ein geeigneter Kandidat, an der Porenbildung beteiligt zu sein. Desweiteren wurden Interaktionspartner vom N-Terminus von Tic110 gesucht. Dabei wurde ein 32 kDa Protein gefunden, dass große Homologien zu sogenannten „short-chain“ Dehydrogenasen aufweist. In der vorliegenden Arbeit wurde über Importversuche und Immunpräzipitationsexperimente eine Zugehörigkeit des Proteins zum Tic Komplex gezeigt. Die Komponente wurde Tic32 genannt. Tic32 ist eine funktionelle Dehydrogenase, deren Beteiligung während des Importprozesses noch zu klären bleibt. T-DNA Insertionslinien von Tic32 ergaben, dass das Protein für die für die Plastidenentwicklung essentiell ist. Da mit Tic32 neben Tic55 und Tic62 nun schon die dritte Tic Komponente gefunden wurde, die Redox Charakteristika aufweist, wurden verschiedene Importexperimente durchgeführt. Dafür wurden zwei chloroplastidäre FNR-Isologe und zwei chloroplastidäre Fd-Isologe in Chloroplasten importiert, deren Redoxzustand vor der Importreaktion mit verschiedenen Metaboliten oder Redoxkomponenten beeinflusst wurde. Sowohl nach Behandlung der Chloroplasten mit HAR, deamino-NAD, Oxalacetat und Kaliumhexacyanoferrat nimmt die Importeffizienz der FNR L2 Form stark ab. Auch für die Ferredoxin-Isologe ließ sich ein unterschiedliches Importverhalten feststellen, wenn auch nicht so eindeutig wie für die FNR-Isologe. Dieser Regulationsmechanismus muß nun in weiteren Experimenten genauer untersucht werden.

Preproptein recognition and translocation across the outer envelope membrane of plastids is catalysed by a proteinaceous machinery, called Toc translocon. The Toc core complex is composed of the pore forming Toc75 and the two GTP-regulated receptors Toc159 and Toc34. A main issue of this work is the nature of preprotein recognition and transfer by this Toc apparatus. It is still under debate, whether Toc34 or Toc159 is the initial receptor. Here, using proteoliposomes with reconstituted either Toc core complex or Toc159 and Toc75 and several in vitro binding analysis Toc34 was shown to act upstream of Toc159. Moreover, a certain set of preproteins engages Toc64 before passing the Toc core complex. The receptor function and the dynamic association with the Toc core complex of this protein are established. Toc64 is the central component of the intermembrane space translocon. This complex migrates at approximately 700 kDa in a BN-PAGE and contains Toc64, Tic22, Toc12 and isHsp70. Toc12 is a novel identified Toc component, which exposes a J-domain toward the intermembrane space. This domain recruits isHsp70 to the intermembrane space translocon in an ATP and preprotein dependent manner. Finally, the work addresses the molecular identity of this isHsp70. Therefore, isHsp70 was purified by chromatographic approaches and analysed by mass spectroscopy. Several peptide masses were obtained, which reveal high similarity of P. sativum isHsp70 to S. oleracea Com70.