GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Abstract

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Figure 1

Effect of ATP and GTP on the proteolysis of the D1 protein in photoinactivated thylakoids. (A) Autoradiogram showing the level of the D1 protein in EDTA-washed thylakoids before and after photoinactivation (PI) for 90 min followed by 90 min of darkness. (B) Time course for the photoinactivation of oxygen evolution (□) and degradation of the D1 protein in the absence (○) or presence of either 1 mM ATP (▴) or 0.2 mM GTP (●). The amount of the remaining D1 protein was normalized to that in the nonirradiated thylakoids and plotted as a function of time. The maximal rate of oxygen evolution was 350 μmol of O₂ per mg of Chl per h in dark-control thylakoids (100%). The arrow indicates the transfer of samples photoinactivated at −1°C to darkness at 22°C.

Figure 2

D1 protein degradation in photoinactivated thylakoids—dependence on nucleotide concentration. (A) Effect on the extent of D1 protein loss. The level of remaining D1 protein was determined by Western blotting in photoinactivated samples that were incubated in darkness for 90 min in the presence of the indicated concentrations of ATP (▴) or GTP (●). (B) Autoradiograms showing the level of D1 protein and its 23-kDa fragment in samples photoinactivated (PI) and subsequently dark-incubated with concentrations of up to 0.2 mM GTP. (C) Effect on the accumulation of the 23-kDa fragment of the D1 protein. The relative amounts of the proteolytic fragment were determined in samples treated as in A.

Figure 3

Time course of D1 protein degradation determined by Western blotting in thylakoids photoinactivated at −1°C and then incubated in darkness for 0, 30, and 90 min at 22°C in the absence (○) and presence of 0.2 mM GTP (●) or 0.2 mM GTP[γS] (■), or 5 units of apyrase per ml (⋄). The arrow indicates the transfer of samples photoinactivated at −1°C to darkness at 22°C.

Figure 4

Effect of ATP and zinc ions on the degradation of the 23-kDa D1 protein fragment. Thylakoids were photoinactivated and subsequently incubated in darkness for 90 min in the presence of the indicated concentrations of ATP and zinc acetate. The level of the proteolytic fragment was expressed relative to the amount detected in the absence of any additions.

Figure 5

Comparative levels of the 23-kDa D1 protein fragment in thylakoids not EDTA-washed before photoinactivation and subsequently incubated in darkness for 90 min in the absence (−) or presence (+) of 0.5 mM ATP, 0.5 mM zinc acetate, 2 mM EDTA, and 0.5 mM ATP[γS].

Figure 6

Effect of GTP, ATP, and zinc ions on D1 protein degradation in photoinactivated PSII core complexes. (A) Extent of D1 protein degradation determined by Western blotting. Isolated PSII core complexes were photoinactivated for 45 min at −1°C and subsequently incubated in darkness for 90 min in the absence and presence of 0.2 mM GTP or GTP[γS]. (B) Levels of the 23-kDa D1 fragment determined in samples of PSII core complexes, treated as in A, incubated in the absence and in the presence of 0.2 mM GTP or 1 mM ATP and 0.5 mM zinc acetate.

Figure 7

Model for the proteolytic events occurring during D1 protein degradation after photoinactivation and photodamage to the PSII reaction center.