Ribosome nascent chain complexes of the chloroplast-encoded cytochrome b6 thylakoid membrane protein interact with cpSRP54 but not with cpSecY.

Małgorzata Piskozub,Bożena Króliczewska,Jarosław Króliczewski

doi:10.1007/s10863-014-9598-0

Abstract

We analysed the interplay between the cpSecY, cpSRP54 and the chloroplast-encoded cytochrome b6 via isolation of chloroplast ribosome nascent chain complexes and the use of cross-linking factors, antibodies and mass spectroscopy analyses. We showed that the cytochrome b6 nascent polypeptide complex is tightly associated with ribosomes and that the translation of cytochrome b6 was discontinuous. The causes of ribosome pausing and the functional significance of this phenomenon may be related to proper protein folding, insertion into thylakoid membranes and the association of cofactors during this process. It was also found that cpSecY was not in the vicinity of cytochrome b6 intermediates during the elongation process and does not act with mature cytochrome b6 after translation. Using the approach of cross-linking during elongation of the cytochrome b6 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain.Electronic supplementary materialThe online version of this article (doi:10.1007/s10863-014-9598-0) contains supplementary material, which is available to authorized users.

Highlights

In the chloroplast the major complexes responsible for electron transport, i.e., photosystem I, photosystem II, the Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.M
Based on a number of observations in chloroplasts, such as run-off translations of thylakoids with bound ribosomes and detection of translation intermediates in the membrane, it can be postulated that insertion of polytopic chloroplast-encoded membrane proteins occurs co-translationally (Klein et al 1988; van Wijk et al 1996)
The observation of functional interaction of cpSecY with chloroplast 70S ribosomes suggested an involvement of cpSecY in the process (Zhang et al 2001)

Summary

Introduction

In the chloroplast the major complexes responsible for electron transport, i.e., photosystem I, photosystem II, the Electronic supplementary material The online version of this article (doi:10.1007/s10863-014-9598-0) contains supplementary material, which is available to authorized users.M. In the chloroplast the major complexes responsible for electron transport, i.e., photosystem I, photosystem II, the Electronic supplementary material The online version of this article (doi:10.1007/s10863-014-9598-0) contains supplementary material, which is available to authorized users. B. Króliczewska Department of Animal Physiology and Biostructure, Wrocław University of Environmental and Life Sciences, Wrocław, Poland cytochrome b6f complex and the ATP synthase complex contain subunits that are encoded from both the nuclear and chloroplast genomes. During the formation of these functional complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. To date import into or across the thylakoid membrane of nuclear encoded proteins has been thought to occur through four independent precursor-specific thylakoid transport pathways (cpTat, cpSec, cpSRP and the spontaneous integration pathway) (Schuenemann 2007). As chloroplasts are of prokaryotic origin, it is not surprising that several of the chloroplast targeting and translocon components share a strong homology with those in E. coli and pronounced differences from bacterial transport mechanisms

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Conclusion