Year of publication
- 2011 (3) (remove)
- English (3) (remove)
- First insights into the phosphorylation of Toc34 proteins (2011)
- The translocation of nuclear-encoded precursor proteins into chloroplasts is a highly ordered process involving the action of several components to regulate this molecular ensemble. Not only GTP hydrolysis and GDP release but also the phosphorylation of TOC GTPases is a widely discussed mechanism to regulate protein import. The receptor component (Toc34) and its isoform of A. thaliana (atToc33) were found to be regulated by phosphorylation. Although the phosphorylation of Toc33 is already known for several years, several questions regarding the molecular components involved in the regulation of the phosphorylation process, precisely what is the protein kinase and where this kinase is initially localized, so far remained unclear. This thesis aimed at the defining of the phosphorylation status of TOC GTPases in monomeric and/or dimeric states, the identification of the nature of Toc33-PK (protein kinase), and in the same context it aimed at gaining first insights into the physiological significance of Toc33 phosphorylation. To this end, (I) An in vitro and in vivo system for investigating of TOC GTPases Phosphorylation (in monomeric or dimeric state) was developed. Since no information is available about the phosphorylation status of the Toc159 isoforms, the second receptor of the TOC complex, it was interesting to investigate whether these isoforms undergo phosphorylation or not. The results indicated that atToc159 isoforms are able to be phosphorylated by the kinase activity in purified outer envelope membranes (OEMs) of pea, but not atToc132. Moreover, an artificial dimer of psToc34 based on the interaction of a C-terminally fused leucine zipper was not phosphorylated. This result reflected the inability of the OEM kinase to phosphorylate the dimers of TOC GTPases. Also, In vivo labeling of atToc33 was developed and occurred in a dose-dependent manner. Therefore, this results evidenced that in vitro phosphorylation of atToc33 (both endogenous wild type and recombinant expressed proteins) is not artificial labeling but represents a physiological relevance. CD (circular dichroism) measurements revealed that recombinant GTPase domain of atToc33 is preferentially phosphorylated in its folded state. Therefore, it could be suggested that folding of atToc33rec is a prerequisite for its phosphorylation and the phosphorylation event occurs as a posttranslational modification most likely after insertion of Toc33 (Toc34) into the OE of chloroplasts. Secondly, (II) Isolation and identification of Toc33-PK from OEMs of chloroplasts was performed. Four independent strategies were developed to identify the Toc33-protein kinase: UV-induced and chemically-based crosslinking, different applied chromatographic techniques, identification of PK-Toc33 interaction by means of HDN-PAGE (histidine- and deoxycholate-based native PAGE), and finally mass spectrometric approaches were performed on fractions including the potential kinase activity. UV-induced crosslinking procedure was developed and resulted in covalent bonding of nine proteins to [a-32P] ATP, while chemically-based one was not significant. The applied chromatographic and HDN-PAGE approaches, including mass spectrometry, have revealed the identification of 13 protein kinases. Of these identified kinases, phototropin2 (Phot2, AT5G58140), leucine-rich repeat PK (LRR-PK, AT4G28650.1), and receptor-like transmembrane PK (RLK, AT5G56040.2) were selected as the most promising candidates (ca. kinase type and one transmembrane helix for membrane localization). (III) The physiological significance of Toc33 phosphoryation was shown to link this process with the environmental changes (especially, the light conditions). Identification of chloroplast OE-located PKs performed by nLC-MALDI-MS/MS resulted in the detection of Phot2. Furthermore, the subcellular localization of Phot2 in OEM of chloroplasts was confirmed by immunoblotting experiments using a-Phot2 antibody. The kinase activity of Phot2 towards TOC GTPases was characterized and revealed that fused GST-KD (kinase domain) protein able to specifically phosphorylate atToc33rec, but not atToc159rec. Also, endogenous atPhot2 was upregulated and heavily detected in the ppi1-S181A plant line (where serine to alanine exchange was performed to abolish the phosphorylation of atToc33). Hence, we suggested that certain signal cascades may directly or indirectly link Toc33 receptor phosphorylation, protein levels of Phot2 (as promising PK candidate), and irradiation conditions (as an inducing signal of the subsequent phosphorylation events). Light-dependent phosphorylation of Toc33 was shown either after de-etiolation conditions or after high light intensities of blue light was performed. Therefore, phosphorylation of Toc33 might be identified as an external regulatory signal to regulate preproteins import into chloroplasts in response to environmental conditions (e.g. light changes) or as a signal of chloroplast biogenesis.
- Functional analysis of human transporter associated with antigen processing (TAP) and its modulation by lipids (2011)
- The adaptive immune system of jawed vertebrates is based on recognition and elimination of cells that are either invaded by intracellular pathogens or malignantly transformed. One essential component of these processes is the cell surface presentation of antigenic peptides via major histocompatibility complex (MHC) class I molecules to cytotoxic T-cells (CTLs). Cells degrade defective ribosomal products and misfolded or unwanted proteins by the ubiquitin-proteasome pathway. The resulting degradation products are recognized and translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER) lumen, where they are loaded onto MHC I molecules. Assembled peptide-MHC complexes are then shuttled by the secretory pathway to the cell surface for antigen presentation to CTLs, leading in the case of viral infection or malignant transformation to lysis and apoptosis of the target cell. Due to the fact that the TAP complex represents a key control point within the antigen presentation pathway, several viruses have evolved sophisticated strategies to evade immune surveillance by interfering with TAP function. Detailed studies of the TAP mechanism or its viral inhibition have been severely impeded by difficulties in expressing sufficient amounts of functional heterodimeric TAP complex. Thus, the overexpression of TAP in the methylotrophic yeast Pichia pastoris was established for functional analysis of this important ABC complex. Biomass production was scaled up by fermentation using classical batch and feed methods. Extensive screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex. Notably, only the very mild detergent digitonin preserved TAP function. Hereby, the optimal solubilization and purification strategy yielded in 30 mg TAP transporter per liter culture. Remarkably, the protein amount was 50-fold increased compared to previously described expression/purification in cultured insect cells. The high yield and quality of TAP produced in P. pastoris allowed an extensive analysis of substrate binding and transport kinetics of the transport complex in the membrane, its solubilized and purified state, as well as the reconstituted state. Thereby, a strong and direct effect of the lipid bilayer on ATP hydrolysis and peptide transport was discovered. These important results were extended further by successful functional reconstitution of the antigen translocation machinery in different lipid environments. For the first time, a stimulation of the transport activity by phosphatidylinositol (PI) and phosphatidylethanolamine (PE) was observed, whereas cholesterol was identified as an inhibitor of TAP activity. Purification of TAP and subsequent thin-layer chromatography (TLC)/liquid chromatography Fourier transform-mass spectrometry (LC FT-MS) fingerprinting of residual lipids exhibited specifically associated glycerophospholipids; mainly PC, PE, and PI species. Strikingly, these lipids not only represent the primary class of phospholipids of the ER but were also shown to be essential for functional reactivation of delipidated, and thus inactive, TAP. The results demonstrate that transport of antigenic peptides by the ABC transporter TAP strictly requires specific glycerophospholipids. In addition to the biochemical characterization of heterologous produced TAP, the soluble domain of the viral inhibitor US6 from human cytomegalovirus was expressed in E. coli. Optimization of the purification and refolding strategy yielded in functional protein, with a 35-fold increased protein amount compared to previous purification procedures. Protein activity was analyzed by specific inhibition of ATP binding to TAP. Furthermore, high protein yields allowed detailed investigation of TAP-dependent spatial and mechanistic separation of MHC I restricted cross-presentation in professional antigen presenting cells (pAPC).
- Substrate binding does not only mean catalysis: internal regulation in the cytochrome bc1 complex from Paracoccus denitrificans (2011)
- The ubiquinol:cytochrome c oxidoreductase is a key component of several aerobic respiratory chains in different organisms. It is an integral membrane protein complex, made up of three catalytic subunits (cytochrome b, cytochrome c1 and Rieske iron sulphur protein) and up to eight additional subunits in mitochondria. The complex oxidizes one quinol molecules and reduces two cytochrome c during the Q cycle, originally described by Peter Mitchell. Electrons are split between the low and the high potential chain and protons are released on the positive side of the membrane, increasing the protonmotive force needed by the ATP-synthase for energy transduction. The cytochrome bc1 complex from P. denitrificans is a perfect model for structural and functional studies. Bacteria are easy to grow and the genetic material is readily accessible for genetic manipulation. Moreover, the P. denitrificans aerobic respiratory chain is very close to the mitochondrial one: the complexes involved in electron transfer resemble the ones found in mitochondria, but lack most of the additional subunits. As a unique feature, P. denitrificans has a strongly acidic domain at the N-terminal region of the cytochrome c1, a sequence of 150 aminoacids which does not correlate with any known protein. An analogous composition can be found in the eukaryotic cytochrome bc1 complex as a part of an accessory subunit, proposed to be involved in facilitating electron transfer between the complex and the electron acceptor cytochrome c. In order to study the function of this domain in the P. denitrificans cytochrome bc1 complex, a deletion mutant has been previously cloned and modified with an affinity tag as a C-terminal extension of cytochrome b. The complex is purified by affinity chromatography and characterized by steady-state kinetics using not only horse heart cytochrome c but also the endogenous electron acceptor, the membrane bound cytochrome c552, employed here as a soluble fragment. Steady–state kinetics indicate that the deletion of the long acidic domain had effects neither on the turnover rate nor on the apparent affinity for the substrate. To understand wether the deletion affects the reaction between the cytochrome bc1 complex and the substrate, laser flash photolysis experiments are performed, showing that the interaction observed was not changed in the complex missing the acidic domain. The results presented in this work confirm the ones previously obtained by Julia Janzon using soluble fragments of the same interaction partners. The deletion, however, affected the oligomerization state of the complex, as shown by LILBID (Laser Induced Liquid Bead Ion Desorption) analysis. The wild type complex has a tetrameric structure, better described as a “dimer of dimers”. The deletion of the acidic domain on the cytochrome c1 results in the separation of the two dimers, yielding the canonical dimer. Therefore, the complex deleted in the acidic domain is used for cloning and expression of a heterodimeric complex, containing an inactivating mutation in the quinol oxidation site in only one monomer, thus allowing a selective switch-off for half the complex. Such a complex is needed for the verification of an internal regulation mechanism, the half-of-the-sites reactivity. According to it, the dimeric structure of the cytochrome bc1 complex has functional implications, since the two monomers can communicate and work in a coordinated manner. This approach confirms that substrate oxidation does effectively take place only in one of the two monomers constituting the dimer, and that the binding of substrate at the Qo and Qi site regulates the switch between active and inactive monomer. Moreover, this mechanism works also as an effective protection against the reaction of quinone intermediates with oxygen and the formation of reactive oxygen species (ROS), responsable for cellular aging. The motion of the ISP head domain is also addressed in this work; in particular the mechanism which regulates the movements towards the cytochrome c1 and the electron bifurcation at the quinol oxidation site. Laser flash kinetics in presence of several inhibitors and the substrate allow studying the response of the ISP to the binding of different species at the quinol oxidation site. The binding of ligand at the Qo site in the complex triggers the conformational switch in the ISP head domain, supporting the mechanism proposed in the literature according to which the Qo site is able to “sense” the presence of substrate and transfer the information to the ISP, regulating its mobility. The internal electron pathway between the ISP and the cytochrome c1 has been analyzed also by stopped-flow kinetics, in presence and absence of inhibitors. The results indicate that two kinetic phases describe the reduction of cytochrome c1 by the ISP, and a model for the simulation of the data is proposed.