dynamics
and function of bioenergetic pathways in oxygenic chloroplast
photosynthesis
Our research is aimed to understand molecular mechanisms which
are involved in assembly, function, maintenance and regulation
of the photosynthetic machinery in oxygenic chloroplast photosynthesis.
For this purpose, we are using the green alga Chlamydomonas reinhardtii
as a primary model system and combining molecular techniques
like reverse genetics and proteomics to elucidate these processes.
Hereby, we are engaged in different research lines. We are currently
investigating the adaptation and remodeling of the photosynthetic
apparatus as a response to iron-deficiency. We are also interested
in an understanding of chloroplast iron-homeostasis as a whole.
The long term goal here is to discover the signaling pathway,
from the sensing mechanism to the target genes and proteins that
determines the operation of the photosynthetic apparatus as a
function of iron nutrition. In another research line we are aiming
to explore electron transfer and binding mechanisms between the
soluble electron transfer proteins plastocyanin or cytochrome
c6 and photosystem I (PSI). Additionally we aim to explore the
molecular recognition between PSI and its light-harvesting protein
complex (LHCI), allowing the formation of the PSI/LHCI complex,
subunit remodeling as well as efficient excitation energy transfer.
In a third research line we are currently establishing the basis
for further systems approaches. Herein we are establishing two-dimensional
protein maps of the thylakoid membrane proteome from wild type
and mutant strains as well as new software tools to search the
Chlamydomonas genomic database using mass spectrometric data.
For proteomic analyses the laboratory is equipped with an ion-trap
mass spectrometer (LCQ Deca XP plus, Thermo Finnigan) that is
coupled to a UltiMate Nano liquid chromatography system (LC-Packings),
containing a Switchos mirco column switching unit, a FAMOS autosampler
and an UliMate Nano HPLC.
1. Iron-homeostasis of the chloroplast and impact of iron-deficiency
on photosynthesis
For the study of the impact of iron nutrition on photosynthesis,
C. reinhardtii is an experimental system of choice, because manipulation
of iron nutrition is facile, marker genes for assessing intracellular
iron status have been described and well-developed methodologies
to investigate altered photosynthetic function in vivo are available.
In a recent study we studied the adaptation of the photosynthetic
machinery with respect to iron-deficiency in Chlamydomonas reinhardtii.
As one of the approaches we analyzed the impact of iron-deficiency
the thylkaoid menbrane proteome by two dimensional gel electrophoresis
(Figures 1 and 2). Our study indicated that iron-deficiency mediated
responses are apparent before chlorosis (diagnostic symptom of
iron-deficiency), which is in contrast to textbook dogma that
attributes chlorosis as a secondary consequence of the inhibition
of chlorophyll synthesis due to iron-deficiency. We concluded
that a regulatory network induces specific responses at a “latent” level
of iron-deficiency before iron-limitation becomes evident (see
Moseley et al., 2002a). In future experiments we aim to explore
this network and identify the key players that define the regulatory
circuits.
Figure 1: 2D gels of thylakoid
membrane proteins. Distinctive spots that increase reproducibly
in –Fe cells
are lettered (e.g. green arrows) while those that decrease are
numbered (e.g. 71 – 75). Reproduced from (Moseley et
al., 2002). Spots B, 6 and 9 correspond to Lhca subunits (Stauber
et al., 2003).
Figure 2: Evolution of the new putative LHCI
protein spot in Box B during adaptation to iron-deficiency (Figure
1). Enlarged box B from silver-stained 2-DE gels of thylakoid
membranes from wild-type cells before and after 1-5 days of growth
after transfer to Fe-free (0 mM Fe) medium.
For further in-depth quantification, relative quantification
experiments are performed using arginine auxotrophic C. reinhardtii
cells, that are grown in the presence of either isotopically
labeled or unlabeled arginine ([13C6]Arg). Thus allowing a comparative
quantification of labeled and unlabeled sister peptides originated
from both conditions by LC-MS/MS and thereby insight in quantitative
change of the chloroplast proteome under iron-sufficient versus
iron-deficient conditions.
2. Bioenergetics of photosystem I
Electron transfer between plastocyanin or cytochrome c6 and photosystem
I
To elucidate binding and electron transfer mechanisms between
PSI and its electron transfer donors plastocyanin or cytochrome
c6 we are using site-directed mutagenesis and biolistic transformation
of Chlamydomonas reinhardtii to alter the chloroplast encoded
reaction-center forming subunits PsaA and PsaB.
Figure 3: Crystal
structures of C. reinhardtii cyt c6 at a resolution of 1.6 Å and
pc at 1.5 Å as
well as S. elongatus PSI at 2.5 Å in a view along the membrane
plane. The numbering for all residues corresponds to the positions
in C. reinhardtii. Positively charged residues as well as the
copper centre of pc are in blue, negatively charged residues
in red, the residues involved in electron transfer - P700, the
heme group of cyt c6 and His87 of pc are in yellow and the two
lumenal helices l and l’ of PsaB and PsaA are in darker
and lighter green, respectively. The PsaF subunit in eukaryotic
PSI contains an N-terminal extension on the lumenal side of PSI
presenting patches of positively charged residues which have
been shown to interact with the negatively charged patches on
the both donors and is orientated through close electrostatic
interactions with PsaB-Glu613. (Taken from Sommer et al., 2004)
The protein-protein interaction between the modified PSI complexes
and endogenous donors plastocyanin or cytochrome c6, all purified
from C. reinhardtii, are analyzed by flash absorption spectroscopy
in cooperation with Prof. W. Hahenel and Dr. F. Drepper from
the University of Freiburg (Germany). Our results demonstrate
that, beside the PsaF subunit, PsaA-W651and PsaB-W627 are crucial
for high affinity binding of plastocyanin or cytochrome c6 to
PSI. However, our results also indicate that both electron transfer
donors bind slightly different to the core of PSI, indicating
that the highly conserved structural recognition motif that is
formed by PsaA-W651 and PsaB-W627 confers a differential selectivity
in binding of both donors to PSI. A finding that will be addressed
in future experiments.
Molecular recognition between PSI and
its light-harvesting protein complex
By integrating reverse genetics and quantitative proteomics
we aim to elucidate the molecular recognition between PSI and
its
light-harvesting protein complex. In a first step we established
a detailed 2-D protein map of Lhca proteins from C. reinhardtii
to determine the Lhca protein composition in this green alga
(Figure 4). In further steps we will tackle putative LHCI binding
sides within the PSI structure by reverse genetics and will
use quantitative proteomics to determine the exact subunits
composition
in the mutants PSI complexes.
Figure 4: 2-D protein map of Lhca proteins from C. reinhardtii.
Enriched PSI particles were separated by two-dimensional gel
electrophoresis and protein spots were identified by LC-MS/MS
analyses. The gel was stained with Coomassie blue. 9 different
Lhca related gene products were identified. Out of this 9, 3
were identified for the first time on protein level.
Studies over the last years have indicated that multi-protein
complexes can be rather dynamic in their polypeptide subunit
composition. It has been estimated that, on average, every fourth
protein in a proteome might be shared between protein complexes
of different function. These dynamic changes may reflect responses
to optimize function in any given physiological context. As an
excellent model to understand subunit remodeling and its impact
on function we aim to elucidate protein dynamics of photosystem
I (PSI) and its associated light-harvesting protein complex (LHCI)
and therefore we will study the protein complex composition isolated
from different physiological conditions or distinct genetic backgrounds.
3.
The Chlamydomonas chloroplast proteome and development of bioinformative
tools for proteomics analyses
To facilitate mass spectrometric data analysis obtained from
protein spots (derived from 2-D protein maps of the chloroplast
subproteome) we devised software that allows the identification
of intron-split peptides as deduced from genomic DNA using
mass spectrometric data (Allmer et al. 2004). Our algorithm
uses small regions of peptide sequence information, which are
automatically deduced from de novo amino acid sequence predictions
together with the molecular mass information of the precursor
ion. The sequence predictions are based on selected collision-induced
mass spectrometric fragmentation spectra. Fragments of the
predicted amino acid sequence are aligned with each of the
six frames of the translated genome and the precursor mass
information is used to assemble the corresponding tryptic peptides
using the gemomic DNA sequence as a matrix. We will continue
to improve this tool, which will prove helpful for Chlamydomonas
gene annotation and in particular for the identification of
peptides that are generated by alternative splicing mechanisms.
Wittstock, U., Agerbirk, N.,
Stauber, E.J., Olsen, C.E., Hippler, M., Mitchell-Olds, T., Gershenzon,
J. and Vogel, H. (2004) Successful
herbivore attack due to metabolic diversion of a plant chemical
defense. Proc Natl Acad Sci U S A, 101, 4859-4864.
Sommer,
F. and Hippler, M. (2003) Photosystem I:
Structure/Function and Assembly of a transmembrane light-driven
Plastocyanin/Cytochrome
c6 – Ferredoxin Oxidoreductase. Handbook of Photochemistry
and Photobiology, Vol. 4, Ed. H.S. Nalwa, American Scientific
Publishers, Stevenson Ranch, California, USA, pages 269-294
Hippler,
M., Rimbault, B. and Takahashi, Y. (2002) Photosynthetic
complex assembly in Chlamydomonas reinhardtii. Protist, 153,
197-220.
Moseley, J.L., Page, M.D., Pergam, N., Eriksson,
M., Quinn, J., Soto, J., Theg, S., Hippler, M. and Merchant,
S. (2002b) Reciprocal
expression of two di-iron enzymes affecting photosystem I and
light-harvesting complex accumulation. Plant Cell, 14, 673-688.
Rochaix, J., Fischer, N.
and Hippler, M. (2000) Chloroplast site-directed
mutagenesis of photosystem I in Chlamydomonas: electron transfer
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Drepper, F., Hippler, M., Nitschke, W. and Haehnel,
W. (1996) Binding dynamics and electron transfer between
plastocyanin and
photosystem I. Biochemistry, 35, 1282-1295.
Hippler, M., Reichert,
J., Sutter, M., Zak, E., Altschmied, L., Schröer, U., Herrmann,
R.G. and Haehnel, W. (1996) The plastocyanin binding domain
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