MINIREVIEW
Biogenesis of the cyanobacterial thylakoid membrane system ^ an
update
Jörg Nickelsen1, Birgit Rengstl1, Anna Stengel1, Marco Schottkowski1, Jürgen Soll2 & Elisabeth Ankele2
1
Molekulare Pflanzenwissenschaften, Biozentrum LMU München, Planegg-Martinsried, Germany; and 2Biochemie und Physiologie der Pflanzen,
Biozentrum LMU München, Planegg-Martinsried, Germany
Correspondence: Jörg Nickelsen,
Molekulare Pflanzenwissenschaften,
Biozentrum LMU München, Großhaderner
Str. 2-4, 82152 Planegg-Martinsried,
Germany. Tel.: 10049 89 2180 74773; fax:
10049 89 2180 997 4773; e-mail:
[email protected]
Received 7 July 2010; revised 30 July 2010;
accepted 2 August 2010.
Final version published online 10 September
2010.
Abstract
Current molecular analyses suggest that initial steps of the biogenesis of cyanobacterial photosystems progress in a membrane subfraction representing a
biosynthetic center with contact to both plasma and thylakoid membranes. This
special membrane fraction is defined by the presence of the photosystem II
assembly factor PratA. The proposed model suggests that both biogenesis of
protein complexes and insertion of chlorophyll molecules into the photosystems
occur in this intermediate membrane system.
DOI:10.1111/j.1574-6968.2010.02096.x
MICROBIOLOGY LETTERS
Editor: Hermann Heipieper
Keywords
Synechocystis; membrane biogenesis;
photosystem II; PratA; Pitt.
Introduction
Cyanobacteria represent the phylogenetic ancestors of chloroplasts from present-day plants and, similar to those, they
contain three major differentiated membrane systems. These
include the outer membrane and the inner or plasma
membrane (PM), which, together with the intervening
periplasm and the peptidoglycan layer, form the cellular
envelope. Interior to the PM is the thylakoid membrane
(TM) system representing the site of the photosynthetic
light reactions coupled to ATP and NADPH generation. All
three membrane systems differ from one another with
regard to their pigment, lipid and protein composition
(Norling et al., 1998; Wada & Murata, 1998). This observation provokes the following questions: Where is TM synthesis initiated in cyanobacteria? How is specificity between
the different membranes achieved and maintained? And
how are these processes organized at the molecular level?
Two excellent reviews have recently summarized the possible
models and key questions of TM biogenesis, which are
controversially discussed (Liberton & Pakrasi, 2008; Mullineaux, 2008 and references therein). In brief, three different
FEMS Microbiol Lett 315 (2011) 1–5
scenarios can be envisioned. (1) Protein, lipid and pigment
synthesis occurs directly on pre-existing TMs. (2) The
components are synthesized and assembled in specialized
thylakoid regions. (3) Initial production of polypeptides and
assembly of protein/pigment complexes occur at the PM,
and these precomplexes are transferred to the thylakoids via
an unknown way (Fig. 1).
Scenario 1 appears rather unlikely, because ultrastructural
cryo-electron microscopy data clearly show that TM layers are
essentially devoid of ribosomes (van de Meene et al., 2006).
This suggests that protein synthesis, and thus biogenesis, does
not occur in direct association with the photosynthetically
active thylakoids. However, ribosome clusters are observed
close to the PM and near TM structures that extend into the
central cytoplasm, favoring models 2 and/or 3 (van de Meene
et al., 2006). Furthermore, TMs appear to converge on the
PM at specific sites (Fig. 2). These convergence sites have been
speculated to eventually mark so-called thylakoid centers,
where TM biogenesis is initiated (van de Meene et al., 2006).
It is still under debate whether at these regions permanent or
transient fusions between PM and TM occur. If so, these
would allow the transfer of lipids and proteins to the
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2
developing TM resembling the situation found in purple
bacteria such as Rhodospirillum rubrum (Collins & Remsen,
1991; Liberton et al., 2006; van de Meene et al., 2006).
Here, we aim at incorporating some very recent findings of
membrane fractionation studies of the model organism
Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803)
into the various abovementioned scenarios. We propose a
novel working model combining scenarios 2 and 3 with TM
Fig. 1. Models for TM biogenesis. Synthesis and assembly of photosystem components occur on (a) pre-existing thylakoids or (b) in
specialized TMs (STM). Alternatively, the PM represents the site of early
photosystem biogenesis steps (c), subsequently precomplexes are transported to the TM via (1) transient connecting regions or (2) vesicle
transport.
J. Nickelsen et al.
convergence sites marking a membrane subfraction with
contact to both the PM and the TM. These sites possibly
represent the regions at which protein/pigment complexes are
assembled and incorporated into photosynthetic membranes.
Protein synthesis/assembly
Three major membrane complexes constitute the basic
apparatus of TMs mediating photosynthetic electron flow,
i.e. photosystem II (PSII), the cytochrome b6f complex and
photosystem I (PSI). PSII functions as a water-plastoquinone oxidoreductase which, in cyanobacteria, consists of 20
protein subunits, 35 chlorophyll a (chl a) molecules and
several additional cofactors including the manganese cluster
catalyzing photosynthetic water splitting (Nelson & BenShem, 2004). PSI comprises only 12 subunits, approximately
80 chlorophylls as well as Fe–S clusters and phylloquinones
(Nelson & Ben-Shem, 2004). While the structures of these
molecular machines have recently been well established
(Stroebel et al., 2003; Ferreira et al., 2004; Loll et al., 2005;
Amunts et al., 2007), to date, only limited information is
available on the molecular details of their biogenesis (Nixon
et al., 2010).
Earlier work based on membrane fractionation studies
initially suggested that precomplexes of both photosystems
are assembled within the PM and not the TM in the
cyanobacterium Synechocystis 6803 (Zak et al., 2001). Using
a combination of sucrose density centrifugation and aqueous two-phase partitioning, protein components of the
core reaction center of PSII (D1, D2, Cyt b559) as well as of
PSI (PsaA and PsaB), were identified in the PM, whereas
more extrinsic proteins such as the inner antenna protein
CP47 of PSII were found in TM preparations only. In
addition, PSII biogenesis factors, such as the D1 C-terminal
protease CtpA or the PSI assembly factors Ycf3 and Ycf4,
were mainly or exclusively detected in the PM (Zak et al.,
2001). Together with the finding that the PM-localized core
complexes contain chlorophyll molecules and can perform
single light-induced charge separations, these data strongly
suggest that the photosystem core complexes found in the
Fig. 2. Ultrastructure of Synechocystis 6803.
Electron micrographs of a Synechocystis
6803 cell (A, scale bar = 200 nm). Sites of TM
convergence are shown at higher magnification
(a, scale bar = 100 nm).
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FEMS Microbiol Lett 315 (2011) 1–5
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Cyanobacterial thylakoid membrane biogenesis
PM, or a specialized section of it, exist in a preassembled
state (Keren et al., 2005; Srivastava et al., 2006).
Further support for the idea that at least PSII biogenesis
begins at non-TM sites was obtained during analysis of the
PratA protein from Synechocystis 6803 (Klinkert et al., 2004).
PratA consists of nine consecutive tetratricopeptide repeat
(TPR) units, a motif that is known to mediate protein–protein interactions. Thereby, it could form a bridge connecting
multiple proteins and serve as a scaffold factor for correct
assembly of PSII (Schottkowski et al., 2009a). PratA directly
interacts with the C-terminus of the D1 reaction center
protein of PSII, and its inactivation affects the C-terminal
processing of D1, an early step of PSII biogenesis. This D1
maturation occurs in almost all photosynthetic organisms,
and it is required for the subsequent docking of the subunits
of the oxygen-evolving complex to the lumenal side of PSII.
Most intriguingly, PratA was shown to be a soluble protein
located in the periplasm, which forms part of a 200 kDa
complex of an as yet unknown composition and function
(Fulda et al., 2000; Klinkert et al., 2004; Schottkowski et al.,
2009a). However, a minor fraction (10–20%) of PratA was
found to associate with membranes in a D1-dependent
manner. Cellular fractionation experiments using two consecutive sucrose gradients revealed that the membranebound PratA is apparently not associated with either the
PM or TMs, but co-sediments with an intermediate membrane subfraction, which was therefore named PratAdefined membrane (PDM) subfraction (Schottkowski et al.,
2009a). Albeit the different density of PDMs as compared
with that of PMs, it cannot be ruled out that PDMs might be
identical to previously described specialized PM subregions,
in which PSII subunits tend to accumulate (Srivastava et al.,
2006). Membrane fractions resembling PDMs with regard to
their density have already been observed in earlier studies,
where they have been postulated to be linked to so-called
thylakoid centers (Hinterstoisser et al., 1993). Based on
electron microscopic analyses, thylakoid centers were initially described in some cyanobacteria as tubular structures
found at the inner face of the PM, at points where thylakoids
extend projections into the cytoplasm (Kunkel, 1982).
Recently, this idea was revisited based on a more detailed
cryo-electron tomography analysis in Synechocystis 6803
(van de Meene et al., 2006).
Interestingly, PratA inactivation and, thus, defective PSII
assembly leads to a significant accumulation of the pD1
precursor protein in PDM fractions (Schottkowski et al.,
2009a). This suggests that PratA function is required for
efficient membrane flow from PDMs to TMs, underlining
the role of PDMs for PSII reaction-center assembly.
Interestingly, related ‘biogenesis regions/centers’ have
recently been observed in the eukaryotic green alga Chlamydomonas reinhardtii, where they are formed by membranes
surrounding the pyrenoid structure of the chloroplast (UniFEMS Microbiol Lett 315 (2011) 1–5
acke & Zerges, 2007). This might indicate an evolutionary
conservation of the molecular principles that underlie TM
biogenesis.
Chlorophyll synthesis
Photosynthesis requires the absorption of light, which is
mediated by photoactive pigments, for example chlorophylls. In chloroplasts of Arabidopsis thaliana, the synthesis
of chlorophyll was described to occur in several plastidic
subcompartments (Eckhardt et al., 2004). While early steps
in synthesis, i.e. the conversion of glutamate to 5-aminolevulinic acid, occur in the chloroplast stroma, the enzymes
required for later steps are associated with the inner
envelope membrane or the TM (Fig. 3). These membraneattached enzymes include the NADPH-protochlorophyllide
oxidoreductase (POR) and the chlorophyll synthase (CS),
which catalyze the reduction of protochlorophyllide a
(pchlide a) to chlorophyllide a (chlide a) and the subsequent
generation of chl a, respectively. Similar to the situation in
higher plants, previous studies revealed that cyanobacterial
chlorophyll biosynthesis also underlies a spatial organization (Peschek et al., 1989; Eckhardt et al., 2004). In
Synechococcus elongatus 7942 (formerly called Anacystis
nidulans), pchlide a and chlide a accumulate in PM preparations and cannot be detected in the TM (Peschek et al.,
1989). Moreover, in Synechocystis 6803, highest chlorophyll
precursor concentrations were found in a membrane fraction suggested to represent the abovementioned thylakoid
center fraction resembling PDMs (Hinterstoisser et al.,
1993). As mentioned, photosynthetic precomplexes already
contain chlorophyll molecules, suggesting that not only the
later steps in chlorophyll synthesis but also the insertion of
pigments occur at the protein assembly sites associated with
the PM or PDMs (Keren et al., 2005).
Fig. 3. Scheme of chlorophyll synthesis in chloroplasts of higher plants
[according to Eckhardt et al. (2004)]. Early steps in chlorophyll biosynthesis occur in the stroma, whereas later steps are performed by enzymes
associated with the thylakoid or inner envelope membranes (EM). For
further details, see text. Glu, glutamate; ALA, 5-aminolevulinic acid;
Protogen, protoporphyrinogen IX; Proto, protoporphyrin IX.
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Further experimental evidence for an important role of
PDMs in chlorophyll synthesis and insertion was recently
provided by the analysis of another TPR protein from
Synechocystis 6803, named Pitt (POR-interacting TPR protein). This TM protein was found to interact directly with
and stabilize the light-dependent POR enzyme (Schottkowski et al., 2009b). Intriguingly, in a pratA mutant, a large
proportion of both Pitt and POR was localized in PDM
fractions. This is in contrast to wild-type cells, where only
minor amounts are found in PDMs and the majority is TM
associated (Schottkowski et al., 2009b). Hence, these two
proteins are affected by the absence of PratA in the same way
as the pD1 precursor protein. Apparently, a defective PSII
assembly and perturbation of membrane flow from PDMs
to TMs causes the retardation of additional PSII biogenesis
factors, including Pitt and POR, at the site of early PSII
assembly, i.e. the PDMs.
However, the question arises as to why in wild-type cells
chlide a is mainly localized in the PM and/or in the
thylakoid centers (Peschek et al., 1989; Hinterstoisser et al.,
1993), whereas the chlide a-synthesizing enzyme POR is
mainly – but not exclusively – detected in the TM (Schottkowski et al., 2009b). One explanation for this discrepancy
could be provided by the localization of CS, which catalyzes
the final esterification of chlide a to chl a. In chloroplasts,
this enzyme has been exclusively localized to TMs (Soll et al.,
1983; Eckhardt et al., 2004; Fig. 3). If this TM localization of
CS also holds for cyanobacteria, TM-synthesized chlide a
could be rapidly converted to chl a, whereas chlide a
synthesized by the minor POR fraction in PDMs would
accumulate due to scarce further processing. However,
previous CS activity measurements in Synechocystis 6803
suggested the presence of CS in both the – putatively PDMrelated – thylakoid centers and TMs (Hinterstoisser et al.,
1993). Hence, higher chlide a synthesis rates in PDMs must
also be considered. These might be due to the activity of the
second, light-independent, POR enzyme (LiPOR) from
Synechocystis 6803, whose localization is still elusive (Armstrong, 1998). Taken together and despite several open
questions, the facts presented draw a picture of PDMs as a
subcompartment, in which not only protein complex biogenesis but also the later steps in chlorophyll synthesis and
its insertion into polypeptides occur.
Conclusion
In conclusion, we propose the following working model for
the biogenesis of TMs in the model organism Synechocystis
6803 (Fig. 4): both protein synthesis/assembly and chlorophyll synthesis/insertion are subject to tight spatial organization. These two processes are localized in a specialized
membrane region, here termed PDMs, which is marked by
the D1-bound form of the PSII biogenesis factor PratA. The
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J. Nickelsen et al.
Fig. 4. Model of PSII biogenesis in Synechocystis 6803. PSII – and
probably also PSI – biogenesis is hypothesized to initiate in a membrane
subfraction (PDM subfraction) at thylakoid convergence sites close to the
PM (compare Fig. 2). Here, the assembly of PSII precomplexes containing
the reaction center proteins D1 and D2 as well as Cyt b599 is coordinated
as indicated by the presence of the assembly factor PratA in its D1-bound
form. In addition, PDMs harbor the chlorophyll synthesis enzyme POR
and its interaction partner Pitt, supporting the idea that chlorophyll
synthesis and insertion into precomplexes occur at the same site.
Subsequently, precomplexes move to thylakoids in an as yet unknown
way, where their assembly is completed. The question mark refers to as
yet unidentified additional components of the assembly machinery. OM,
outer membrane; Chlide, chlorophyllide a; Chl, chlorophyll a; pD1,
precursor of D1.
fact that non-D1-bound PratA is a soluble periplasmic
protein strongly argues for at least temporary contacts of
PDMs with the PM. These areas of contact are likely to be
identical to the previously described thylakoid centers,
which are located at the cell periphery, between PM and
TMs (Hinterstoisser et al., 1993; van de Meene et al., 2006).
Hence, the existence of such structures close to both the PM
and the TM could easily explain the involvement of the
periplasmic PratA factor in TM biogenesis. Furthermore,
the finding that pD1, Pitt and POR are all localized to a
higher amount in PDMs upon inactivation of PratA
strongly suggests an essential role of PratA in the functional
and/or structural organization of these biogenesis centers
and, thus, membrane flow from PDMs to TMs.
Although the described model seems to apply to PSII
biogenesis, less evidence is available concerning the spatial
organization of the PSI assembly process. Nevertheless, the
detection of the PSI reaction center proteins PsaA and PsaB
in PM or PM-related fractions suggests that also PSI
biogenesis is initiated in the PM or even in PDMs similar
to PSII (Zak et al., 2001).
Future work will be directed toward the visualization of
the biogenesis process, for instance by time-resolved studies
with green fluorescent protein-tagged proteins. The ultrastructural localization of the various factors involved, especially the PratA protein, will unambiguously answer the
question whether, indeed, PDMs and thylakoid centers are
directly linked. The identification of additional PDM-marker proteins will enable one to elucidate the molecular
FEMS Microbiol Lett 315 (2011) 1–5
5
Cyanobacterial thylakoid membrane biogenesis
details of membrane trafficking mechanisms underlying TM
and PM synthesis and maintenance in more detail.
Acknowledgement
Our work on the biogenesis of cyanobacterial membranes is
supported by the Deutsche Forschungsgemeinschaft SFBTR1/C10.
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