The role o f the PSII-H protein within the PSII complex has yet to be determined. A Synechocystis sp PCC 6803 deletion mutant for this subunit (Mayes et al. 1993) was shown to have a functional PSII but electron flow between the two quinones was impaired. The effect o f deletion o f some subunits has been shown to be more severe in eukaryotes than in prokaryotes such as Synechocystis sp PCC 6803. An example o f this is the PSII-K subunit. The cyanobacterial deletion mutants are still capable o f autotrophic growth whereas the C. reinhardtii deletion strain is unable to grow in the absence o f acetate. This shows that while the complexes are basically similar in structure and function there are significant differences in the eukaryotic system.
A series o f mutants were created in the psbYi gene o f C. reinhardtii with the aim o f refining the knowledge o f the function o f this protein. Two deletion mutants were created by inserting the aadK cassette into an restriction site within the coding sequence. The extraneous DNA was added in both the same and opposite orientations as the coding sequence. The deletion mutants were found to lack any active PSII and hence were incapable o f photoautotrophic growth. This differs from the situation found in Synechocystis but is not a unique example o f this phenomenon. We attempted to confirm that the PSII-H protein was not expressed in these mutants, by using western blots, with PSII-H specific antibodies. We used an antibody raised against the spinach protein and one which we ordered against the C. reinhardtii sequence. Although numerous assays were performed neither antibody ever reacted to a protein present in any C. reinhardtii
sample.
Two further mutants were created in C. reinhardtii to try and discover the significance o f the phosphorylation o f this subunit. The aadK sequence was inserted into a site downstream o f the gene, in the opposite orientation (Mlul). This mutant was to act as a control for all others in this region. The position o f the psbYi
gene, in an operon with a number o f others, led to the possibility that inserting DNA may disrupt the expression o f the other genes. The results o f all the experiments to characterise this strain showed that the phenotype o f this mutant did not differ significantly from the wild type control so it is unlikely that any alteration in the other mutants is due to the presence o f the aadK sequence.
The last PSII-H mutant constructed carried a site directed change in addition to the M lul addition o f the aadA cassette. The altered residue was the threonine at position two in the mature protein. This residue is known to be phosphorylated in a light dependent manner but the role o f this modification has yet to be conclusively determined. The site directed change altered the threonine to an alanine which cannot be modified by post translational phosphorylation. The initial characterisation o f this mutant did not lead to any new conclusions. The cells were photosynthetically competent and capable o f oxygen evolution but the rate was slightly lower than that seen in wild type or M lul cells.
The next experiments carried out on this mutant used an in vitro
phosphorylation assay to determine whether any effect on the phosphorylation profiles could be seen. In vitro experiments were carried out on two different preparations. The first was a purified thylakoid membrane fraction and the second consisted o f all the membrane fractions from C. reinhardtii cells. The difference between the two experiments in the T3A sample was dramatic. In the broken cell preparation phosphorylation o f proteins was clearly obvious while in the thylakoid membrane sample no phosphorylation was observed. The wild type thylakoid sample behaved in the manner expected with significant phosphorylation o f the LHCII and PSII-H proteins in the light. This implies that the preparation does not cause the effect observed in the T3A sample but some change inherent in the cells. There are two possible modifications which could have caused the effect; the presence o f the aadA cassette and the site directed change. The M lul cells would act as a control in determining which o f these factors is the most important in producing this result. The changes in the T3A cells must alter the PSII complex in some way that when the thylakoid membranes are purified some extra factors are lost. These could be involved at some stage in the activation o f the kinase or could even be due to the direct loss o f the kinase.
No further conclusions can be drawn from any o f the phosphorylation assays carried out. It is possible that the PSII-H protein is still phosphorylated in the site directed mutant but this has not been confirmed. The results from the broken cell in vitro experiments did not show the characteristic strong phosphorylation patterns o f LHCII and PSII-H in the light incubated samples so the possible phosphorylation o f the mutant inferred from the same gel is suspect.
Further analysis has been commissioned in other laboratories which may help further our understanding o f the phosphorylation o f this protein.
The psbH gene lies in an operon with those o f three other subunits o f PSII, PSII-B, PSII-T and PSII-N. It was decided that while we were investigating one gene in this operon it would be easy to extend this work to another. The PSII-N subunit was chosen as mutants had previously been created in the other two genes. Four different mutants were created in the region around the psb\^ gene. Two were insertion mutants with the aadK cassette interrupting the coding sequence while in the other two the extra DNA was introduced outside o f the coding region. The PSII-N null mutant, only one o f the two created reached homoplasticity, is able to grow autotrophically but further analysis has shown that the level o f PSII activity is not as great as in the wild type strain. The result from the deletion mutants is compared to that seen from the control mutants which contain the aadA cassette but contain an intact coding sequence. These cells were also photoautotrophic but were capable o f higher levels o f oxygen evolution than the deletion mutants. Despite this improvement the rate is still only 50% o f that seen for wild type cells. This suggests that introducing DNA into the region around p sb N is disrupting the expression o f a protein which is required for optimal function. The psbN gene is on the opposite strand to all the others in the operon, and lies between psb T and psbYi,
The introduction o f the aadA cassette may be influencing the expression o f the genes on this other strand, most likely psbYi as this lies downstream. The phenotype is not as severe as that seen for the PSII-H deletion mutants which tells us that PSII-H is transcribed but that the level o f protein present m ay be less than seen in wild type cells.
When the p sbN gene is disrupted the rate o f oxygen evolution falls and the cells appear to be more susceptible to high light damage. This leads us to the conclusion that at low levels o f light the PSII complex can function efficiently in the absence o f this protein but at higher levels o f illumination it is required to stabilise the complex. The exact role o f PSII-N cannot be determined.