Part 5 Discusion (cont.)

Overlay of the PSI monomer projection from the 2.5 Å x-ray coordinates of cyanobacterial PSI (2) onto the Chlamydomonas LHCI-PSI supercomplex shows that there are additional densities (Fig. 4b). We assign the majority of this density to Lhca proteins. With the reaction center core, it has been found that higher plant PSI does not contain the PsaM and PsaX proteins (9) as does cyanobacterial PSI (2). However it does contain the additional low molecular weight subunits PsaG, PsaH, PsaN and possibly PsaO (9, 39). It has been suggested that PsaH is located close to PsaL, and in Arabidopsis it was shown that inactivation of the psaH gene inhibited the State 1 to State 2 transition suggesting that phosphorylated LHCII proteins bind to PsaH (40). The PsaN subunit is extrinsically located on the lumenal surface, while PsaG, like PsaH, is an intrinsic protein possibly located on the opposite side of the complex to the PsaH, PsaL, and PsaI proteins (9). Studies by Boekema et al. using spinach (10) concluded that Lhca proteins bind to one side of the PSI monomer in the membrane where the PsaF, PsaJ, PsaG, and PsaK are thought to be located (9). This suggestion is supported by deletion studies using Arabidopsis, which have shown that in the absence of the PsaK and PsaG proteins the Lhca proteins associate less strongly with the PSI reaction center core (41, 42). Similarly, in Chlamydomonas PsaK has been implicated in the association of LHCI with the PSI reaction center core (44). The idea that the LHCI subunits are located on the outer PsaF, PsaJ, PsaG, PsaK-containing side of PSI is in line with the recent discovery that iron stressinduced IsiA or CP43 proteins of cyanobacteria (6, 7, 43) or the Chl a/Chl b-binding Pcb proteins of Prochlorococcus SS120 (8) associate with this side of the PSI reaction center complex. More recently Germano et al. have reported a top view projection map of the LHCI-PSI supercomplex of Chlamydomonas derived from single particle analysis (32). The size and shape of the PSI particle they studied is similar to that reported here. Based on an overlay of the x-ray structure of the cyanobacterial PSI and comparison with the LHCI-PSI supercomplex isolated from spinach, they have concluded that 14 Lhca proteins are bound to the PSI reaction center core. If each Lhca subunit binds 10 Chls, as reported for higher plant Lhca (11) then this would increase the antenna size of the PSI reaction center by an additional 140 Chls thus giving a total of 240 Chls per P700, assuming that the PSI reaction center binds about 100 Chl. This, they argued, contrasts with the 200 Chl per P700 of the LHCI-PSI supercomplex isolated from spinach (10) and accounts for the smaller size of the latter. In coming to their conclusion they not only placed Lhca proteins along the outer side, as in the case of spinach, but also identified possible locations of Lhc proteins closer to the region assigned to the PsaL, PsaH, and PsaI cluster (9). They noted in a small number of their class averages that some of the extra density in this region, amounting to a surface area of 40 nm2, was missing. They argued that this missing density seemed to be close to PsaH and because PsaH has been implicated in the formation of State II (40) considered the possibility that these Lhc subunits could in part be Lhcb of LHCII.