Part 5 Discusion

First, we conclude that using mild isolation conditions we have not identified a trimeric form of PSI in Chlamydomonas as typically found in cyanobacteria (1, 2) and other types of oxyphotobacteria (6-8). The monomeric nature of PSI in higher plants and green algae has also been concluded by others (9, 10, 32). On the other hand Chlamydomonas has a dimeric PSII reaction center core similar to that found in higher plants and cyanobacteria (30, 33-35). Despite the apparent absence of a trimeric PSI complex in Chlamydomonas we have isolated a LHCI-PSI supercomplex suggesting that our isolation procedures are sufficiently mild to maintain oligomeric organization of PSI. The LHCI-PSI supercomplex was present in F3 of the sucrose density gradient, and its emission spectrum indicates that chlorophylls bound to the Lhca proteins are functionally coupled since there was no significant fluorescence at 674 nm as found with the detergentsolubilized, isolated Lhca proteins (Fig. 3b). However, it has been suggested previously in Refs. 9, 11, and 12 that when the Lhca proteins associate with the PSI reaction center core their long wavelength absorption and emission spectra undergo a significant red shift. In the case of the long wavelength absorption maxima the overall shift is from 671 nm (Fig. 3a) to beyond 677 nm since the LHCI-PSI supercomplex absorbs maximally at 680 nm (Fig. 1a). The Chlamydomonas PSI reaction center core without LHCI present has a red absorption peak at 677 nm as reported by Bassi et al. (12) and confirmed here (data not shown). According to Bassi et al. (12) aggregated forms of Chlamydomonas Lhca proteins can be isolated as two populations, consisting of the same Lhca proteins but absorbing at 673 nm and 680 nm and having 77 K fluorescence peaks at 685 nm and 705 nm, respectively. They also isolated a Lhca fraction having an absorption maximum at 670 nm and low temperature emission at about 675 nm, which probably consisted of non-aggregated protein, in agreement with our findings. Similar aggregations of Lhca proteins leading to red shifts in absorption and emission have been found for higher plants (11). In Chlamydomonas there seems to be at least 10 Lhca proteins (12, 36) encoded by different genes, but just how many gene products are contained in the F3 fraction is difficult to assess. Immunoblotting with a polyclonal LHCI antibody indicates the presence of four or more different forms of Lhca proteins (Fig. 2b). Importantly we did not detect LHCII proteins in the F3 fraction by immunoblotting with Lhcb1 and Lhcb2 although the antibodies were effective in detecting LHCII in the F1 and F2 fractions (Fig. 2b). The absence of LHCII proteins in the F3 fraction suggests that Chlamydomonas cells were in State 1 when the thylakoid membranes were isolated. It is generally believed that the State 1 to State 2 transition involves transfer of Lhcb subunits from PSII to PSI in response to N-terminal phosphorylation of these Chl a/bbinding proteins (37). To check this further we conducted an immunological analysis using antibody specific for phosphorylated threonine (purchased from Zymed Laboratories Inc.). As can be seen in Fig. 6 we did not detect LHCII phosphorylation in F1 or F3. In F2 clear signals were identified for CP29 (Lhcb4) and PsbH proteins that are known to dephosphorylate significantly more slowly than the LHCII proteins involved in State transitions (38).