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Firlej-Kwoka, Ewa (2008): Protein import into the inner envelope membrane of chloroplasts. Dissertation, LMU München: Fakultät für Biologie



Most of the proteins localized in the chloroplast inner envelope membrane are synthesized on cytosolic ribosomes with a cleavable N-terminal chloroplast transit peptide. Most of them reach their final localization via the so called general import pathway consisting of the Toc complex at the chloroplast outer envelope membrane and the Tic complex at the chloroplast inner envelope membrane. Recent studies characterized precursor proteins which are targeted into the chloroplast inner envelope membrane by two different import pathways. The first route, called “conservative sorting”, was described for Tic40 and Tic110, which prior to inner envelope membrane insertion reach the stroma. The second route, called “stop-transfer” was proposed for ARC6, which is arrested at the level of the inner envelope membrane and probably laterally inserted into the lipid bilayer. Taking into consideration both import mechanisms we characterized import pathways of nine chloroplast inner envelope membrane proteins containing cleavable transit peptides and a different number of hydrophobic -helices. On the basis of the results observed in the stromal processing assays as well as results obtained in the pulse-chase experiments, within investigated precursor proteins two classes could be distinguished. The first class consisted of precursors processed once to their mature forms, i.e. containing a “single” transit peptide, whereas the second class consisted of precursors processed twice to the intermediate and the mature form, i.e. containing a bipartite transit peptide. In the processing of almost all precursor proteins stromal processing peptidase (SPP) was involved. Most probably at least one protein containing a bipartite transit peptide was also processed by another peptidase present not in the stromal compartment. We showed that despite of the differences in the number of hydrophobic transmembrane segments and different types of transit peptides, all investigated proteins had similar import properties. Their import was dependent on outer envelope membrane receptors and mediated by the general import pathway at least in the initial import phase. All investigated proteins required energy for import. 200 M ATP was sufficient for proteins used in this study to achieve the maximal import rate. Interestingly, neither intermediates nor mature proteins were extractable from the membrane by urea treatment and all proteins seemed not to possess a soluble import intermediate. Therefore we claim that all investigated precursor proteins were imported via the “stop-transfer” pathway. Moreover, most probably at least some components of the Tic complex were involved in the transport of precursor proteins at the level of the inner envelope membrane and the process was Ca2+/calmodulin regulated.