| Summary: | 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.
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