Compartmentalization of the protein repair machinery in photosynthetic membranes

A crucial component of protein homeostasis in cells is the repair of damaged proteins. The repair of oxygen-evolving photosystem II (PS II) supercomplexes in plant chloroplasts is a prime example of a very efficient repair process that evolved in response to the high vulnerability of PS II to photooxidative damage, exacerbated by high-light (HL) stress. Significant progress in recent years has unraveled individual components and steps that constitute the PS II repair machinery, which is embedded in the thylakoid membrane system inside chloroplasts. However, an open question is how a certain order of these repair steps is established and how unwanted back-reactions that jeopardize the repair efficiency are avoided. Here, we report that spatial separation of key enzymes involved in PS II repair is realized by subcompartmentalization of the thylakoid membrane, accomplished by the formation of stacked grana membranes. The spatial segregation of kinases, phosphatases, proteases, and ribosomes ensures a certain order of events with minimal mutual interference. The margins of the grana turn out to be the site of protein degradation, well separated from active PS II in grana core and de novo protein synthesis in unstacked stroma lamellae. Furthermore, HL induces a partial conversion of stacked grana core to grana margin, which leads to a controlled access of proteases to PS II. Our study suggests that the origin of grana in evolution ensures high repair efficiency, which is essential for PS II homeostasis.Repair of damaged protein complexes is essential for the survival of all living organisms. One of nature’s most efficient repair machineries is localized in photosynthetic thylakoid membranes of plants, which can turn over the total pool of the water-splitting photosystem II (PS II) supercomplex in less than 1 h (1). This remarkable potential for protein repair is crucial for the survival and fitness of plants because photodamage by reactive oxygen species is an inherent feature of PS II photochemistry. Significant knowledge gained over the past decade identifies individual steps involved in PS II repair. This progress has led to the formulation of a repair cycle that describes the life cycle of PS II, proceeding from its damage, disassembly, degradation, and resynthesis to its reassembly (2–4).To appreciate the challenges for repairing damaged PS II, it is essential to understand two structural features of the thylakoid membrane system. The first is that functional PS II is organized as a dimeric 1.4-MDa supercomplex in which each monomer consists of at least 28 subunits with two trimeric light harvesting complexes II (LHC II) attached to the core (5, 6). Within this huge supercomplex, the main target of photodamage is the central D1 subunit (7). The PS II repair cycle is therefore mainly designed for specific replacement of the damaged D1, which requires disassembly and reassembly of the whole supercomplex. The second characteristic is that in plants, some of thylakoid membranes pile up to strictly stacked cylindrical grana membranes (8, 9). The consequence of grana formation is four distinct domains: The flat, strictly stacked grana core membranes are connected to unstacked stroma lamellae via highly curved grana margins that are located at the periphery of the grana cylinder. The grana cylinder is sealed at the top and bottom by the flat so-called “end membranes.” Recently, a curvature thylakoid (CURT) protein family has been identified that facilitates the physical bending of grana margins (10). Consequently, CURT proteins are highly enriched in grana margins (10). These structural boundary conditions realized in thylakoid membranes have important consequences for the PS II repair cycle. The damage to the supercomplex occurs in stacked grana, where most of PS II is concentrated. The replacement of damaged D1, however, takes place in stroma lamellae. Thus, PS II repair depends on brisk protein trafficking between grana core, margins, and stroma lamellae, which is challenged by very low PS II mobility in grana core membranes caused by the macromolecular crowding (11).The mainstream model for the order of events of the PS II repair cycle starts with the phosphorylation of PS II core subunits (D1, D2, CP43, and PsbH) in the supercomplex, catalyzed mainly by the protein kinase Stn8 (12). PS II core phosphorylation triggers disassembly of the supercomplex (13, 14), which is required for mobilization of PS II from the rather immobile protein matrix in grana core (15). After migration of the trimmed down PS II complex to the unstacked thylakoid regions, it is dephosphorylated by the PS II core phosphatase (PBCP) (16) and by other putative PS II phosphatases. This dephosphorylation step is followed by the degradation of the damaged D1 by FtsH and Deg proteases. A new D1 subunit, which is de novo synthesized at the plastidial 70S ribosomes and processed by the CtpA peptidase, is inserted into the truncated PS II complex. The PS II repair cycle is completed by the reassembly of the supercomplex and its back-migration to grana core. The role of PS II core phosphorylation has been controversial. However, there is now consensus that it is required for ultrastructural changes of the thylakoid membrane system and for the mobilization of PS II in grana core (13, 14, 17).A crucial open question in the PS II repair cycle is how the specific order of events is established and controlled. The significance of this question is demonstrated by the fact that some steps are reverse reactions of the preceding steps (e.g., dephosphorylation follows phosphorylation, D1 synthesis follows D1 degradation). Uncontrolled intermixing of these reactions could lower the efficiency of the whole repair process. As for all multistep metabolic pathways, coordination and fine-tuning of the individual steps are required for the PS II repair cycle. In this study, we show that confining enzymes involved in PS II repair to distinct subcompartments in thylakoid membranes is a strategy to avoid uncontrolled back-reactions. We identify that the different thylakoid membrane subcompartments have distinct roles in the PS II repair cycle. In particular, the grana margins play a pivotal role.