In vitro oligomerization of a membrane protein complex - Liposome-based reconstitution of trimeric photosystem I from isolated monomers

Many membrane proteins can be isolated in different oligomeric forms. Photo system I (PSI), for example, exists in cyanobacteria either as a monomeric or as a trimeric complex. Neither the factors responsible for the specific trimerization process nor its biological role are known at present. In the filamentous cyanobacterium Spirulina platensis, trimers in contrast to mono mers show chlorophyll fluorescence emission at 760 nm. To investigate the o ligomerization process as well as the nature of the long wavelength chlorop hylls, we describe here an in vitro reconstitution procedure to assemble tr imeric PS I from isolated purified PS I monomers. Monomers (and trimers) we re extracted from S. platensis with n-dodecyl p-D-maltoside and further pur ified by perfusion chromatography steps. The isolated complexes had the sam e polypeptide composition as other cyanobacteria (PsaA-PsaF and PsaI-PsaM), as determined from high resolution gels and immunoblotting. They were inco rporated into proteoliposomes, which had been prepared by the detergent abs orption method, starting from a phosphatidylcholine:phosphatidic acid mixtu re solubilized by octylglucoside. After the addition of monomeric PSI (lipi d:chlorophyll, 25:1), octylglucoside was gradually removed by the stepwise addition of Biobeads. The 77 K fluorescence emission spectrum of these prot eoliposomes displays a long wavelength emission at 760 nm that is character istic of PS I trimers, which indicates for the first time the successful in vitro reconstitution of PS I trimers, In addition, a high performance liqu id chromatography analysis of complexes extracted from these proteoliposome s confirms the formation of structural trimers, We also could show with thi s system 1) that at least one of the stromal subunits PsaC, -D, and -E is n ecessary for trimer formation and 2) that the extreme long wavelength emitt ing chlorophyll is formed as a result of trimer formation.