separation-spinach-thylakoid-protein-complexes-native-green-gel

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and ...

Michigan State University

Photosynthetic organisms rapidly adjust the capture, transfer and utilization of light energy to optimize the efficiency of photosynthesis and avoid photodamage. These adjustments involve fine-tuning of expression levels and mutual interactions among electron/proton transfer components and their associated light-harvesting antenna. Detailed studies of these interactions and their dynamics have been hindered by the low throughput and resolution of currently available research tools, which involve laborious isolation, separation and characterization steps. To address these issues, we developed an approach that measured multiple spectroscopic properties of thylakoid preparations directly in native polyacrylamide gel electrophoresis gels, enabling unprecedented resolution of photosynthetic complexes, both in terms of the spectroscopic and functional details, as well as the ability to distinguish separate complexes and thus test their functional connections. As a demonstration, we explore the thylakoid membrane components of Chlamydomonas reinhardtii acclimated to high and low light, using a combination of room temperature absorption and 77K fluorescence emission to generate a multi-dimensional molecular and spectroscopic map of the photosynthetic apparatus. We show that low-light-acclimated cells accumulate a photosystem I-containing megacomplex that is absent in high-light-acclimated cells and contains distinct LhcII proteins that can be distinguished based on their spectral signatures.

Spectroanalysis in native gels (SING): rapid spectral analysis of pigmented thylakoid membrane complexes separated by CN-PAGE.

Photosystem II is known to be a highly dynamic multi-protein complex that participates in a variety of regulatory and repair processes. In contrast, photosystem I (PSI) has, until quite recently, been thought of as relatively static. We report the discovery of plant PSI-LHCII megacomplexes containing multiple LHCII trimers per PSI reaction center. These PSI-LHCII megacomplexes respond rapidly to changes in light intensity, as visualized by native gel electrophoresis. PSI-LHCII megacomplex formation was found to require thylakoid stacking, and to depend upon growth light intensity and leaf age. These factors were, in turn, correlated with changes in PSI/PSII ratios and, intriguingly, PSI-LHCII megacomplex dynamics appeared to depend upon PSII core phosphorylation. These findings suggest new functions for PSI and a new level of regulation involving specialized subpopulations of photosystem I which have profound implications for current models of thylakoid dynamics.

Photosystem I-LHCII megacomplexes respond to high light and aging in plants.

The funding statement in the last sentence of the Acknowledgements section in the original publication is incorrect. The corrected Acknowledgements section is printed below.

Article	Year
Spectroanalysis in native gels (SING): rapid spectral analysis of pigmented thylakoid membrane complexes separated by CN-PAGE. The Plant journal : for cell and molecular biology\| PubMed ID: 28865165	2017
Photosystem I-LHCII megacomplexes respond to high light and aging in plants. Photosynthesis research\| PubMed ID: 28975583	2018
Correction to: Photosystem I-LHCII megacomplexes respond to high light and aging in plants. Photosynthesis research\| PubMed ID: 29404825	2018

Eliezer Schwarz

Biography

JoVE Journal Publications

Other Publications