Protein Import into Chloroplasts

Import of a hybrid construct consisting of the transit sequence of SSU, the N-proximal part of mature Tic110 and the mature SSU into chloroplasts led to the appearance of a soluble stromal import intermediate and the proposal that Tic110 might use a re-export pathway from the stroma to the inner envelope membrane. For full length Tic110 no soluble intermediate has been observed yet. One of the goals of this work was to investigate the import pathway of Tic110 in more detail. In this research the soluble stromal intermediate of Tic110 was observed, its re-export to the membrane was followed, and finally, the intermediate was isolated and co-immunoprecipitated with the stromal chaperones Hsp93, Hsp70 and to a lesser extent Cpn60. The obtained results indicate that Tic110, as proposed, uses a re-export pathway (conservative sorting) during its import into the chloroplast inner envelope membrane. Tic110 also requires stromal chaperones for achieving its native conformation, prior to the insertion into the inner envelope membrane. The pathway for targeting to the intermembrane space of chloroplasts had not been intensively studied yet. For this reason, the analysis of two intermembrane space localized proteins was conducted: Tic22, a 22 kDa Tic-complex protein component, and MGD1, synthase of MGDG, the most abundant galactolipid in nature. Both proteins are nuclear-encoded and synthesized on cytosolic ribosomes with a cleavable N?terminal chloroplast targeting presequence. Tic22 was found to be associated with the outer face of the inner envelope membrane, as well as with the inner face of the outer envelope membrane, even though at a lower level. MGD1 was proposed to be associated with one of the envelopes by weak electrostatic interactions. Import properties of Tic22 and MGD1 and the localization of MGD1 were investigated in this research. Results presented in this thesis show that import of MGD1 is dependent on, and that of Tic22 is enhanced by, but not dependent on, addition of external ATP. Both preproteins need thermolysin sensitive components on the chloroplast surface for successful import. Chemical crosslinking and immunoprecipitation have demonstrated that Tic22 and MGD1 interact with the components of the Toc translocon of the chloroplast outer envelope during their translocation. Import competition experiments showed that both proteins use the Toc machinery of the general import pathway. Therefore, proteins targeted to the intermembrane space seem to use the same translocation mode across the outer envelope as stromal proteins

Effects of TROL Presequence Mutagenesis on Its Import and Dual Localization in Chloroplasts

Thylakoid rhodanase-like protein (TROL) is involved in the final step of photosynthetic electron transport from ferredoxin to ferredoxin: NADP+ oxidoreductase (FNR). TROL is located in two distinct chloroplast compartments—in the inner envelope of chloroplasts, in its precursor form ; and in the thylakoid membranes, in its fully processed form. Its role in the inner envelope, as well as the determinants for its differential localization, have not been resolved yet. In this work we created six N-terminal amino acid substitutions surrounding the predicted processing site in the presequence of TROL in order to obtain a construct whose import is affected or localization limited to a single intrachloroplastic site. By using in vitro transcription and translation and subsequent protein import methods, we found that a single amino acid exchange in the presequence, Ala67 to Ile67 interferes with processing in the stroma and directs the whole pool of in vitro translated TROL to the inner envelope of chloroplasts. This result opens up the possibility of studying the role of TROL in the chloroplast inner envelope as well as possible consequence/s of its absence from the thylakoids

Protein Import into Chloroplasts

Import of a hybrid construct consisting of the transit sequence of SSU, the N-proximal part of mature Tic110 and the mature SSU into chloroplasts led to the appearance of a soluble stromal import intermediate and the proposal that Tic110 might use a re-export pathway from the stroma to the inner envelope membrane. For full length Tic110 no soluble intermediate has been observed yet. One of the goals of this work was to investigate the import pathway of Tic110 in more detail. In this research the soluble stromal intermediate of Tic110 was observed, its re-export to the membrane was followed, and finally, the intermediate was isolated and co-immunoprecipitated with the stromal chaperones Hsp93, Hsp70 and to a lesser extent Cpn60. The obtained results indicate that Tic110, as proposed, uses a re-export pathway (conservative sorting) during its import into the chloroplast inner envelope membrane. Tic110 also requires stromal chaperones for achieving its native conformation, prior to the insertion into the inner envelope membrane. The pathway for targeting to the intermembrane space of chloroplasts had not been intensively studied yet. For this reason, the analysis of two intermembrane space localized proteins was conducted: Tic22, a 22 kDa Tic-complex protein component, and MGD1, synthase of MGDG, the most abundant galactolipid in nature. Both proteins are nuclear-encoded and synthesized on cytosolic ribosomes with a cleavable N?terminal chloroplast targeting presequence. Tic22 was found to be associated with the outer face of the inner envelope membrane, as well as with the inner face of the outer envelope membrane, even though at a lower level. MGD1 was proposed to be associated with one of the envelopes by weak electrostatic interactions. Import properties of Tic22 and MGD1 and the localization of MGD1 were investigated in this research. Results presented in this thesis show that import of MGD1 is dependent on, and that of Tic22 is enhanced by, but not dependent on, addition of external ATP. Both preproteins need thermolysin sensitive components on the chloroplast surface for successful import. Chemical crosslinking and immunoprecipitation have demonstrated that Tic22 and MGD1 interact with the components of the Toc translocon of the chloroplast outer envelope during their translocation. Import competition experiments showed that both proteins use the Toc machinery of the general import pathway. Therefore, proteins targeted to the intermembrane space seem to use the same translocation mode across the outer envelope as stromal proteins

Balancing chloroplast redox status – regulation of FNR binding and release

Working in synchrony, photosynthetic charge separation, electron transfer, and redox reactions generate proton motive force necessary for the synthesis of ATP and funneling of electrons toward stromal reducing equivalent NADPH. The last step of electron transfer from ferredoxin to NADP+ is catalyzed by ferredoxin-NADP+ oxidoreductase (FNR). Two proteins, TROL (thylakoid rhodanese-like) and Tic62 (62 kDa component of the translocon at the inner envelope of chloroplasts), have been characterized and shown to form dynamic complexes with FNR. Inactivation of TROL leads to changes in efficiency of electron transfer and induction of non-photochemical quenching. TROL-deficient plants have changed nuclear gene expression with up-regulation of NADPH-dependent malic enzyme, which can form NADPH in an alternative pathway. Thus, NADPH synthesis, mediated by FNR-TROL interaction, may be the source element in metabolic retrograde signal-transduction pathway linking light reactions with nuclear gene expression

Requirements for a conservative protein translocation pathway in chloroplasts

AbstractThe chloroplast inner envelope translocon subunit Tic110 is imported via a soluble stromal translocation intermediate. In this study an in-organellar import system is established which allows for an accumulation of this intermediate in order to analyze its requirements for reexport. All results demonstrate that the re-export of Tic110 from the soluble intermediate stage into the inner envelope requires ATP hydrolysis, which cannot be replaced by other NTPs. Furthermore, the molecular chaperone Hsp93 seems prominently involved in the reexport pathway of Tic110, because other stromal intermediates like that of the oxygen evolving complex subunit OE33 (iOE33) en route to the thylakoid lumen interacts preferentially with Hsp70

Beginners guide to sample preparation techniques for transmission electron microscopy

Background purpose: The revolution in microscopy came in 1930 with the invention of electron microscope. Since then, we can study specimens on ultrastructural and even atomic level. Besides transmission electron microscopy (TEM), for which specimen preparation techniques will be described in this article, there are also other types of electron microscopes that are not discussed in this review. Materials and methods: Here, we have described basic procedures for TEM sample preparation, which include tissue sample preparation, chemical fixation of tissue with fixatives, cryo-fixation performed by quick freezing, dehydration with ethanol, infiltration with transitional solvents, resin embedding and polymerization, processing of embedded specimens, sectioning of samples with ultramicrotome, positive and negative contrasting of samples, immunolabeling, and imaging. Conclusion: Such collection of methods can be useful for novices in transmission electron microscopy

Balancing chloroplast redox status – regulation of FNR binding and release

Working in synchrony, photosynthetic charge separation, electron transfer, and redox reactions generate proton motive force necessary for the synthesis of ATP and funneling of electrons toward stromal reducing equivalent NADPH. The last step of electron transfer from ferredoxin to NADP+ is catalyzed by ferredoxin-NADP+ oxidoreductase (FNR). Two proteins, TROL (thylakoid rhodanese-like) and Tic62 (62 kDa component of the translocon at the inner envelope of chloroplasts), have been characterized and shown to form dynamic complexes with FNR. Inactivation of TROL leads to changes in efficiency of electron transfer and induction of non-photochemical quenching. TROL-deficient plants have changed nuclear gene expression with up-regulation of NADPH-dependent malic enzyme, which can form NADPH in an alternative pathway. Thus, NADPH synthesis, mediated by FNR-TROL interaction, may be the source element in metabolic retrograde signal-transduction pathway linking light reactions with nuclear gene expression