Thylakoids from Chlamydomonas reinhardtii

In PS I, as in PS II, there are a number of Chi alb protein complexes having lightharvesting and energy-transfer functions. Such complexes most probably exist in direct contact with the RC (part of the core complex), and certainly exist as peripheral LHC I antenna complexes further removed from the RC. A native PS I complex (80-180 Chi per RC, 100 kDa) with at least 6 polypeptides was isolated by solubilization of the thylakoid membrane with nonionic detergents (for example, Triton X-100) [177,178]. With further detergent treatment, the PS I complex dissociated into the core complex (CC I with the RC) and the peripheral antenna complex (LHC I) (spinach, barley, pea, Chlamydomonas reinhardtii [179-183]. The peripheral antenna complex (pea, spinach Chi alb ratio 4.0 1, typical fluorescence at 730 nm) contains 3-4 antenna polypeptides (19-25 kDa) [181,184,185]. This complex was also dissociated into two different antenna complexes - LHC la (2 polypeptides of 22 and 23 kDa) and LHC Ib (1 polypeptide, 20 kDa) - which differ in their fluorescence characteristics (680 nm and 730 nm) [184]. No structural data on these polypeptides are available at present. It was postulated that in C. reinhardtii, in addition to the peripheral antenna complex, an antenna system (with 4 polypeptides) exists, which connects the peripheral antenna energetically with the core complex CC 1 [183]. 

Chlamydomonas reinhardtii double mutant npq2/lorl lacks the P,e-carotenoids lutein and loroxanthin as well as all P,P-epoxycarotenoids derived from zeaxanthin (e g. violaxanthin and neoxanthin). Thus, the only carotenoids present in the thylakoid membranes of the npq2/lorl cells are P-carotene and zeaxanthin. The effect of these mutations and the lack of specific xanthophylls on the Chi antenna size of the photosystems was investigated [16]. In cells of the mutant strain, the Chi antenna size of PSII was substantially smaller than that of the wild type (Table 1). In contrast, the Chi antenna size of PSI was not truncated in the mutant. This analysis showed that absence of lutein, violaxanthin and neoxanthin specifically caused a smaller functional Chi antenna size for PSII but not for that of PSI. Thus, xanthophyll-bios5mthesis genes, such as lycopene e-cyclase and zeaxanthin epoxidase may be targets for a truncated Chi antenna size in PSII. 

Table 1. Photosystem Chi antenna size in wild type and three Chlamydomonas reinhardtii mutant strains. The cbs3 strain lacks Chi b and was isolated upon DNA insertional mutagenesis [15]. The npq2/ lorl strain lacks all P,e-carotenoids as well as the P,P-epoxycarotenoids. It contains zeaxanthin but lacks lutein, violaxanthin and neoxanthin from its thylakoid membranes [16]. The tlal strain was isolated upon DNA insertional mutagenesis [17]. Note that the tlal transformant has the smallest combined Chi antenna size of the three mutants described.

In the present study we have used the chi b-less mutant of Chlamydomonas reinhardtii strain cbnl-48 to clarify the role of chi b and carotenoids in the assembly of LHC II. The thylakoids of the chi b-less mutant present a distinct pigment composition and a specific LHC II polypeptide pattern. LHC II from thylakoid membranes of the mutant could not be resolved by mildly dissociating SDS-PAGE. However we reconstituted the LHC II from delipidated chi b-less mutant thylakoids with the pigment extract derived from wildtype thylakoids. Similar to the native LHC II this reconstituted LHC II bind chi b and transfer light energy from chi b to chi a. 

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