Compartmentalization of the protein repair machinery in photosynthetic membranes

Abstract

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.

Keywords: PS II repair cycle; grana margin; photoinhibition; photosynthesis; thylakoid membrane.

Fig. 1.

Identification and characterization of the grana margin. (A) Photograph of the two pellets after ultracentrifugation. The solid pellet corresponds to stroma lamellae, and the loose pellet corresponds to grana margins. (B) Coomassie-stained SDS/PAGE urea gel of the membrane fractions. G.c., grana core; G.m., grana margin; S.I., stroma lamellae; T., thylakoid. Major protein bands (Right) and molecular mass (Left) are indicated. Chlorophyll a/b ratios are given below the figure. (C) Control immunoblot of the grana margin marker protein CURT1A. An arrow on the right indicates the CURT1A band. Note that this band is missing in the curt1abcd quadruple mutant. (D) CURT1A immunoblot shows enrichment of the CURT1A protein in grana margins. (E) AtpF (ATPase subunit I) immunoblot shows enrichment of the ATPase in grana margins.

Fig. 2.

HL induces conversion of grana core to margins. (A) Electron micrograph of the thylakoid membrane system of an HL-treated leaf. The bent and expanded grana margins are highlighted with dotted red boxes. Arrowheads indicate additional lumen swellings in the expanded margins. (B) Electron micrograph of a dark-adapted leaf. (Scale bar: 200 nm.) (C) Graphical representation of the HL-induced membrane interconversion. (D) Yield of membranes in dark- and HL-treated protoplasts as deduced from the chlorophyll amount. The grana core shrinks and margins expand after HL treatment.

Fig. 3.

Localization of the enzymes and reactions reveals subcompartmentalization of the PS II repair cycle. (Top) Membrane yield in dark-adapted and HL-treated conditions is graphically represented. (Bottom) Amount and concentration of FtsH, D1, Stn8, phospho-PS II (P), and PS II in different membrane subdomains are plotted on a scale of 0–1. The percentage of the yield of FtsH and Stn8 is also shown on a scale of 0–100.

Fig. 4.

Analysis of grana core membranes reveals constancy of PS II organization. (A–F) Representative AFM images of dark- and HL-treated grana membranes. Green dots (B, C, E, and F) represent PS II protrusions as graphically rendered using the particle recognition program. (Scale bar: 200 nm.) (G) Nearest neighbor distribution functions of PS II in dark- and HL-incubated grana membranes are plotted. The x axis shows the distance between the nearest PS II neighbors, and the y axis shows the cumulative frequency. (H) Representative image of the sucrose density gradients shows distribution of PS II complexes in dark- and HL-treated samples. (I) Line profile of the chlorophyll density identifies major PS II bands on the gradient. (J) Amounts of various PS II complexes in the gradients are graphically represented. The numbers on the x axis correspond to bands identified in the line profile.

Fig. 5.

Scheme of the PS II repair cycle showing temporal and spatial segregation of the enzymes and the reactions they catalyze. The thylakoid subdomains—grana core, grana margins, and stroma lamellae—are demarcated. A key representing various assembly states and modifications of PS II is given below the figure.