Isolation and characterization of photosystem I and the fucoxanthin-chlorophyll a/c proteins of the chromophyte alga Heterosigma carterae
Creators: name: Harnett, Julia Patricia
Publisher: name: University of British Columbia
Year: 2009

description: The chromophyte algae are a large and diverse group of organisms. They are unique in that they possess four membranes surrounding the chloroplast, as well as chlorophyll c. Compared to higher plants relatively little is known regarding the photosynthetic proteins of the chromophytes. In particular, much less is known regarding the light-harvesting proteins (FCPs) and the photosystems with which they are associated. FCPs (fucoxanthin-chl ale proteins) are the light-harvesting proteins found in chromophytes and they are part of an extended light-harvesting protein family which includes the chl a/b proteins (CABs) of chlorophytes and rhodophytes, as well as the peridinin-chlorophyll- a/c proteins (PCPs) of dinoflagellates. In the chromophytes it is not known how the different FCPs are related to each other or where FCPs are located within the thylakoids, i.e. if there are any specific associations with either PSI or PSII. In order to understand a little more about these photosynthetic proteins in chromophytes, I have developed several methods, and modified others, to separate out photosynthetic proteins of the chromophyte Heterosigma carterae. Using sucrose density gradients and FPLC (perfusion chromatography) to separate out dodecyl-p-D-maltoside- or digitonin-solubilized thylakoids, I obtained different FCP fractions, as well as several different PSI fractions with associated FCPs. With each type of detergent solubilization a large fraction of FCPs were released. The FCP fraction of the digitonin sucrose gradient contained mostly the main FCP (19.5 kDa). This fraction was further purified and an antibody raised to the main FCP. Immunological analyses with the anti-FCP antibody and the anti-CPIa antibody (known to cross-react with several H. carterae FCPs) showed further evidence supporting a monophyletic origin of the light-harvesting proteins in the different photosynthetic eukaryotes. The FCP band from the DM-gradient contained several FCPs, some of which could be further purified through FPLC. Two minor FCPs were purified for N-terminal sequencing. However, only weak homology was found with any other photosynthetic proteins, none of these being light-harvesting proteins. There was one main PSI fraction found with each of the detergent solubilizations, as well as a few minor PSI complexes in the digitonin-solubilized thylakoids. These fractions were characterized by polypeptide analysis (SDS-PAGE and Western blotting), pigment analysis (absorbance measurements), activity measurements (chl a/P700), dimensions (electron microscopy), and extrinsic protein determination (salt washes). A sucrose gradient PSI sample was further purified by FPLC to see if any other PSI proteins could be removed. The general characteristics of chromophyte PSI, particularly the polypeptides, were quite similar to higher plant and cyanobacteria. There also appeared to be several FCPs, including the main FCP, associated with PSI. However, whether these FCPs were exclusive to PSI could not be determined at this time.