Regulation of translation elongation in cyanobacteria: membrane targeting of the ribosome nascent-chain complexes controls the synthesis of D1 protein.

The photosystem II reaction centre protein D1 is encoded by the psbA gene. The D1 protein is stable in darkness but undergoes rapid turnover in the light. Here, we show that, in cyanobacterium Synechocystis sp. PCC6803, the synthesis of the D1 protein is regulated at the level of translation elongation in addition to the previously known transcriptional regulation. When Synechocystis sp. PCC6803 cells were transferred from light to darkness, the psbA mRNA remained abundant for hours. Cytosolic ribosomes were attached to psbA transcripts in the dark, and translation continued up to a distinct pausing site. However, ribosome nascent D1 chain complexes were not targeted to the thylakoid membrane, and no full-length D1 protein was produced in darkness. The arrest in translation elongation was released in the light, concomitantly with targeting of ribosome D1 nascent-chain complexes to the thylakoid membrane, allowing the synthesis of the full-length D1 protein. Downregulation of membrane targeting of ribosome complexes was also observed in the light if damage to the D1 protein was slow. This novel type of regulation of prokaryotic translation functions to balance the synthesis and degradation of the rapidly turning over photosystem II D1 protein in Synechocystis sp. PCC6803.

Exposure of Synechocystis 6803 cells to series of single turnover flashes increases the psbA transcript level by activating transcription and down-regulating psbA mRNA degradation.

Exposure of Synechocystis sp. PCC 6803 cells to series of single turnover flashes increases specifically the level of psbA and psbD2 messages, encoding the D1 and D2 proteins of photosystem II, as compared to light exposed cells. This increase is due to maintenance the transcription rate as high as in growth light and to the down-regulation of transcript degradation as in darkness. Inhibition of the plastoquinone pool reduction by DCMU or its oxidation by DBMIB does not diminish the transcription of the psbA gene under growth conditions. However, the degradation rate of psbA transcript, as well as of other transcripts encoding proteins of thylakoid complexes, is down-regulated in all conditions leading to the oxidation of the plastoquinone pool. We conclude that single turnover flashes are sensed as 'light' by transcription machinery of the cells irrespective of the plastoquinone pool reduction state and as 'dark' by the transcript degradation system.

Mutagenesis of the D-E loop of photosystem II reaction centre protein D1. Function and assembly of photosystem II.

The sequence connecting alpha-helices D and E of the D1 protein in photosystem II (PSII) is longer than that found in the corresponding loop of the L subunit in the rhodobacterial reaction centre. This sequence was mutated in order to determine its role in oxygenic photosynthesis. Site-specific mutants, including point mutations and deletions of different size, of the PEST-like region and the putative cleavage area in the D-E loop of the D protein were constructed in Synechocystis sp. PCC 6803. The effects of mutations on the functional and structural properties of PSII and turnover of the D1 protein were examined. Our results demonstrate that deletion of either the PEST-like sequence (deltaR225-F239) or the putative cleavage region (deltaG240-V249, deltaR225-V249) of the D1 protein resulted in severe perturbations on the function of the QB electron acceptor of PSII. However, PSII centres of the mutant with deleted PEST region remained functional enough to support autotrophic growth whereas deletions of the putative cleavage region prevented autotrophic growth. Although enhanced degradation rates of the mutant D1 proteins under low-light growth conditions demonstrate that neither the PEST-like sequence nor the putative cleavage region are required for D1 proteolysis, it became clear that the extension in the D-E loop of the D1 protein is essential for proper PSII assembly and photoautotrophic growth. Moreover, modifications of the D-E loop resulted in transcriptional activation of the psbA gene, indicating that neither light intensity, as such, nor the activity of the electron transfer chain are the only determinants in regulation of psbA gene transcription.

Constructed deletions in lumen-exposed regions of the D1 protein in the cyanobacterium Synechocystis 6803: Effects on D1 insertion and accumulation in the thylakoid membrane, and on Photosystem II assembly.

Modified forms of the D1 protein with deletions in lumen-exposed regions, were constructed in the cyanobacterium Synechocystis 6803 using site-directed mutagenesis. Integration and stability of the mutated D1 proteins in the thylakoid membrane were studied by immunoblot and pulse-chase analyses. It was found that in Δ(N325-E333), the D1 protein with a deletion in the C-terminal tail, could insert in the thylakoids to normal amounts but its stability in the membrane was dramatically reduced. Insertion of D1 in Δ(V58-D61) or Δ(D103-G109);G110R, with deletions in the A-B loop, was severely obstructed, For Δ(P350-T354), with a deletion in the processed region of the C-terminus of D1, no phenotypic effects were observed. The effects of failed D1 insertion or accumulation on Photosystem II assembly was monitored by immunoblot analysis. The conclusions from these experiments are that the extrinsic 33 kDa protein, CP43, and the ß subunit of cytochrome b559 accumulate in the thylakoid membrane independently of the D1 protein, and that accumulation of the D2 protein and CP47 requires insertion but not necessarily accumulation of the D1 protein.

D1 polypeptide degradation may regulate psbA gene expression at transcriptional and translational levels in Synechocystis sp. PCC 6803.

Light has been suggested to regulate both synthesis and degradation of the Photosystem II (PS II) reaction centre polypeptide D1, encoded by the psbA gene. The modified degradation rate of the D1 polypeptide in site-directed Synechocystis sp PCC 6803 D1 mutants CA1 [del(E242-E244);Q241H], E243K and E229D has provided a tool to determine whether the rate of D1 polypeptide synthesis is directly regulated by light-intensity-related factors or by a control mechanism mediated by light-dependent degradation of the D1 polypeptide. In vivo accumulation of [(35)S] methionine into the D1 polypeptide was found to correlate with D1 polypeptide degradation rather than with incident irradiance. This suggests that the degradation rate of the D1 polypeptide regulates its own synthesis at translational level. Furthermore, several fold differences in the psbA mRNA levels were measured between D1 mutant strains, indicating that the psbA gene transcription is not solely under light control.

A Mutation in the D-de Loop of D1 Modifies the Stability of the S2QA- and S2QB- States in Photosystem II.

Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three site-specific mutants of the D1 polypeptide of Synechocystis PCC 6803: E243K, E229D, and CA1 (deletion of three glutamates 242-244 and a substitution, glutamine-241 to histidine). The phenotypes of the E229D and E243K mutants were similar to that of the control strain (AR) in all of the studied aspects. The characteristics of CA1 were very different. Formate, which inhibits the QA- to QB- reaction, was severalfold less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, G. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson [1993] Plant Mol Biol 22: 1-12). A change in the redox potential of the QA/QA- couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA1. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D1 could influence the properties of the couple QA/QA- in D2 and the mechanism of recovery from photoinhibition.

Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II.

The degradation rate of the D1 polypeptide was measured in three Synechocystis PCC 6803 mutants in vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [delta (E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mumol photons m-2 s-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t 1/2 = 35 min) was about twice as long as in AR (control strain) cells (t 1/2 = 19 min). In growth light (40 mumol photons m-2 s-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t 1/2 approximately 5 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.