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Biochemistry. Thylakoid membranes solubilized with beta-dodecyl
maltoside were subjected to sucrose density centrifugation with
the view of isolating high molecular weight supercomplexes containing
Pcb protein. Fig. 1a shows that
four fractions (F1-F4) were resolved. SDS-PAGE analyses (Fig. 1b)
suggested that PSI and PSII proteins were present in fractions F2,
F3, and F4 as confirmed by immunological blotting analyses (Fig.
1c) by using Abs raised to the reaction center proteins of PSI (PsaA)
and PSII (PsbA-D1 protein). The immunological blotting, however,
indicated that F2 was enriched in PSI, whereas F4 was highly enriched
in PSII. The F1 fraction was dominated by a protein having an apparent
Mr of ~34 kDa, and N-terminal sequencing identified this as a Pcb
protein. This Pcb protein was also present in the other three fractions.
Spectroscopic Analyses. Pigment extraction and HPLC analysis
gave a Chl a/b ratio of ~5 for the isolated thylakoids and
therefore the long-wavelength room temperature (RT) absorption peak
was at 674 nm and dominated by Chl a (Fig.
2a).
This agrees with the findings of Christen et al. (19), but higher
Chl b levels can be found in this organism depending on the
light intensity to which the cells are exposed in the ascidian host
(31). The Chl a/b ratio for the Pcb-rich F1 fraction was
3 and the absorption peak was blue-shifted to 669 nm (Fig. 2a).
This free Pcb fraction had an emission peak at 676 nm at RT (data
not shown) and at 679 nm at 77 K (see Fig. 2b). In contrast, the
fluorescence emission from thylakoids was maximum at 682 nm at RT
(data not shown) and 684 and 696 nm at 77 K (see Fig. 2b). The low
temperature peaks are typical of PSII emission, 685 nm from CP43
and 697 nm from CP47 (32). As noted (19), no significant low-temperature
emission band for PSI was present, contrasting with cyanobacterial
and higher plant PSI. The absence of significant 679-nm Pcb fluorescence
at 77 K from thylakoids suggests that the Pcb proteins transfer
energy efficiently to reaction centers in line
with their role as a light-harvesting system. The heavy fraction
(F4) had a red absorption maximum at 671 nm (Fig. 2a) and a RT emission
maximum of 679 nm (data not shown). Importantly the low-temperature
fluorescence spectrum of this heavy fraction peaked at 684 nm with
no significant peak or shoulder at 679 nm due to free or excitonically
uncoupled Pcb proteins (Fig. 2b). Moreover a shoulder at 697 nm
confirms the presence of PSII in this fraction. The long wavelength
absorption maxima of F2 and F3 were at 671 and 672 nm, respectively,
and their RT and 77 K emission spectra were
dominated by fluorescence from free Pcb protein or Pcb proteins
not fully functionally associated with reaction centers.
Electron Microscopy. The Pcb–PSI supercomplex isolated
from Prochlorococcus SS120 with its 18-Pcb subunits is a giant circular
particle having a diameter of 320 Å (17). Despite using the
same solubilization and isolation procedures for Prochloron as used
for Prochlorococcus SS120, we observed no such particle in the heaviest
fractions, F4 or F3, or any particle that could have been derived
from a partial degradation of this PSI supercomplex. We did, however,
identify a circular particle having a diameter of ~220 Å (circled
in Fig. 3a), which was at low frequency in the F4 fraction but
common in F3. We assign this particle to a PSI trimer based on its
similarity to the PSI trimer of cyanobacteria (28) (see below and
Fig. 4 a and b). However, an oblong-shaped
particle having dimensions of 210 x 290 Å was observed in
F4 (boxed in Fig. 3a). This represented the largest and most common
particle in F4. Based on past experience of viewing PSII core dimers
(29) and PSI supercomplexes containing Pcb proteins (17) in the
electron microscope, we assign the large Prochloron structures boxed
in Fig. 3a as being PSII supercomplexes. We interpret these supercomplexes
to consist of a central PSII dimer and associated Pcb proteins (Fig.
3b). By overlaying the outlines for the PSII dimer of cyanobacterial
PSII and for CP43 based on x-ray models [the latter as an analog
of a Pcb protein (15, 30)], the large particles are seen to consist
of a core dimer with a flanking region accommodating five Pcb proteins
along each edge, making a total of 10 Pcb subunits per PSII dimer
(Fig. 3c). Assuming a Mr of 850 kDa for the PSII dimer and 46 kDa
for the Pcb protein, to include the bound Chls, then the estimated
Mr of this large particle is ~1,500 kDa.
Although the structure described above is representative of the
majority of particles in fraction F4 of the sucrose density
gradient, there were other PSII particles within the F3 band that
seemed to have lost some or all of their flanking Pcb
subunits. We interpret the particle shown in Fig.
4c, e.g., as a PSII dimer with 5-Pcb subunits missing from one
side, having
dimensions of 210 x 210 Å. This interpretation is supported
by overlaying the outline of the x-ray structures (Fig. 4d) and
is consistent with the assignments within the complete Pcb–PSII
supercomplex as shown in Fig. 3c. Fig. 4 e and f shows the
top view of a PSII core dimer present in F3 with no Pcb subunits
attached. Also contained within the F3 fraction, as
mentioned above, were PSI trimers observed at a higher frequency
than in F4, shown as a processed image in Fig. 4a
with an overlay of the outline of the x-ray structure of a PSI trimer
from S. elongatus (28) in Fig. 4b. When viewed in the
electron microscope, F2 seems to consist mainly of monomers of PSI
although some dimeric PSII particles were present in
this fraction.
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