In Vitro Oligomerization of a Membrane Protein Complex

Abstract

Many membrane proteins can be isolated in different oligomeric forms. Photosystem I (PSI), for example, ex- ists in cyanobacteria either as a monomeric or as a tri- meric 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 monomers show chlorophyll fluorescence emission at 760 nm. To investigate the oligomerization process as well as the nature of the long wavelength chlorophylls, we describe here an in vitro reconstitution procedure to assemble trimeric PS I from isolated purified PS I monomers. Monomers (and trimers) were extracted from S. platen- sis with n-dodecyl !-D-maltoside and further purified by perfusion chromatography steps. The isolated com- plexes had the same polypeptide composition as other cyanobacteria (PsaA–PsaF and PsaI–PsaM), as deter- mined from high resolution gels and immunoblotting. They were incorporated into proteoliposomes, which had been prepared by the detergent absorption method, starting from a phosphatidylcholine:phosphatidic acid mixture solubilized by octylglucoside. After the addition of monomeric PS I (lipid:chlorophyll, 25:1), octylglu- coside was gradually removed by the stepwise addition of Biobeads. The 77 K fluorescence emission spectrum of these proteoliposomes displays a long wavelength emis- sion at 760 nm that is characteristic of PS I trimers, which indicates for the first time the successful in vitro reconstitution of PS I trimers. In addition, a high per- formance liquid chromatography analysis of complexes extracted from these proteoliposomes confirms the for- mation of structural trimers. We also could show with this system 1) that at least one of the stromal subunits PsaC, -D, and -E is necessary for trimer formation and 2) that the extreme long wavelength emitting chlorophyll is formed as a result of trimer formation.