Download Differential assembly of polypeptides of the light

Photosynth Res (2012) 111:125–138
DOI 10.1007/s11120-011-9707-4
REGULAR PAPER
Differential assembly of polypeptides of the light-harvesting 2
complex encoded by distinct operons during acclimation
of Rhodobacter sphaeroides to low light intensity
Kamil Woronowicz • Oluwatobi B. Olubanjo •
Hee Chang Sung • Joana L. Lamptey • Robert A. Niederman
Received: 27 June 2011 / Accepted: 10 August 2011
Ó Springer Science+Business Media B.V. 2011
Abstract In order to obtain an improved understanding
of the assembly of the bacterial photosynthetic apparatus,
we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced
incident light intensity. Photoheterotrophically growing
cells were shifted from 1,100 to 100 W/m2 and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were
subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)LH1 complexes were excised and subjected to in-gel
trypsin digestion followed by liquid chromatography (LC)mass spectroscopy (MS)/MS. The results revealed that the
LH2 band contained distinct levels of the LH2-a and -b
polypeptides encoded by the two puc operons. Polypeptide
subunits encoded by the puc2AB operon predominated
under high light and in the early stages of acclimation to
low light, while after 24 h, the puc1BAC components were
most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in
This is a re-publication of the original article in the September 2011
issue of this journal. When referring to this article in future
publications, please cite the original article, as follows: Woronowicz
K, Olubanjo OB, Sung HC, Lamptey JL, Niederman RA (2011)
Differential assembly of polypeptides of the light-harvesting 2
complex encoded by distinct operons during acclimation of
Rhodobacter sphaeroides to low light intensity. Photosynth
Res108(2–3):201–214. doi: 10.1007/s11120-011-9681-x
K. Woronowicz O. B. Olubanjo H. C. Sung J. L. Lamptey R. A. Niederman (&)
Department of Molecular Biology and Biochemistry, Rutgers
University, Busch Campus, 604 Allison Road, Piscataway,
NJ 08854-8082, USA
e-mail: [email protected]
Puc1A, was a protein of major abundance. A predominance
of Puc2A components in the LH2 complex formed at high
light intensity is followed by a [2.5-fold enrichment in
Puc1B levels between 3 and 24 h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This
indicates that the puc1BAC operon is under more stringent
light control, thought to reflect differences in the puc1
upstream regulatory region. In contrast, elevated levels of
Puc2 polypeptides were seen 48 h after the gratuitous
induction of ICM formation at low aeration in the dark,
while after 24 h of acclimation to low light, an absence of
alterations in Puc polypeptide distributions was observed in
the upper LH2-enriched gel band, despite an approximate
twofold increase in overall LH2 levels. This is consistent
with the origin of this band from a pool of LH2 laid down
early in development that is distinct from subsequently
assembled LH2-only domains, forming the LH2 gel band.
Keywords Light-harvesting complexes Light regulation puc operon Proteomics Rhodobacter sphaeroides
Abbreviations
AFM Atomic force microscopy
b-OG n-octyl b-D-glucopyranoside
DOC
Deoxycholate
CM
Cytoplasmic membrane
COGs Clusters of orthologous groups
ICM
Intracytoplasmic membrane
LH
Light harvesting
LH1
Core pigment-protein light-harvesting complex
LH2
Peripheral pigment-protein light-harvesting
complex
RC
Photochemical reaction center
UPB
Upper pigmented band
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Introduction
The photosynthetic units of the facultative photoheterotroph
Rhodobacter sphaeroides, a member of the a-3 subclass of
Proteobacteria, contain two integral membrane light-harvesting complexes, designated as LH1 and LH2, which
function as core and peripheral antennae, respectively.
Radiant energy harvested by LH2 is transferred to LH1,
which directs these excitations within the photosynthetic unit
cores to the photochemical reaction center (RC), where the
excitation energy is trapped as a stable transmembrane
charge separation (Hunter et al. 2009). The apoproteins of
both LH complexes consist of heterodimers of a- and bpolypeptides containing 50–60 amino acid residues (Theiler
et al. 1984, 1985), which in LH1, bind two molecules of BChl
(B875 BChl with Qy absorption band at 876 nm and two
carotenoids (Broglie et al. 1980), and in LH2, two B850
BChls, one B800 BChl (with Qy absorption bands at 850 and
800 nm, respectively) and one to two carotenoids (Hunter
et al. 1988). The atomic-resolution structure of the LH2
complex from the related bacterium Rhodoblastus acidophilus has been determined by X-ray crystallography
(Papiz et al. 2003) and consists of a circular arrangement of
18 B850 BChls sandwiched between two concentric cylinders of transmembrane helices, with the outer and inner
nonameric cylinders made up of b- and a-polypeptides,
respectively. The B850 BChls form a ring strucutre with
overlapping bacteriochlorin moieties in which non-covalent
associations alternate with the a- and b-polypeptides, while
nine discrete B800 BChls form an outer ring. A nine-membered ring of ab-heterodimers has also been suggested for the
Rba sphaeroides LH2 complex, based on a 6-Å projection
structure obtained from negatively stained 2-dimensional
crystals (Walz et al. 1998) and AFM topographs of isolated
membranes (Bahatyrova et al. 2004).
In Rba. sphaeroides, the LH2 polypeptides are encoded
by the pucBA structural genes of the puc operon (Kiley and
Kaplan 1987) which contains an additional gene (pucC),
immediately downstream of pucA. The pucC gene, which
encodes a member of the major facilitator superfamily, is
cotranscribed with pucBA, acting posttranscriptionally in
the membrane assembly of the LH2-a and b apoproteins
(Lee et al. 1989; Gibson et al. 1992). Upon the availability
of the complete genome sequence of Rba. sphaeroides
(Mackenzie et al. 2001), it was found that in addition to this
previously described puc operon (subsequently designated
as puc1BAC), which encodes the respective LH2-b (51
amino acid residues) and LH2-a (54 residues) apoproteins
subunits, a second puc operon exists (designated as
puc2BA) which is located outside the photosynthesis gene
cluster (Zeng et al. 2003). Expression studies suggested
that while the Puc2B polypeptide (51 residues, 94%
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Photosynth Res (2012) 111:125–138
identity to Puc1B) enters into LH2 complex formation,
neither the full-length 263-residue Puc2A gene product,
nor its N-terminal 48-amino acid Puc1A homolog (58%
identity), were present in LH2 complexes. It thus appeared
that puc1A was the sole source of the a–polypeptide for the
LH2 complex, whereas *30% of the LH2 complexes were
found to contain the puc2B-encoded b-polypeptide. Major
differences in the regulation of the two operons were also
revealed in which puc1BAC was shown to be under more
stringent regulatory control than puc2BA. In addition, it
was demonstrated that the presence of LH2 was dependent
upon puc1BAC, while the ultimate cellular level of LH2
was also dependent upon puc2BA.
In this report, we describe a proteomic analysis of the
levels of the various puc1BAC and puc2BA operon-encoded polypeptides in the LH2 complexes assembled in Rba.
sphaeroides exposed to a lowering of both incident light
intensity and oxygen tension. For the light regulation study,
cells grown photoheterotrophically at high incident light
intensity were shifted to low light, circumstances under
which an extensive remodeling of the intracytoplasmic
membrane (ICM) occurs. This process is characterized by
preferential synthesis and assembly of the LH2 complex,
which results in marked expansion of the absorption crosssection largely by the addition of LH2 to the RC-LH1 core
complex-enriched ICM formed under high illumination
levels (Woronowicz et al. 2011). In the second approach,
ICM development was induced gratuitously at low oxygen
tension in concentrated suspension of pigment-depleted,
chemoheterotrophically grown cells (Niederman et al.
1976). Under these conditions, photosynthetic units are
sequentially assembled, with RC-LH1 core structures
inserted initially into the cytoplasmic membrane (CM) in a
form that is largely inactive in forward electron transfer
(Koblı́zek et al. 2005). This is followed by the activation of
functional electron flow, together with the addition of LH2,
which results in further invagination and vesicularization
of the membrane to form the ICM. During each of these
ICM developmental processes, LH2 is thought to pack
initially between linear arrays of dimeric core complexes
observed by atomic force microscopy (AFM) (Bahatyrova
et al. 2004). When these regions become fully occupied,
the LH2 complex may ultimately form a light-responsive
peripheral antenna complement by clustering into
LH2-only domains (Hunter et al. 2005).
Intracytoplasmic membrane vesicle (chromatophore)
preparations and an upper pigmented band (UPB) containing
ICM growth initiation sites were isolated from cells undergoing acclimation to low light intensity and gratuitous
induction of ICM formation after the lowering of oxygen
tension. The isolated membrane preparations were subjected
to non-denaturing polyacrylamide gel electrophoresis (clear
Photosynth Res (2012) 111:125–138
native electrophoresis, CNE) which gives rise to four wellresolved pigmented bands (Woronowicz and Niederman
2010). The slowest migrating (top) and fastest migrating
(bottom) bands contained spectrally homogeneous preparations of the native RC-LH1 and LH2 complexes, respectively, while associations of these core and peripheral
complexes were present in the two bands of intermediate
migration. Surprisingly, a detailed proteomic analysis of the
LH2 gel bands of chromatophores from cells acclimating to
reduced light intensity revealed distinct alterations in the levels
of the LH2-a and -b polypeptides encoded by the two separate
puc operons (Zeng et al. 2003). These results demonstrate that
two puc operons respond differently to changes in illumination
levels during photoheterotrophic growth. (A preliminary report
of this work was presented at the Photosynthetic Light Harvesting Satellite Meeting of the 15th International Congress of
Photosynthesis, Tianjin, China, 2010).
Materials and methods
Cell growth and membrane isolation
Rba. sphaeroides wild-type strain NCIB 8253 was grown
photoheterotrophically at 30°C in l-l Roux bottles, in the
medium of Cohen-Bazire et al. (1956). Cells were initially
grown at high light intensity (1,100 W/m2) in a water-cooled
plastic aquarium illuminated by two opposed 150 W tungsten lamps controlled by a rheostat. Exponentially growing
high light cells were transferred to a refrigerated incubator
and at a reduced light intensity (100 W/m2) provided by
40-W tungsten bulbs. Light intensities were monitored with a
radiometer (YSI-Kettering). High light cells were also
transferred to indirect weak illumination (30 W/m2) as
described by Woronowicz et al. (2011). In addition, aerobically grown batch cells were concentrated and subjected to
gratuitous induction of ICM formation at low-aeration as
described by Koblı̀zek et al. (2005).
For membrane isolation, which was performed in 1 mM
Tris, pH 7.5, at 4°C, cells were centrifuged at 12,0009g,
washed and resuspended. A few crystals of DNAseI
(Roche) and protease inhibitor cocktail (Roche) were
added and the washed cells were passed twice through a
French pressure cell. Debris and unbroken cells were
removed by centrifugation at 16,0009g. The supernatant
was layered onto a 5–35% (w/w) sucrose gradient prepared
over a 60% sucrose cushion and subjected to rate-zone
ultracentrifugation for 3 h at 28,000 rpm in Beckman SW28 rotor. Two bands were collected: the UPB, containing
membrane growth initiation sites and the main pigmented
band, consisting of chromatophores.
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Clear native electrophoresis
For the isolation of pigment-protein complexes, chromatophore samples were solubilized with 15 mM each of
n-octyl b-D-glucopyranoside (b-OG) and deoxycholate
(DOC), or with 2 g digitonin/g total protein, and applied to
a 5–10% polyacrylamide clear native gel as described by
Wittig et al. (2007). For clear native electrophoresis (CNE)
of the UPB fraction, solubilization was performed with
digitonin. Protein concentration was determined using the
BSA protein assay (Thermo) and 312 lg of total protein
was loaded onto each gel lane. Electrophoresis was performed in a Vertical Slab Unit Model SE-400 (Hoeffer
Scientific Instruments) at a constant current of 10 mA, until
current was no longer measurable (*6 h). Unstained gels
were scanned using a Typhoon imager (GE Healthcare) set
in the fluorescence mode with a blue laser excitation source
(488 nm) and a 670-nm emission filter; a plastic transparency (Tektronix) was placed on top of the gel to provide
uniform background fluorescence. Gels were also scanned
with a Canon visible light scanner before and after staining
with Coomassie-blue (Gel Code blue safe, Thermo). Up to
four pigmented bands were identified and their pigmentprotein complex composition was determined from
absorption spectra of the excised bands obtained with a
DU-640 spectrophotometer (Beckman), using excised gel
containing no sample as a blank.
Proteomic analysis
Gel bands resolved in CNE were excised and fixed for at
least 30 min in a 40% methanol, 10% acetic acid solution
and subjected to in-gel digestion with trypsin, followed by
LC-MS/MS using a Dionex U-3000 nanoflow liquid
chromatography system in line with a Thermo LTQ linear
ion trap mass spectrometer, operated in the nanoLC mode
to obtain sub-femtomol sensitivity. Database analysis of
peaks generated from LTQ data was performed as described by Sleat et al. (2009) in which tandem mass spectrometry data files were searched against the NCBI
assembly of the annotated Rba. sphaeroides 2.4.1 genome
(Mackenzie et al. 2001). The data were quantified by
spectral counting, which measures protein abundance on
the basis of the number of tandem mass spectral observations for all constituent peptides (Liu et al. 2004). Spectral
count sampling statistics have been shown to have high
technical reproducibility and can be successfully applied to
relative protein abundance and differential protein
expression studies in mass label-free LC-MS/MS (Zeng
et al. 2007; D’Amici et al. 2010).
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Results
Membrane isolation from cells acclimating
to low-intensity illumination and separation of integral
pigment–protein complexes by clear native
electrophoresis
Figure 1 shows the near-IR absorption spectra of cell
extracts and membrane fractions isolated after 0, 3, and
24 h of acclimation from high (1,100 W/m2) to low
intensity illumination (100 W/m2). Only low levels of LH2
were present (LH2/LH1 molar ratio = 0.39) in the initial
exponential-phase high light cells (time = 0), with the
predominance of the RC-LH1 core complex indicated by
the LH1 absorption maximum at 875 nm and the presence
of a small band appearing at *750 nm, especially notable
in the isolated 0 h UPB membrane fraction (see also
Fig. 2b), attributable to the RC bacteriopheophytin component. LH2/LH1 was initially 2.2-fold lower in the UPB
relative to chromatophores and was maintained at 1.7- and
1.5-fold lower levels after 3 and 24 h, respectively, consistent with a role for these CM invagination sites as hotspots for initial assembly of LH1-RC cores and thereafter
as LH2 assembly sites prior to ICM vesicularization. The
gradual increase in molar ratios of LH2/LH1 resulted in a
4.3-fold higher level after 24 h in chromatophores, as
compared to 6.1-fold in the UPB. Overall, these dramatic
increases in LH2 levels demonstrated that the essential
membrane fractions can be isolated during a light-intensity
downshift to critically test the proposed mode for expansion of the functional absorption cross-section and
remodeling of the LH apparatus during acclimation to
lowered light intensities (Hunter et al. 2005).
In order to assess the assembly of the LH2 and RC-LH1
complexes within the developing ICM, a non-denaturing
electrophoresis procedure (CNE) was established for their
isolation from the purified membranes (Fig. 2). This method
was adapted from the high-resolution CNE procedure of
Wittig et al. (2007) for direct in-gel functional and fluorescence assays of well-separated mitochondrial inner membrane protein complexes. Coomassie blue, which interferes
with catalytic activity and fluorescence determinations in
blue-native electrophoresis (Schägger and von Jagow 1991),
was replaced with clear mixtures of anionic and neutral
detergents in the cathode buffer. Membrane protein solubility during electrophoresis is enhanced in this manner, and
like the Coomassie dye, a charge shift is imposed to augment
their anodic migration. Protein aggregation and band
broadening are prevented, resulting in superior resolution,
while facilitating in-gel functional assays. In the current
studies, migration of the pigment-protein complexes can be
directly visualized, and their absorption spectra can be
determined in the gel slices containing the resolved bands.
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Photosynth Res (2012) 111:125–138
Figure 2a shows that after b-OG/DOC solubilization of
chromatophores isolated from the adapting cells, four
pigmented bands were resolved: a top RC-LH1 band of
near spectral homogeneity (Fig. 2b); a bottom, spectrally
homogeneous LH2 band (Fig. 2c); and two bands of
intermediate migration containing mixtures of RC-LH1
and LH2. The upper intermediate complex is enriched in
LH2 (designated LH2–LH1), while the lower one is enriched in RC-LH1 (designated LH1–LH2). These intermediate complexes may represent detergent-protein micelles
arising from ICM regions seen in AFM where the LH2 and
LH1 complexes are associated (Woronowicz and Niederman, 2010) (see below for further evidence of authentic
LH2–LH1 associations). Relative increases in the intensity
of the two LH2-enriched bands can be seen as the low-light
acclimation process proceeds (Fig. 2a). It can be seen in
Fig. 2d that the bottom LH2 band is free of contamination
by RC and LH1 polypeptides, essentially confirming the
absorption spectrum. Importantly, this clear native gel
procedure has provided the basis for a critical proteomic
analysis of the assembly of the pigment-protein complexes
under these conditions.
Protein composition of the LH2 CNE bands
After the separation of intact pigment-protein bands by
CNE, they were excised from the gel and subjected to
direct in-gel trypsin digestion followed by LC-MS/MS as
described in ‘‘Materials and methods’’ section. It should be
emphasized that data obtained here by proteomic analysis
in the absence of mass isotope labeling, only reflect the
ability of trypsin to act at potential cleavage sites and are
therefore semiquantitative in nature. This means that proteins that are not highly abundant with a large number of
accessible cleavage sites give rise to higher spectral counts
than more abundant proteins with far fewer sites; in
extreme cases where no sites are available, no counts are
found in the resulting pivot table, viz., pufB (LH1-b
polypeptide) and Puc1A (LH2-a); however, the latter
polypeptide could be detected after chymotrypsin cleavage
(see below). Nevertheless, valid comparisons can be made
of the same protein between different gel lanes, which were
loaded with equal amounts of total protein, thereby providing valid data for protein expression studies of the
clusters of orthologous groups as described here (Fig. 3).
Differences in the number of trypsin cleavage sites and
their accessibility explain the lack of congruence between
the apparent absence of cross contamination of the LH2 gel
band by RC-LH1 core complexes as observed in the nearIR absorption spectrum (Fig. 2c) and the presence of 7% of
total spectral counts in this orthologous group cluster as
demonstrated for the 3-h LH2 bands in Fig. 3. Accordingly, the total counts for all four CNE bands analyzed in
Photosynth Res (2012) 111:125–138
A
Crude Extract
Upper Pigmented Band
0h
Absorbance
Fig. 1 a Near-IR absorption
spectra of the crude cell
extracts, UPB, and
chromatophore fractions after
isolation by rate-zone
sedimentation on sucrose
density gradients from cells
undergoing acclimation to low
light intensity. Crude extracts
represent the supernatant
material obtained from cell
lysates after French-press
treatment and centrifugation at
16,0009g. b LH2/LH1 molar
ratios calculated after spectral
deconvolution as described by
Sturgis et al. (1988)
129
Chromatophores
LH1 (B875)
800
875
850
800
875
850
3h
LH2
(B800-850)
24 h
0.3 A
800
750
850
950
750
850
950
750
875
850
850
950
Wavelength, nm
B
2
UPB
LH2/LH1, mol/mol
Chromatophores
1.5
Crude
1
0.5
0
0h
this experiment were 3,224 for the RC-LH1-PufX core
complex and 1,138 for the LH2 complex, giving at least a
2.4-fold bias for the cores, which are present at near
equimolar levels with LH2 as judged from near-IR
absorption of the 3-h chromatophores (Fig. 1). This would
reduce the actual contamination by core complex protein to
\3.0%, thereby accounting for the apparent lack of an
obvious LH1 contribution to the absorption spectrum of the
3-h LH2 band (Fig. 2c).
An analysis of the photosynthetic membrane proteome of
Rba. sphaeroides 2.4.1 by Zeng et al. (2007) identified 98
3h
24 h
proteins associated with ICM vesicles; among these, 70 were
enriched relative to the outer membrane and soluble fractions. In a nondenaturing clear gel electrophoresis study of an
n-dodecyl-b-D-maltoside solubilized ICM fraction purified
from the carotenoidless Rba. sphaeroides strain 26.1
(D’Amici et al. 2010), proteomic analysis resulted in the
identification of 52 proteins arising from 10 different bands
containing multiprotein complexes. Among these were LH2
dimers, trimers, and high molecular mass supercomplexes,
RC-LH1-PufX monomers and dimers, succinate dehydrogenase trimers and monomeric forms of ATP synthase, and
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Photosynth Res (2012) 111:125–138
Fig. 2 Separation of intact pigment-protein complexes by CNE of c
b-OG/DOC solubilized chromatophores isolated from cells undergoing acclimation to low-light intensity. a Left panel Typhoon
fluorescence scanner (GE Healthcare) profile of unstained gel
observed against a plastic film providing uniform background
emission at 670-nm; pigments absorbing at the excitation wavelength
(488 nm) appear as white images, defining the positions of the
original pigmented gel bands. Note marked increases in the density of
the bottom (LH2) band and upper intermediate (LH2–LH1 band) as a
function of acclimation time. Right panel Scan of a typical unstained
gel lane obtained with a visible light scanner (Canon). Approx.
molecular masses of the pigmented bands are shown at right, obtained
from plot of Rf versus Mr for native electrophoresis high molecular
weight calibration kit (GE Healthcare) as described in Table 1. b,
c Absorption spectra determined directly on indicated gel slices. In
the top (RC-LH1) band, note the presence of the RC bacteriopheophytin absorption band at *750 nm and the RC monomeric BChl
band at 803 nm. The blue shift of the LH1 B875 absorption band to
872 nm and the shoulder on the blue side indicate some contamination with LH2 (LH2:LH1 molar ratio = 0.21 determined as described
by Sturgis et al. 1988); however, it has also been possible to isolate
the RC-LH1 band in a spectrally homogeneous form (Woronowicz
and Niederman 2010). Note the high level of spectral purity of the
LH2 complex in the bottom band (maxima at 849 and 799 nm;
LH2:LH1 molar ratio = [300:1). Comparison of this spectrum with
that of the 24-h crude cell extract (Fig. 1a) indicates that during the
isolation procedures, some diminution in the absorbance of the labile
B800 band has occurred. Also noteworthy are the differences in
carotenoid contents of the complexes, reflecting the BChl:carotenoid
molar ratios near 1.0 reported for LH1 and *2.0 for LH2, as well as
the blueshift in the position of the redmost vibrational splitting of
LH1-bound carotenoid absorption (the S2(1Bu?) state of spheroidene)
in comparison to that of LH2, with maxima at *507 and 510 nm,
respectively (Hunter et al. 1988). From near-IR absorption spectra of
the upper and lower intermediate bands, ratios LH2:LH1 ratios of
2.39–2.77 and 0.63–0.76, respectively, were obtained. d Portion of
silver-stained gel of CNE bottom bands subjected directly to SDSpolyacrylamide gel electrophoresis, showing an absence of the RCLH1 apoproteins, essentially confirming the observed absorption
spectra. The band above the LH2-a band apparently represents the
11.4-kDa hypothetical protein RSP6124; levels of RSP6124 were
correlated with LH2 levels after purification of the chromatophore
band by two-phase partitioning, accounting for 5% of clusters of
orthologous groups (COGs) at 3 h and 12% at 24 h (Woronowicz and
one of these (RSP1760) demonstrated that the RSP1760 gene
Niederman 2010)
the cytochrome bc1 complex. In both of these studies, a
considerable number of proteins of unknown function were
encountered, and an in-frame deletion of the gene encoding
product is essential for both photosynthetic growth and ICM
formation (Zeng et al. 2007). This has served to illustrate that
these proteomic strategies show considerable promise for
identification of new ICM proteins with unique functions in
maintaining and optimizing photosynthesis.
Table 1 Apparent aggregation states of pigment-protein complexes as estimated from electrophoretic migration in clear native electrophoresis
Band
Designation
Apparent Mr (910-3)a
Aggregation stateb
Aggregate identity
Top
Upper intermed.
RC-LH1
LH2–LH1
748
600
2.31
1.02
[RC-LH1-PufX]2
[RC-LH1-PufX][LH2]2
Lower intermed.
LH1–LH2
463
1.02
[RC-LH1-PufX][LH2]
Bottom
LH2
375
2.85
[LH2]3
a
Obtained from an exponentially fitted plot of Rf versus Mr for native electrophoresis high molecular maker proteins run in a separate gel lane
(molecular masses of 669, 440, 232, 140 and 67 kDa)
b
Calculated using monomer molecular weights based on polypeptide sequences and pigment contents. For RC-LH1-PufX, molecular mass of
324.0 kDa included RC Fe and two ubiquinone molecules, the RC-H, -M, and -L polypeptides (101.8 kDa total/monomeric RC), 14 LH1-a and -b
subunits (molecular mass = 213.1 kDa) and PufX (9.1 kDa). For LH2, the molecular mass of 131.4 included nine LH2-a and -b subunits
123
Photosynth Res (2012) 111:125–138
131
Fig. 3 Proteomic analysis of
CNE bottom (LH2) band of
chromatophores isolated from
cells undergoing acclimation to
low light intensity. Distribution
of spectral counts after in-gel
trypsin digestion of
chromatophore LH2 band
isolated from cell samples taken
3 and 24 h after the light
intensity downshift. The
distributions shown are for
COGs (Tatusov et al. 1997), in
which the usual energy
production and conversion
category has been divided into
subgroups to account for the
distinct metabolic capabilities
unique to
photoheterotrophically grown
Rba. sphaeroides
In this study, the LH2 band of chromatophores isolated
from the ICM undergoing acclimation from high to low
intensity illumination (Fig. 3), showed a nearly threefold
increase in the relative spectral counts attributable to LH2
between 3 and 24 h of acclimation, while RC-LH1
remained at 7% of the total counts obtained for the clusters
of orthologous groups. In addition, a large array of comigrating proteins was revealed and in particular, the cytochrome bc1 complex and several F1FO-ATP synthase
subunits gave unexpectedly high counts, given the inability
to detect these components in AFM (Bahatyrova et al. 2004,
Sturgis et al. 2009). Significant levels of general membrane
assembly factors were also encountered, as well as
numerous hypothetical proteins of unknown function,
including high spectral counts for the RSP6124 that were
maintained throughout the acclimation process. It is also
noteworthy that a comparison of the 3- and 24-h LH2 bands
showed a twofold reduction in spectral counts attributable
to transport proteins, while those of the cytochrome bc1
complex increased by threefold. Thus, as expected, a higher
level of these permease components, which serve as CM
markers, are present in the vesicular ICM early during lowlight acclimation and are diluted out by LH and RC protein
as membrane remodeling proceeds. On the other hand,
cytochrome bc1 components catalyzing light-driven cyclic
electron flow become preferentially localized in the
remodeled vesicular ICM during the acclimation process,
conditions under which the levels of the electron contributing RC-LH1 complex undergoes a [9-fold increase.
Proteomic analysis reveals distinct light regulation
of the puc1BAC and Puc2BA operons
In addition to the puc1BAC operon, which encodes the
respective LH2-b (51 amino acid residues) and LH2-a apoproteins (54 residues), as well as the PucC polypeptide
involved in LH2 assembly, the Rba. sphaeroides genome
contains a second puc operon, designated as puc2BA (Zeng
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132
et al. 2003). These investigators also reported that while the
LH2-b polypeptide encoded by the puc2B structural gene
was assembled into LH2 complexes, neither the putative fulllength 263-residue Puc2A gene product, nor its N-terminal
48-amino acid Puc1A homolog, appeared in assembled LH2
complexes. In contrast, the present proteomics data demonstrate that the Puc2A polypeptide is actually a major component of LH2-containing complexes isolated in CNE
(Fig. 4). Moreover, this LH2-a component was the second
most abundant protein among the [170 proteins that gave
rise to significant spectral counts after trypsin digestion of the
CNE gel bands, and significant levels were also encountered
in LH2 bands digested with chymotrypsin (Fig. 4a).
Proteomic analysis of the LH2 CNE band showed a
predominance of the Puc2A polypeptide in LH2 complexes
formed at high light intensity, as reflected in chromatophores isolated at the outset (0 h) and 3 h into the shift
from high to low light (Fig. 4a), which is followed by an
enrichment in Puc1B levels after 24 h. As seen in the relative distribution of spectral counts (Fig. 4a, bottom panel),
this amounted to [2.5-fold increase in Puc1B spectral
count levels between 3 and 24 h that was accompanied by a
nearly 1.8-fold decline in those of Puc2A. These data
provide support for the suggestion by Zeng et al. (2003)
that the puc1BAC operon is under more stringent light
control than the puc2BA operon, thought to reflect differences in the puc2 upstream regulatory region. Unlike
trypsin, chymotrypsin digestion resulted in cleavage of
Puc1A and also revealed high levels of both Puc1A and
Puc1B along with lower levels of Puc2A in the 24-h chromatophore LH2 band (Puc1AB/Puc2A = 5.6) (Fig. 4a). Note
also that the presence of some Puc1B in the 3-h chromatophore preparation, which is apparently due to the reported
dependence of puc2BA expression on the expression of all
three puc1BAC genes (Zeng et al. 2003).
Also noteworthy are the similarities in the ratios of the
Puc1/Puc2 polypeptides for the 24-h UPB and chromatophore bands (1.6 and 1.1 for trypsin digestion and 5.0 and
5.6 for chymotrypsin, respectively), showing that LH2
assembly occurs in both of the distinct inner membrane
domains that give rise to these membrane fractions. This is
also supported by the similarities in the increases in molar
ratios of LH2/LH1 after 24 h (6.1- and 4.3-fold in the UPB
and chromatophores, respectively) (Fig. 1). Figure 4b
demonstrates that during ICM induction under semiaerobic
conditions, or during acclimation from high-intensity illumination to indirect diffuse light, no differential synthesis of
Puc1A and Puc1B polypeptides has apparently occurred
(Puc1B/Puc2AB ratios were 0.48 after 48 h of induction and
0.31 after 11 days of acclimation to diffuse light, respectively). This indicates that light regulation of the pucAB
operons is confined to rapidly dividing cells acclimating to
physiologically relevant downshifts in light intensity.
123
Photosynth Res (2012) 111:125–138
Fig. 4 Proteomic analysis of LH2 polypeptides in CNE bands of c
membrane fractions isolated from cells undergoing ICM development.
Top panels in Figs. 4a–c show overall spectral count distributions,
while bottom panels show % distribution of spectral counts. a Low-light
acclimation profiles of LH2 polypeptides in bottom (LH2) band
resolved from chromatophores and UPB. Because of the limited
spectral counts in the 0-h chromatophores, it was not possible to
calculate their % distribution. Note the 6.0-fold increase in spectral
counts of Puc1B between 3 and 24 h as compared to the only 1.5-fold
increase for Puc2A. In addition to revealing the presence of all four LH2
polypeptides encoded by the puc1BAC and pucBA operons through
digestion with both trypsin and chymotrypsin, trypsin digestion of the
24-h UPB LH2 gel band yielded a single peptide sequence arising from
the putative Puc1C gene product, involved in the assembly of LH2
polypeptides (not shown). b LH2 polypeptide profiles in bottom (LH2)
band resolved from chromatophores isolated during ICM induction at
low aeration and during acclimation from high-intensity illumination to
indirect diffuse light as described by Woronowicz et al. (2011). c Lowlight acclimation profiles of LH2 polypeptides in CNE intermediate
bands resolved from chromatophores
Figure 4c shows the Puc1/Puc2 ratios arising from LC/MSMS of the CNE bands of intermediate migration, after
digestion with trypsin. The Puc1B/Puc2AB for the LH2enriched LH2–LH1 upper intermediate band was virtually
unchanged between 3 and 24 h following the light-intensity
downshift (0.13 and 0.17, respectively), despite a [10-fold
increase in LH2 cellular levels over this time period. These
results have considerable bearing on the interpretation of the
origins of the LH2-containing complexes isolated by CNE
(Fig. 2), insofar as these essentially unchanged LH2 polypeptide ratios suggests that the LH2–LH1 band arises from
regions of LH1–LH2 associations, thought to be laid down
mainly during high light growth. These appear in AFM topographs as rows of aligned dimeric RC-LH1 core complexes
interspersed with narrow rows of LH2 (Fig. 5a) (Bahatyrova
et al. 2004; Hunter et al. 2005). On the other hand, the origin of
the bottom LH2 band with its increased levels of new Puc1BA
polypeptides at 24 h (Fig. 4a) can be attributed largely to the
LH2-only domains that arise when LH2–LH1 associations are
complete (Fig. 5b), which represent the light-responsive LH2
complement formed as the cells adapt to low intensity illumination. In contrast, for the LH1-enriched LH1-LH2 lower
intermediate band (Fig. 4c), the Puc1B/Puc2AB ratio
increased from 0.21 at 3 h to 0.58 at 24 h, suggesting that this
CNE band arises in part from regions of RC-LH1 core-LH2
interactions at the outer RC-LH1 edges (Fig. 5a).
Discussion
Multigene families of puc operons
The existence of a second operon encoding LH2 polypeptides in Rba. sphaeroides was not surprising in view of the
presence of multiple puc operons in a variety of other purple
bacteria, including Rhodopseudomonas palustris in which
Photosynth Res (2012) 111:125–138
133
B Additional ICM induction protocols
A Low light adaptation
Bottom band – LH2
160
Puc1A – LH2-
150
Puc2A – LH2-
125
Puc1B – LH2Puc2B – LH2-
100
Chymotrypsin
75
50
25
0
80
Low O 2 induction
Puc2A – LH2-
+ C-term. ext.
Puc1B – LH2-
140
+ C-term. extension
Spectral counts
Spectral counts
175
Puc2B – LH2-
120
100
Diffuse light
80
60
40
20
0h
3h
24 h
Chromatophores
24 h
24 h
UPB Chrom
0
24 h
UPB
Bottom band – LH2
60
24 h
48 h
Chromatophores
11 d
Bottom band – LH2
50
60
Spectral counts (%)
Spectral counts (%)
70
50
40
30
20
10
0
0h
3h
24 h
Chromatophores
24 h
24 h
UPB Chrom
24 h
UPB
40
30
20
10
0
24 h
48 h
Chromatophores
11 d
C Low light adaptation -- Chromatophores
150
LH2-LH1
Puc2A – LH2-
Spectral counts
125
+ C-terminal extension
Puc1B – LH2Puc2B – LH2-
100
LH1-LH2
75
50
25
0
3h
24 h
0h
3h
24 h
Low light adaptation -- Chromatophores
Spectral counts (%)
100
LH2-LH1
LH1-LH2
75
50
25
0
3h
24 h
0h
3h
24 h
123
134
Photosynth Res (2012) 111:125–138
LH2 ring
A
LH2-LH1 band
50 nm
B
25 nm
C
--RC-LH1
[RC-LH1-PufX] 2
--LH2-LH1 [RCLH1-PufX][LH2] 2
RC-LH1
band
--LH1-LH2 [RCLH1-PufX][LH2]
LH2-only
domain
LH1-LH2 band
--LH2 [LH2] 3
LH2 band
LH2 ring
Fig. 5 Model for proposed origins of CNE gel bands. a Local
arrangement of photosynthetic complexes in an ICM patch showing
highly ordered linear arrays of the RC-LH1 core complex with LH2
rows interspersed between (Bahatyrova et al. 2004). Existing
structural data were used to model AFM topograph of cytoplasmic
surfaces of ICM vesicle patch. A dimeric RC-LH1 complex ([RCLH1-PufX]2) is outlined in red, which is proposed to be the source of
the RC-LH1 band. The LH2–LH1 (LH2-enriched upper intermediate)
band ([RC-LH1-PufX][LH2]2), outlined in orange, is shown arising
from a monomeric RC-LH1 complex in association with two LH2
rings, while the LH1–LH2 (RC-LH1-enriched upper intermediate)
band ([RC-LH1-PufX][LH2]), outlined in green, is shown arising
from a monomeric RC-LH1 complex in association with a single LH2
ring. b Tapping mode AFM topograph showing cytoplasmic surface
of ICM vesicle patch adsorbed onto mica in a liquid cell (Bahatyrova
et al. 2004; Sturgis et al. 2009). The blue circle outlines an LH2-only
domain from which the LH2 band is thought to arise in an [LH2]3
aggregation state. c Scan of 24-h unstained gel lane. The molecular
masses for the gel bands shown in Fig. 2 support the aggregation
states of each of the four bands as depicted here
structural genes are present for encoding five ab-apoprotein
pairs (Tadros and Waterkamp 1989; Tadros et al. 1993; Gall
and Robert 1999), Rbl. acidophilus, capable of encoding at
least four different ab-apoproteins (Gardiner et al. 1996) and
Phaeospirillum molischianum in which evidence for multiple puc operons has also been reported (Mascle-Allemand
et al. 2010). In these organisms, the various puc structural
genes are expressed differentially in response to changes in
light intensity, which in Rps. palustris is manifested in the
formation of a nonameric B800-850 LH2 complex at high
light intensity and an octameric 800-nm absorbing LH4
complex under low light (Evans et al. 1990; Scheuring et al.
2006).
The peripheral LH4 complex (Hartigan et al. 2002)
which is regulated by a specific phytochrome (Evans et al.
2005; Giraud et al. 2005), is present under high-intensity
illumination at approximately the same level as the nonameric LH2 complex, while a 10-fold increase in the LH4
level relative to LH2 is found under low light. Recently,
evidence obtained by single molecule spectroscopy by
Brotosudarmo et al. (2009) has suggested that a B820 band
is also present in the purified low-light peripheral antenna
protein of Rps. palustris. These individual low-light
antenna complexes arise as a result of a heterogeneous
polypeptide composition provided by the multigene family
of puc operons, resulting from the assembly of LH2 rings
from polypeptides encoded from the distinct genes of the
different operons (Tharia et al. 1999). Moulisová et al.
(2009) explained the splitting of long-wavelength exciton
bands by a heterogeneous composition of LH2 apoproteins
that provides some of the BChls in the B850 ring with
B820-like site energies, accounting for the disorder in the
LH2 structure, as reflected in a strong inhomogeneity of
B850 excitons in the low-light samples.
In Rbl. acidophilus, besides the structurally characterized
LH2 complex (Papiz et al. 2003), an additional peripheral
antenna with absorption bands at 800 and 820 nm (LH3) is
formed when grown under low-light conditions (Bissig et al.
1988; Gardiner et al. 1993, McLuskey et al. 2001). Using
Psh. molischianum, Mascle-Allemand et al. (2010) showed
that octameric LH2 rings (bands at 802 and 849 nm) (Koepke et al. 1996) form the predominant pigment-protein
complex in the normal-light membranes, while in cells
adapting to low-light, an LH3 complex clearly predominated, ultimately replacing LH2. Mascle-Allemand et al.
(2010) also demonstrated antenna mixing in Psh. molischianum, in which this modulation can result in a complete shift
between the production of octameric LH2 complexes
absorbing maximally near 850 nm to an LH3-type complex
with a long-wavelength absorption band near 820 nm. The
ab-apoproteins making up the individual LH2 rings contained polypeptides that were derived from different operons, demonstrating a lack of strict coupling of polypeptide
synthesis, cofactor addition and insertion into the membrane.
Since the LH3-type complex serves to increase the spectral
range of solar energy capture, polypeptide chain mixing
could be important in maintaining photosynthetic efficiency
during acclimation to low light intensity.
123
Possible distinct roles for Puc1 and Puc2 LH2
complexes
It is tempting to speculate on a physiological role for the
low-light intensity responsive differential synthesis of Puc
Photosynth Res (2012) 111:125–138
polypeptides in Rba. sphaeroides, in which a predominantly Puc2 LH2 complex would function at high light
intensity and a predominantly Puc1 LH2 complex would be
required for optimal photon collection and excitation
energy transfer under low intensity illumination. However,
this awaits proteomic analysis of a functional, highly
purified LH2 protein preparation isolated from high light
cells, since the Puc2A peptide detected here arose from the
215 amino acid residue C-terminal extension. This large
protein fragment does not contain any apparent membrane
spanning regions, and it is possible that it is not part of
the functional complex and instead, has arisen from in vivo
enzymatic cleavage, representing an adventitious attachment
to the CNE LH2 band.
The recent study of Wang et al. (2009), however, has
demonstrated that when the puc2BA genes were amplified
and cloned into an appropriate plasmid vector under control
of the puc promoter, both the Puc2A and Puc2B polypeptides are expressed normally. The puc2BA-encoded polypeptides were assembled into a membrane-associated Puc2BA
LH2 complex in which a large puc2A-encoded polypeptide
was present. The puc2BA genes were also expressed when
integrated into the Rba. sphaeroides genome and Puc1C was
necessary for synthesis of LH2 complexes from puc2BA.
The resulting LH2 was spectroscopically distinct from the
Puc1BA LH2 with a blue shifted B850 absorption band at
846 nm but the functional activity of the Puc2BA LH2
complex, especially under high light intensity, remains to be
determined. Nevertheless, these results make it likely that in
the present study, a Puc2BA complex is indeed formed in
our wild-type strain and that it is this authentic complex that
predominates under high light intensity. It is noteworthy that
in the Puc2A-encoded polypeptide, the essential conserved
Tyr residues (Tyr44 and Tyr45) are present in the conserved
48-residue N-terminal region. These residues have been
shown to be involved in binding the 2-acetyl carbonyl
groups of the B850 BChls (Fowler et al. 1992), suggesting
that the Puc2BA complex can be assembled in a fully
functional state.
In contrast to this study and the report of Wang et al.
(2009), Zeng et al. (2003) reported that the Puc2A-encoded
polypeptide was not found in assembled LH2 complexes.
For these studies, the PucA and PucB polypeptides were
tagged by constructing PhoA fusion proteins and expressed
in a puc1BAC deletion strain, which did not produce LH2
complexes. Fusion proteins containing full-length PucB
and PucA polypeptides were formed, indicating that Puc2A
had not undergone processing. While expression of
puc2BA did not require the puc1BAC operon, and Puc2B
and Puc2A passed through the membrane in an apparently
normal manner, immunoprecipitation with an anti-PhoA
antibody showed that in a puc1 operon background, LH2
spectral complex were formed with the chimeric
135
polypeptides containing Puc2B and the N-terminal 48
amino acid puc1A orthologue encoded by puc2A, but not in
the case of the chimera with the full length Puc2A polypeptide. Moreover, the LH2 complex was not assembled
when the C-terminal Puc2A extension was added to
Puc1A, suggesting that it must be removed for LH2-a
assembly. The differences between these results and those
of Wang et al. (2009) in which Puc2B and the full length
puc2A-encoded polypeptides were assembled into a membrane-associated LH2 complex are presently not
understood.
Regulation of Puc polypeptide expression
in Rba. sphaeroides
In the initial puc operon expression studies (Kiley et al.
1988: Lee et al. 1989), 0.5- and 2.3-kb puc-specific transcripts were detected with respective abundance levels of
up to 25:1. Both of these transcripts started at the same 50 end, 117 nucleotides upstream of the pucB start codon and
their expression was regulated by oxygen tension and light
intensity. After deletion of the puc1BA genes (Lee et al.
1989), a highly homologous 1.1- to 1.3-kb transcript
appeared. In this pucBA- strain, no LH2 complexes were
assembled from the puc2B and puc2A transcripts that were
formed, which can now be explained by the requirement
for Puc1C in the assembly of the Puc2BA polypeptides
(Zeng et al. 2003). The mRNA from the puc2B start codon
to the termination puc2A codon has a length of *963 bp,
substantially longer than the corresponding length of the
puc1BA mRNA (337 bp), accounting for the size of the
1.1- to 1.3-kb transcript detected by using a homologous
puc1BA probe.
Putative regulatory regions upstream of the puc2B gene
include two PpsR binding motifs located at positions
similar to the same sequence motifs upstream of puc1B. In
addition, an FnrL binding sequence was located upstream
in a unique region closer to the puc2B start codon than for
puc1B (Zeng et al. 2003). This has suggested that like the
puc1 operon, expression of the puc2 operon is under regulatory control by both PpsR and FnrL. PpsR forms part of
the AppAPspR antirepressor-repressor system in which
AppA integrates both redox and light signals and serves as
a blue-light photoreceptor, in addition to functioning as a
redox sensor of the quinone pool redox state (Masuda and
Bauer 2002; Braatsch et al. 2002). FnrL is a positive,
global anaerobic regulator required for enhanced expression of puc and selective structural genes encoding
oxidoreductases and enzymes of pigment biosynthesis
(Zeng and Kaplan 2001). It was suggested by Zeng et al.
(2003) that the difference in location of the FnrL sequence
upstream of puc1BAC may be responsible for the more
123
136
stringent light control of this operon than that seen for the
puc2BA operon.
Puc polypeptide composition of LH2-containing CNE
gel bands supports structural model for their origin
in native ICM
Prompted by the elucidation of the supramolecular surface
arrangement of ICM vesicles by AFM (Bahatyrova et al.
2004) together with membrane fractionation and radiolabeling studies of BChl insertion into developing pigmentprotein complexes, Hunter et al. (2005) have proposed that
during acclimation to lowered light intensity, new LH2
rings initially cluster between expanding linear arrays of
dimeric LH1-RC core complexes at membrane invagination sites (Fig. 5a). As the membrane matures further and
these regions become fully occupied with rows of LH2
rings, ICM domains consisting exclusively of LH2 are
formed by the packing of newly synthesized complexes
into membrane regions that represent the light-responsive
peripheral antenna complement (Fig. 5b). For the isolation
of the developing pigment-protein complexes by Hunter
et al. (2005), the isolated UPB and chromatophore membranes were fractionated into their constituent LH2, LH1,
and RC components by solubilization in lithium dodecyl
sulfate at 4°C in the dark, followed by polyacrylamide gel
electrophoresis at this temperature (Broglie et al. 1980).
This procedure was sufficiently gentle to preserve regions
of contact between LH2 and LH1, giving rise to series
complexes of intermediate migration, containing variable
LH2:LH1 stoichiometries. In contrast, clear native electrophoresis as described here affords a less complex native
electrophoresis procedure to assess membrane regions of
putative LH2/LH1 associations, insofar as only two intermediate complexes appear, an upper one enriched in LH2
and lower LH1-enriched one.
Accordingly, from the relative distribution of Puc1- and
Puc2-encoded LH2-a and -b polypeptides during acclimation to reduced light intensity, it is possible to make a
more explicit interpretation of the membrane origins of the
CNE gel bands. Because the Puc2AB/Puc1B polypeptide
ratio was largely unchanged in the LH2-enriched LH2–
LH1 upper intermediate band during the light intensity
downshift (Fig. 4c), it is proposed that this band arises
from early LH2–LH1 association represented by the narrow rows of LH2 rings between the linear arrays of dimeric
LH1-RC core complexes (Fig. 5a). Since these are thought
to represent the first regions to be assembled, they do not
subsequently exchange with the new LH2 complexes
formed during the remainder of the membrane remodeling
process. On the other hand, for the LH1-enriched LH1–
LH2 lower intermediate band, the nearly three-fold
increase in the Puc1B/Puc2AB ratio (Fig. 4c) suggests that
123
Photosynth Res (2012) 111:125–138
this band arises from isolated regions of dimeric RC-LH1
core-LH2 associations at the outer edges of the rows of
dimeric RC-LH1 core complexes (Fig. 5a). Finally, suggested origins for the RC-LH1 top and the bottom LH2
bands are also presented in Fig. 5b.
Acknowledgments We thank Prof. Peter Lobel and Dr. Haiyan
Zheng of the Center for Advanced Biotechnology and Medicine,
University of Medicine and Dentistry of New Jersey, for conducting
the proteomics analysis. This work was supported by the Aresty
Research Center for Undergraduates at Rutgers University (OBO,
HCS, JTL) and U. S. Department of Energy Grant No. DE-FG0208ER15957 from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science
(RAN).
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