Defining the heterogeneous composition of Arabidopsis thylakoid membrane

Thylakoid membrane (TM) of land plants is organized into an appressed domain (grana), enriched in photosystem (PS) II and a non-appressed domain (stroma lamellae) enriched in PSI. This ultrastructure controls the exciton spillover from PSII to PSI. The bulky machinery required for the biogenesis and repair of TM protein complexes is located in the non-appressed membranes. Thus, the connecting domain (CD) between grana and stroma lamellae is the key player in both the structural and functional integrity of the photosynthetic machinery. In addition, both the grana domain and the stroma lamellae are highly curved at their edges due to the action of the CURVATURE1 (CURT1) proteins, forming a domain distinct from the CD, called the curvature. Here we elucidate the biochemical properties and proteome composition of different thylakoid domains. To this end, the TM of Arabidopsis thaliana (Arabidopsis), isolated both in the natural stacked configuration and in an artificially unstacked configuration to induce a homogeneous protein composition, was solubilized and fractionated, using the mild detergent digitonin (DIG). Using mass spectrometry-based proteomics, we characterize composition, distribution and interaction of proteins involved in TM function in grana, CD and stroma lamellae domains. We find that a subset of thylakoid protein complexes are readily solubilized into small vesicles by DIG and accumulate in a loose pellet (LP) together with CURT1. By combining an extensive biochemical and proteome characterization of the TM fractions we provide an optimized protocol and proteome maps that can be used as a basis for experimental design in photosynthesis research.