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Organism. At present P. didemni cannot be cultured
(5, 18). Therefore colonies of the ascidian Lissoclimum patella
containing the Prochloron cells as symbionts were collected
from a depth of 2–4 m in the ‘‘Blue pools’’
area of the Heron Island Reef (Great Barrier Reef, Australia). Prochloron
cells were squeezed out of cut ascidian colonies into buffered seawater
(0.1 M Tris, pH 9.2). The seawater was taken directly from the Heron
Island Reef and passed through a sterile filter before use. The
cells were collected by low-speed centrifugation (1,000g) and washed
with buffered seawater (50 mM Mops, pH 7.5). The concentrated cells
were then transferred within 24 h on ice to the University of Sydney
for the extraction of thylakoids.
Isolation of Thylakoid Membranes. The procedure used was
as described (19). Freshly harvested cells were suspended in seawater
(50 mM Hepes, pH 7.8) or aqueous buffer (50 mM Hepes, pH 7.8, 15
mM NaCl, 5 mM MgCl2, 20% glycerol) and passed twice through a prechilled
French press at a pressure of 100 MPa. The resulting unbroken cells
and debris were removed from the French press extracts by centrifugation
with a JA-10 Beckman rotor at 1,000g for 10 min at 4°C. By using
a Ti70 Beckman rotor, thylakoid membranes were pelleted at 10,000g
and stored in 50 mM Mes, pH 6.0 at -70°C. For experiments samples
were rapidly thawed and washed twice with an aqueous buffer (50
mM Mes-NaOH, pH 6.0, 10 mM CaCl2, 5 mM MgCl2, 20% glycerol, 1 mM
PMSF).
Isolation and Purification of Supercomplexes. The thylakoid
membrane fraction of Prochloron was solubilized for 10 min
in the dark at 4°C by using 1% beta-dodecyl maltoside. The solubilized
complexes were separated on continuous sucrose density gradi-ents
generated by the freeze-thawing technique (20) containing 50mMMes-NaOH
(pH 6.0), 500mM betaine, 20mMCaCl2, 2.5 mMMgCl2, and 0.03% beta-dodecyl
maltoside and ultracentrifuged at 90,000g for 16 h at 4°C (SW28
Beckman rotor). The procedure was similar to that used previously
for isolating PSI supercomplexes from Synechocystis PCC 6803
(14, 15) and Prochlorococcus SS120 (17). The green pigment-containing
fractions were removed carefully by using a syringe.
Spectral Analysis. Room temperature absorption spectra were
performed by using a Shimadzu UV-1601 spectrophotometer. The room
and low temperature (77 K) fluorescence spectra were recorded by
using a Perkin–Elmer LS50 luminescence spectrometer (5-nm slit
width for emission) with an excitation wavelength of 430 nm.
Chl Analysis. HPLC was used to determine Chl a/b ratios following
a published procedure (21). Pigment was extracted into 100% acetone
and subjected to HPLC analysis by using a Kontron Spherisorb ODS-1
C18 reverse-phase column (Zurich) and calibrated with purified Chl
a and Chl b obtained from Sigma.
SDS-PAGE Analysis. The polypeptide composition of supercomplexes
isolated by sucrose density gradient centrifugation was resolved
by Tricine-SDS-PAGE (10%) with 6 M urea (22). Before the samples
were loaded onto the gels, they were
denatured by incubating with 50 mM Tris/HCl (pH 6.8), 4% (wt/vol)
SDS, 12% sucrose, and 100 mM DTT at room temperature for 1 h. Low
molecular weight makers (Bio-Rad) were used to estimate polypeptide
size.
Immunoblotting and N-Terminal Sequencing. Western blotting was
conducted by using a published procedure (20) with Abs raised to
the PSII reaction center PsbA (D1) protein of Synechocystis 6803
and to the reaction center proteins of PSI (PsaA) of Chlamydomonas
reinhardtii (kindly provided by P. Nixon, Imperial College). Polypeptides
separated by SDS-PAGE were transferred to poly(vinylidene difluoride)
membrane before identification by N-terminal sequencing conducted
by J. Keen (University of Leeds).
Electron Microscopy and Image Processing. Electron microscopy
analyses were carried out by negatively staining the samples with
2% (wt/vol) uranyl acetate and imaging at room temperature by using
a Philips CM100 electron microscope set to a calibrated magnification
of x51,500. The 14 best micrographs for each sample, displaying
minimal or no discernible
drift or astigmatism, were digitized on a Leafscan 45 densitometer
at a step size of 10 um. The resultant sampling
frequency of 1.94 Å per pixel on the specimen scale was coarsened
to 3.88 Å per pixel to aid in the speed of subsequent
processing by using the IMAGIC-5 software environment (23, 24).
On calculation of the contrast transfer function, the first
zero-crossing for each micrograph was consistently found to be in
the range of 18.6–21.3 Å. We identified and analyzed
populations of particles in the heaviest sucrose gradient fractions.
Application of classification procedures (25) enabled a
number of subpopulations within each dataset to be identified, differing
in both size and orientation. Each subpopulation was
treated de novo and iteratively refined, leading to improved 2D
class averages.
Modeling Studies. All studies were conducted by using the O
software package (26), with structural coordinates derived from
the 3.8-Å resolved model of PSII (27) designated under Protein
Data Bank ID code 1FE1 (www.rcsb.org) elucidated from the x-ray
diffraction of crystals of PSII isolated from Synechococcus elongatus.
Subunits attributed to Pcb protein were modeled by using the coordinates
assigned to the six transmembrane helices of CP43 in 1FE1 (27),
given the high structural homology known to exist between these
two protein families (15).
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