Part 2 - Introduction (contd.)

In higher plants, green algae, and red algae, the outer lightharvesting system associated with PSI is made up of Lhca proteins that are encoded by the cab genes and known collectively as LHCI (light-harvesting complex I) (3). The oligomeric state of Lhca proteins in vivo is as yet not well understood but is often assumed to be dimeric, with each Lhca monomer having a structure similar to that of the related Lhcb proteins of the LHCII complex of photosystem II (PSII), which has been determined to 3.4 Å (4). Note that in cyanobacteria another type of outer light-harvesting system is present, composed of phycobiliproteins encoded by apc and cpc genes (5), which may, under some circumstances, attach to the stromal surface of the PSI core complex, although this has yet to be shown conclusively. Under conditions of iron deficiency, however, cyanobacteria form an additional outer light-harvesting system, composed of an 18-subunit membrane-intrinsic antenna "ring" around a PSI trimeric reaction center core (6, 7). The subunit is the Chl a-binding protein encoded by the isiA gene, which is homologous to the CP43 Chl a-binding protein of PSII. A similar protein, known as Pcb, which binds both Chl a and Chl b has also been found to form an 18-subunit light-harvesting antenna ring around trimeric PSI of the prochlorophyte Prochlorococcus marinus SS120 (8). To date all studies of the structure of PSI have concluded that unlike cyanobacteria, PSI is monomeric in higher plants and green algae (9). Some studies have explored the structural basis by which LHCI acts as the outer light-harvesting system of PSI. Indeed, Boekema et al. (10) concluded that in spinach the Lhca proteins were bound to one side of the PSI monomer. The increase in antenna size by 100 Chl a would correspond to the association of eight subunits of Lhca proteins with the PSI reaction center assuming that each subunit binds 12 Chl molecules as does the Lhcb monomer (4). However, unlike LHCII, the Chl a/b ratio is higher for LHCI, often assumed to be 3.3 or more (11, 12) compared with 1.4 for LHCII (4). Also it has been found that higher plant Lhca proteins bind 10 Chls (11). Boekema et al. (10) also noted that according to their interpretation of images derived from electron microscopy and single particle analysis, the positioning of LHCI on the outer side of the PSI reaction center would not preclude the possibility that under some conditions PSI of higher plants and green algae could form trimers similar to those found in cyanobacteria. Indeed PSI from these eukaryotic organisms contains the PsaL subunit, which in cyanobacteria is required for the stabilization of trimeric PSI (13). Using Chlamydomonas reinhardtii we set out to examine the oligomeric state of PSI in this green alga and establish structural details of the arrangement of LHCI with the reaction center core. A LHCI-PSI supercomplex preparation has been isolated using a His-tagged mutant. Projection maps of PSI with and without LHCI components bound have been obtained by electron microscopy and single particle image processing of negatively stained preparations. By difference mapping and by modeling in higher resolution structures of the underlying intrinsic proteins derived from x-ray diffraction studies (2) and electron crystallography (4), these maps have provided information about the number and positioning of the Lhca proteins within the supercomplex.