Deciphering Thylakoid Sub-compartments using a Mass Spectrometry-based Approach

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. Molecular & Cellular Proteomics 13: 10.1074/mcp.M114.040923, 2147-2167, 2014.As primary producers, plants are at the basis of the food chain for most ecosystems and control the planet atmosphere via photosynthesis. In eukaryotes, this process, responsible for carbon fixation and for the release of gaseous oxygen into the atmosphere, takes place in a specialized compartment, the chloroplast. This semiautonomous organelle plays a number of essential functions, including photosynthesis, nitrogen assimilation, sulfur reduction and assimilation, synthesis of amino acids, fatty acids, and many secondary metabolites (1-6). The chloroplast is not the only type of plastids. Plastids are indeed found in every plant tissue (with a very few exceptions such as angiosperm pollen grains) and in...

The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants

“…In flowering plants, levels of HCF222 mRNA declined by a factor of 5 relative to the maximum expression in apical meristems of 1-d-old seedlings. This is consistent with the proteome study, where HCF222 was measured with only a few counts in chloroplasts from 4-to 5-weekold seedlings (Tomizioli et al, 2014). Together, these observations suggest that HCF222 shows highest expression levels in young leaf stages.…”

“…6 and 7). The localization in chloroplasts is supported by a recent semiquantitative proteome study, where HCF222 was detected for the first time in this cell compartment (Tomizioli et al, 2014). Furthermore, our successful complementation of the knockout mutant with a chloroplast-directed HCF222 strongly suggests that the main site of action of this protein is located to the chloroplast (Table II; Fig.…”

“…In the proteome study of Tomizioli et al (2014), who analyzed thylakoid subfractions from Arabidopsis chloroplasts, HCF222 was detected exclusively in the fraction of the stromal thylakoid membrane system. Our salt and detergent treatment of the cell membrane fraction also identified HCF222 as a membrane-bound protein (Supplemental Fig.…”

A Thylakoid Membrane Protein Harboring a DnaJ-type Zinc Finger Domain Is Required for Photosystem I Accumulation in Plants

“…PSA2 was detected in two independent proteomic analyses of thylakoid membranes in Arabidopsis, the latter of which reported its enrichment specifically in non-appressed membranes (28,38). We confirmed that PSA2 is localized to non-appressed membranes by probing immunoblots of leaf extracts, intact chloroplasts, and chloroplast subfractions with the PSA2 antibody (Fig.…”

TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii

“…2B). Consistently, proteomics approaches on thylakoid membranes separated into grana and stroma lamellae identified the Arabidopsis thaliana TEF30 homolog (AT1G55480) exclusively in stroma lamellae (Tomizioli et al, 2014).…”