Breakdown of the photosystem II reaction center D1 protein under photoinhibitory conditions: identification and localization of the C-terminal degradation products

Abstract

Illumination of a suspension of thylakoids with light at high intensity causes inhibition of the photosystem II electron transport activity and loss from the membrane of the D1 protein of the photosystem II reaction center. Impairment of the electron transport activity and depletion of D1 protein from the thylakoid membrane of pea were investigated with reference to the presence or absence of oxygen in the suspension. The breakdown products of the D1 protein were identified by immunoblotting with anti-D1 polyclonal antibodies which were proven to recognize mainly the C-terminal region of the protein. The results obtained show that (i) the light-induced inactivation of the photosystem II electron transport activity under anaerobic conditions is faster than in the presence of oxygen; (ii) depletion of D1 protein is observed on a longer time scale with respect to loss of electron transport activity and is faster when photoinhibition is performed in the presence of oxygen; (iii) C-terminal fragments of D1 are only observed when photoinhibition is carried out anaerobically and are mainly localized in the stroma-exposed regions; and (iv) the fragments observed after anaerobic photoinhibition are quickly degraded on further illumination of the thylakoid suspension in the presence of oxygen.

Photoinduced degradation of the D1 protein in isolated thylakoids and various photosystem II particles after donor‐side inactivations Detection of a C‐terminal 16 kDa fragment

“…2 was reacted with our antiDie polyclonal antiserum previously shown to recognize the C-terminal region of the protein [16]. Therefore, the 16 kDa fragment we detected must represent the C-terminal region of the protein.…”

“…The properties of the two polyclonal anti-Ill antisera (anti-D I c and anti-D 1 s, recognizing, respectively, the C-and N-terminal regions of the protein) have previously been described [16,19]. Proteolysis of wheat thylakoids with the Lys-e-specific endoprotease was performed at a chl concentration of 200/.tgml wi'.E !…”

“…Therefore, the 16 kDa fragment we detected must represent the C-terminal region of the protein. Since the DI protein from wheat contains a single lysine residue at position 238 we may compare the photoinduced 16 kDa fragment with the two polypeptides obtained by digestion of wheat DI protein with the highly specific Lys-C endopeptidase [16]. Of these two fragments the N-terminal one was recognized by our anti-D IN (Fig.…”

“…Different patterns for D1 degradation have also been reported under different experimental conditions and various PSII preparations [8,[13][14][15][16]19]. Thus during in viva light-induced turnover DI protein is thought to be cleaved at or close to the QEEE sequence [15,17] or at residue 238 [18], bath located in the stroma-exposed loop between the fourth and fifth putative transmembrane helices thereby producing an N-terminal fragment with an apparent mol.…”

“…Two main mechanisms have been proposed: the first, referred to as the 'aeceptor-side' mechanism, implies modifications at the level of either QB or QA plastoquinones [5,9,10] thereby preventing exchange with the plastoquinone pool. The second, or 'donor-side' mechanism, implies the accumulation of highly oxidant species such as Tyr~ and/or P6~0 [11,12].Different patterns for D1 degradation have also been reported under different experimental conditions and various PSII preparations [8,[13][14][15][16]19]. Thus during in viva light-induced turnover DI protein is thought to be cleaved at or close to the QEEE sequence [15,17] or at residue 238 [18], bath located in the stroma-exposed loop between the fourth and fifth putative transmembrane helices thereby producing an N-terminal fragment with an apparent mol.…”