Abstract
Photosynthesis has shaped atmospheric and ocean chemistries and probably changed the climate as well, as oxygen is released from water as part of the photosynthetic process. In photosynthetic eukaryotes, this process occurs in the chloroplast, an organelle containing the most abundant biological membrane, the thylakoids. The thylakoids of plants and some green algae are structurally inhomogeneous, consisting of two main domains: the grana, which are piles of membranes gathered by stacking forces, and the stroma-lamellae, which are unstacked thylakoids connecting the grana. The major photosynthetic complexes are unevenly distributed within these compartments because of steric and electrostatic constraints. Although proteomic analysis of thylakoids has been instrumental to define its protein components, no extensive proteomic study of subthylakoid localization of proteins in the BBY (grana) and the stroma-lamellae fractions has been achieved so far. To fill this gap, we performed a complete survey of the protein composition of these thylakoid subcompartments using thylakoid membrane fractionations. We employed semiquantitative proteomics coupled with a data analysis pipeline and manual annotation to differentiate genuine BBY and stroma-lamellae proteins from possible contaminants. About 300 thylakoid (or potentially thylakoid) proteins were shown to be enriched in either the BBY or the stroma-lamellae fractions. Overall, present findings corroborate previous observations obtained for photosynthetic proteins that used nonproteomic approaches. The originality of the present proteomic relies in the identification of photosynthetic proteins whose differential distribution in the thylakoid subcompartments might explain already observed phenomenon such as LHCII docking. Besides, from the present localization results we can suggest new molecular actors for photosynthesis-linked activities. For instance, most PsbP-like subunits being differently localized in stroma-lamellae, these proteins could be linked to the PSI-NDH complex in the context of cyclic electron flow around PSI. In addition, we could identify about a hundred new likely minor thylakoid (or chloroplast) proteins, some of them being potential regulators of the chloroplast physiology.
Highlights
Be enriched in either the BBY or the stroma-lamellae fractions
Thylakoid Grana and Stroma-lamellae Proteomes composed of soluble proteins, which is the site where CO2 assimilation takes place thanks to the consumption of reducing equivalents and ATP produced by light-driven electron flow, and [3] the thylakoid membrane, which is a highly organized internal membrane network formed of flat compressed vesicles and which is the center of oxygenic photosynthesis
Thylakoids have been subjected to numerous proteomic investigations, mostly focusing on two biochemically different thylakoid protein populations: soluble proteins located in the lumen and more hydrophobic proteins present in the thylakoid membranes
Summary
Be enriched in either the BBY or the stroma-lamellae fractions. Overall, present findings corroborate previous observations obtained for photosynthetic proteins that used nonproteomic approaches. High throughput analysis techniques, combined with genome sequencing data, have largely modified the experimental approaches to study protein localization within a cell and changed our perception of the functional consequence of this localization [11,12,13] This is true for the chloroplast, where several proteome catalogs have been produced so far (14 – 17). MS analysis of tryptic peptides released from the surface of Arabidopsis thylakoid membranes was used to characterize the reversible phosphorylation of chloroplast thylakoid proteins [27] These studies confirmed earliest data demonstrating that various subunits of the PSII and light-harvesting polypeptides (LHCII) are phosphorylated. We performed large-scale analyses that aimed at identifying the whole chloroplast proteome In this context, we focused, in the same set of experiments, on the localization of proteins in the stroma, thylakoids, and envelope membranes [14]. Among the 500 proteins that were identified in the thylakoid fraction, 220 could be assigned to the thylakoid compartment (see AT_Chloro database at http://www.grenoble.prabi.fr/at_ chloro/)
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