Abstract
Thylakoids are a formation of flattened membrane vesicles and protein complexes found in cyanobacteria, algae and plants. In the chloroplasts of land plants the thylakoid membrane systems form a network of densely packed stacks called grana lamellae, which are connected by unstacked stroma lamellae. Photosystem II is mainly localized in the appressed grana region, while photosystem I and the ATP synthase complexes are enriched in the stroma lamellae. The cytochrome b6/f complex is distributed laterally throughout both stacked and unstacked membrane regions. The photosynthetic complexes consist of integral and peripheral proteins. The first part of this protocol (A) shows how to fractionate thylakoids into grana and stroma lamellae. The second part of this protocol (B) shows how to distinguish between strong hydrophobic integral membrane associations and weak electrostatic membrane and/or membrane complex associations. As it is necessary to specifically detect the protein of interest in the fractions, a specific antibody raised against the protein of interest or a complemented null mutant of a structural component expressing a tagged fusion protein would be of great advantage. The last part of this protocol (C) shows, how to investigate the topology of integral and peripheral proteins. This method requires a specific antibody for the protein of interest. For integral membrane proteins peptide-specific antibodies or epitope-tagged versions are required. The protocol is suitable for the investigation of low molecular weight proteins (LMW) below 5 kDa (Torabi et al., 2014).
Keywords: Stroma, Grana, Photosystem, Topology, Thylakoid
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Thylakoid system Figure 2. Scheme of the fractionation procedure (A) Figure 3. Coomassie staining of the thylakoid fractionation (A). Thylakoid membranes (T) were fractionated into stroma lamellae (S), intermittent fraction (I), and grana lamellae (G), and the separated proteins were subsequently stained with Coomassie to judge the purity of the fractions. PSI proteins PsaA and PsaB as well as the ATP synthase subunits CFo α/β were highly enriched in the stroma lamellae and only small amounts were present in the grana membranes. The PSII proteins CP47, CP43, D1, D2 and the antenna proteins of the light harvesting complex of PSII (LHCII) were predominantly present in grana fractions but almost lacking in the stroma lamellae fraction. The intermittent fraction contained proteins of both photosystems. An equal amount of chlorophyll (5 µg) was loaded. Figure 4. Washing experiment (B). Thylakoid membranes treated with different salt-containing buffers were fractionated into pellet (P) and supernatant (S) and separated by SDS-PAGE. The separated proteins were analyzed by Coomassie staining and immunoblot analysis. The dissociation of the hydrophilic ATP synthase subunits CFo α/β from the membrane can be identified by Coomassie staining under all salt conditions used. In contrast the hydrophobic light harvesting complex of PSII (LHCII) and the PSI core subunits PsaA/B could not be released from the pellet fraction. Specific antisera were used to identify the peripheral lumenal PsbO protein, which is associated with PSII. The PsbO protein could be released completely from the membrane only under stringent salt conditions (2 M NaSCN and 0.1 M NaOH). Figure 5. Scheme of the protease treatment (C) Figure 6. Topology studies (C). Untreated and sonicated thylakoids were incubated with thermolysin and subjected to immunodecoration using PsbO and PsaE antisera. In the untreated thylakoids the lumenal PsbO is protected from thermolysin treatment, while the stromal exposed PsaE is degraded. In the sonicated thylakoids, which form about 50% inside-out vesicles, PsbO is partially degraded, while PsaE is partially protected.
Notes
Recipes
Acknowledgments
This protocol was adapted or modified from previous work by Karnauchov et al. (1997). This research was supported by the German Science Foundation (Deutsche Forschungsgemeinschaft (ME 1794) to J. M.
References
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