In Vitro Synthesis and Assembly of Photosystem II Core Proteins

Abstract

The D1 reaction center protein of the membranebound photosystem II complex (PSII) has a much higher turnover rate than the other PSII proteins. Thus, the D1 protein has to be replaced while the other PSII components are not newly synthesized. In this study, this D1 protein replacement into PSII complexes was followed in two in vitro translation systems: isolated chloroplasts and a homologous run-off translation system consisting primarily of isolated thylakoids with attached ribosomes. The incorporation of newly synthesized radiolabeled products into different (sub)complexes was analyzed by sucrose density gradient centrifugation of n-dodecyl ␤-D-maltoside-solubilized thylakoid membranes. This analysis allowed us to follow the release of the nascent polypeptide chains from the ribosomes and identification of at least four assembly steps of the PSII complex, as shown below.(i) Both in isolated chloroplasts and in thylakoids, newly synthesized D1 protein is predominantly incorporated into existing PSII subcomplexes, indicating that synthesis and import of nuclear-encoded factors is not needed for D1 protein replacement.(ii) In chloroplasts, D1 protein incorporation into PSII core complexes is more efficient than during translation in isolated thylakoids. In the thylakoid translation system, a large percentage of radiolabeled D1 protein is found in smaller PSII subcomplexes, like PSII reaction center particles, and as unassembled protein in the membrane. This indicates that stromal factors are required in the replacement process of the D1 protein.(iii) Both in isolated chloroplasts and in thylakoids, the other PSII core proteins D2, CP43, and CP47 are also synthesized and released from the membrane-bound ribosomes, but incorporation into PSII complexes occurs to a much smaller extent than the D1 protein. Instead they accumulate predominantly as unassembled proteins in the thylakoid membrane.(iv) In chloroplasts, synthesis of the D1 protein seems to be adjusted according to the possibilities of incorporation into PSII complexes, while synthesis of the D2 protein, CP43, and CP47 is less regulated and their accumulation as unassembled protein in the membrane is abundant. Photosystem II (PSII)1 is a multiprotein complex of more than 25 different proteins in the thylakoid membrane and catalyzes the light-driven reduction of plastoquinone by electrons derived from water (see Refs. 1 and 2 for review). The heart of the PSII complex can be isolated as the PSII reaction center particle and is composed of a heterodimer of two homologous proteins, the D1 and D2 protein, the two cytochrome b 559 subunits (4 and 9 kDa), and at least one small protein (4.8 kDa, psbI gene product) of unknown function (see Ref. 3). Recently also a nuclear-encoded, small (6.1 kDa) protein has been suggested to be part of the PSII reaction center (4). Based on immunological data (5) and structural similarities between the bacterial reaction center and the reaction center of higher plants (6), the D1 and D2 proteins are predicted to contain five memb...

Genome-wide analysis of thylakoid-bound ribosomes in maize reveals principles of cotranslational targeting to the thylakoid membrane

“…2B) either as a ratio (upper plot) or separately (lower plot). All ORFs encoding proteins that were shown previously to integrate cotranslationally into the thylakoid membrane (PsbA, PsbB, PsbC, PsbD, PsaA PsaB, and PetA) (10)(11)(12)(13)22) are represented by prominent membrane-associated peaks, validating the method. Additional peaks revealed the thylakoid-associated translation of 12 proteins that had not been assayed in prior studies: AtpF, AtpI, PetB, NdhA, NdhB, NdhC, NdhD, NdhE, NdhF, NdhG, Ycf4, and CcsA (Fig.…”

“…Pioneering studies demonstrated that some chloroplast ribosomes are attached to the thylakoid membrane by the nascent peptide, implying a cotranslational integration mechanism (8,9). Several specific plastid-encoded proteins have been shown to integrate cotranslationally: the PSII subunits PsbA (also known as D1), PsbB, PsbC, and PsbD; the PSI subunits PsaA and PsaB; and the cytochrome b 6 f subunit PetA (also known as cytochrome f) (10)(11)(12)(13)(14). The insertion of PetA into the membrane requires cpSecA (12,15,16), whereas PsbA integrates independent of both the cpSecA and cpTAT systems (17).…”

Light is required for efficient translation elongation and subsequent integration of the D1‐protein into Photosystem II

“…Newly synthesized D1 protein is reassembled into existing PSII complexes. In two recent studies it has been shown that such replacement of the D1 protein can be achieved both in isolated chloroplasts and in isolated thylakoids [4,5]. The sequence of synthesis and assembly was partially resolved by pulse-chase experiments in these in vitro systems [5].…”

“…The distribution of PSII proteins and PSII (sub)complexes in the sucrose gradient fractions was determined by several biophysical and biochemical techniques as described in [4].…”

“…In vitro translation in isolated chloroplasts was carried out in the dark or in the light (approximately 50 gmol photons m -2 s 1) at 23°C, as described in [4]. The chloroplasts (0.5 gg chlorophyll ~tl 1) were incubated for 5 rain at 23°C in a translation mixture consisting of 330 mM sorbitol, 50 mM HEPES-KOH (pH 8.0), 10 mM dithiothreitol, 10 mM MgATP and 40 ~tM of each amino acid except methionine.…”