ANALISIS MICROBIOLÓGICO

The coding sequence for the mature PSII-W protein was cloned into the expression vector pMAL-c2; antibodies were raised to a purified fusion protein MBP~PSII-W. Once optimised, no non-specific bands were observed following immunological probing o f whole cell WT CC-1021 samples with the SK26 antibodies. This observations shows that there are no MBP-like proteins present in C reinhardtii, and further that the pMAL-c2 expression system is highly suitable for raising antibodies to PSII and other C reinhardtii proteins.

Western analysis with the SK26 antibody was successful in the detection o f the PSII-W protein in WT CC-1021, PSII" and PSI" cell extracts, in WT CC-1021 and PSI" thylakoid preparations, and in PSII preparations. The presence of this protein could not be detected in PSII" thylakoid preparations or PSI preparations. A weak band corresponding to the mature PSII-W protein was detected in the PSII" cell extracts. This suggests that the PSII-W pre-protein is still expressed and processed and that low levels of unassembled mature protein are present even in the absence o f an assembled PSII complex. One explanation for the decreased levels relative to the WT and PSI" samples is that, in the absence o f PSII, the turnover o f the mature PSII-W subunit is accelerated. The PSII-W band was not detected in thylakoid preparations from the PSII" cells. It is unclear why the presence o f the PSII-W protein could be detected in PSII" whole cells samples but not in the PSII" thylakoid samples, since import into the membrane precedes thylakoid processing (Figure 3.8). This may indicate that the protein detected in the whole cell samples is not located in the thylakoid membrane and is lost during thylakoid purification.

Future investigations could be performed to establish if the unassembled protein is detectable in the PSII" cells and whether processing intermediates can be detected. Initially work using northern analysis would establish if the psbW transcript accumulated in the PSII" mutant or whether a feedback mechanism operates to down-regulate psbW

expression in the absence o f PSII assembly. If this was the case the next step would be to detect the presence o f the protein in isolated chloroplasts from WT CC-1021 and PSII" cells (Siiltemeyer et al., 1988; Sültemeyer et al., 1995).

To analyse the PSII-W processing intermediates, western analysis o f isolated chloroplasts could be performed to determine if the partially processed PSII-W protein is

present in the stroma. Alternatively immunoprécipitation o f the lysate o f either whole cells or chloroplasts which had been briefly pulse-radiolabelled could be employed. This strategy, described by Howe and Merchant (1993), would be used to detect the presence and/or absence o f the pre-protein in WT CC-1021 and P S ir cells and chloroplasts.

The PSI-A subunit was detected, as expected, in WT CC-1021 and PSIF cell extracts. It was also present in PSI" cell extracts. Analysis o f the thylakoid and photo system II preparations with aPSI-A antibodies confirmed the presence o f the PSI-A protein, and by inference the PSI complex, in WT CC-1021 and PSII" cells and thylakoids, and in PSI preparations. As expected the PSI-A protein could not be detected in PSI" thylakoids and in the PSII preparation, the latter confirming the purity o f the PSII preparation.

Western analysis o f the thylakoid samples was also performed with the anti-Dl antibody. The presence o f this protein, and by inference the presence o f the PSII complex, was detected in both WT CC-1021 and PSI" thylakoid samples. No D1 band was produced for the PSII" thylakoid samples. This result confirmed the integrity o f both o f the mutants. It also established that while the levels o f D1 (PSII) loaded in WT CC-1021 and PSI" thylakoids were not equivalent, they were similar.

Work by Hiyama et al. (2000) proposed that the PSII-W protein is also present within PSI. It should be noted that the presence of the PSII-W protein was not monitored with antibodies by Hiyama et al. (2000). Furthermore, the PSI preparations used were not well characterised. From the sequence analysis of PSII-W, it can be concluded that it is a strongly bound membrane protein that may bind to crude preparations o f thylakoid complexes through hydrophobic interactions.

Using the SK26 antibodies raised to the mature PSII-W protein o f C. reinhardtii

western analysis was undertaken to investigate if PSII-W could be detected in PSI. Preliminary western analysis with the SK26 antibodies o f purified PSII pre-treated with acetone demonstrated that the PSII-W protein binds strongly to either itself and/or to other subunits o f the PSII complex (Section 4.3. liv). Our explanation o f the detection o f PSII-W in PSI preparations by Hiyama et al. (2000) is therefore that this represents protein released from PSII that has adventitiously bound to their PSI. From our results, it can be concluded that the PSII-W protein is detected when PSII is present and PSI absent in a sample. The protein could not be detected in samples with PSI solely or when PSII

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fwas lacking. These results show that PSII-W is present exclusively in the PSII complex o f C reinhardtii, and is absent from the PSI complex.

Examination o f the expression o f the PSII-W yielded some interesting results. Cells were initially grown under three light regimes at normal growth temperature. When the light intensity is changed from darkness to low light (lOpE/m^/s) and then moderate light (50pE/m^/s) there is a gradual increase in the levels o f PSII-W. A similar pattern is observed when the same Ught conditions are studies at the stress temperature o f 37°C. These results support the findings at 25°C. Thus the expression and/or accumulation o f this protein appear to occur in a light dependent manner.

The levels o f the PSII-W protein at the same light conditions for the two different temperatures, 25°C and 37°C, can also be compared. There are two possible explanations for the results observed. Firstly, the expression and/or accumulation o f PSII-W under stress temperature (37°C) may be down regulated when cells are grown in the dark or under low light (lOpE/m^/s) relative to the same light conditions at 25°C. Alternatively, under these light conditions at 37°C the rate of PSII turnover may be increased, relative to the PSII turnover rate at 25°C. Under moderate light conditions (SOpE/m^/s) the levels are similar for the two temperatures examined. While it is tempting to speculate why this might occur, more experiments are required to examine this question.