Qualitative and quantitative analyses of the composition and dynamics of light harvesting complex I in eukaryotic photosynthesis [Elektronische Ressource] / von Einar Jamandre Stauber

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2007
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Qualitative and quantitative analyses of the composition and dynamics of
light harvesting complex I in eukaryotic photosynthesis




Dissertation

zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.)



vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena




von

Diplombiologe Einar Jamandre Stauber
geboren am 22. August 1973 in Moscow (Idaho, USA)




November 2007 Abbreviations

ADP adenosine diphosphate
ATP adenosine triphosphate
CAB chlorophyll a/b binding protein
CC424 arginine auxotrophic Chlamydomonas reinhardtii strain
CV coefficient of variation
DEAE (diethylamino)ethyl
1-DE one-dimensional gel electrophoresis
2-DE two-dimensional gel electrophoresis
EST expressed sequence tag
IEF-PAGE isoelectic focusing/SDS-PAGE
IRLhca3 RNAi mutant of Lhca3
LC-MS liquid chromatography-mass spectrometry
LC-MS/MS liquid chromatography-tandem mass spectrometry
Lhc light harvesting complex protein
Lhca light harvesting complex protein of photosystem I
Lhcb light harvesting complex protein of photosystem II
LHCI light harvesting complex of photosystem I
LHCII light harvesting complex of photosystem II
MS mass spectrometry
NADP nicotinamide adenine dinucleotide phosphate
P inorganic phosphate i
PSI photosystem I
PSI-LHCI holocomplex of photosystem I and light harvesting complex I
PSII photosystem II
SILAC stable isotope labelling by amino acids in cell culture
RNAi ribonucleic acid interference technology
SDS-PAGE sodium dodecyl sulfate – polyacrylamide gel electrophorhesis
Table of contents

1. Introduction ……………………………………………………………...…... 1
2. Background ………………………………………………………………….. 3
2.1. PSI is a light-driven plastocyanin or cytochrome c -ferredoxin oxidoreductase 3 6
2.2. Light-harvesting complex I delivers excitation energy to photosystem I ….... 4
2.3. Light-harvesting complex I plays an important role in acclimation of the
photosynthetic apparatus to iron deficiency …………………………………. 7
2.4. Chlamydomonas and tomato as eukaryotic model organisms to study light-
harvesting complex I …………………………………………………...……. 9
2.5. Proteomics and mass spectrometry as tools for qualitative and quantitative
analyses of protein complexes ………………………………….……………. 11
3. Aims of the study ……………………………………………...……….……. 15
4. Published papers and manuscripts in submission ………………...…………. 16

Manuscript 1. E.J. Stauber, M. Hippler (2004) Chlamydomonas reinhardtii
proteomics. Plant Physiology and Biochemistry 42, 989-1001 …….….. 19
Manuscript 2. Y. Takahashi, T. Yasui, E.J. Stauber, M. Hippler (2004)
Comparison of the subunit compositions of the PSI-LHCI
supercomplex and the LHCI in the green alga Chlamydomonas
reinhardtii. Biochemistry 43, 7816-7823 ……………...……………… 33
Manuscript 3. S. Storf, E.J. Stauber, M. Hippler, V.H.R. Schmid (2004)
Proteomic analysis of the photosystem I light-harvesting antenna in
tomato (Lycopersicon esculentum). Biochemistry 43, 9214-9224 .......... 42
Manuscript 4. B. Naumann*, E.J. Stauber*, A. Busch, F. Sommer, M. Hippler
(2005) N-terminal processing of Lhca3 is a key step in remodeling of
the photosystem I-Light-harvesting complex under iron deficiency in
Chlamydomonas reinhardtii. Journal of Biological Chemistry 280,
20431-20441, * Both authors contributed equally. …………………..... 54
Manuscript 5. E.J. Stauber, A. Busch, B. Naumann, A. Svatoš, M. Hippler:
Proteotypic profiling of LHCI from Chlamydomonas reinhardtii
provides new insights into structure and function of the complex.
Manuscript in preparation for Proteomics. …………...………............. 66
5. Discussion ………………………………………………………...…………. 99
5.1. Heterogeneity of light-harvesting complex I in plants ………………..……... 99
5.1.1. Structure of oligomeric light-harvesting complex I in Chlamydomonas and its
association with photosystem I …………………………………...…………. 99
5.1.2. Stoichiometry of light-harvesting complex I proteins in Chlamydomonas .…. 101
5.1.3. Composition of light-harvesting complex I in tomato …………………....…. 104
5.2. Remodelling of Chlamydomonas photosystem I - light-harvesting complex I
under iron deficiency …………………………………………….………..…. 107
5.3. Stable isotope labelling and isotope dilution allow mass-spectrometric protein
quantitation …………………………………………………………………... 112
5.4. Conclusions and perspectives ………………………………………….…….. 114
6. Summary …………………………………………...…………………….….. 117
7. Zusammenfassung …………………………………...…………………….… 119
References ……………………………………………...……………………………... 121

1. Introduction

The accessory light-harvesting complexes (LHCs) enable land plants and green algae to live
in highly variable environments. The LHCs form an antenna around photosystem I (PSI)
(called LHCI) and photosystem II (PSII) (called LHCII) that gather solar energy and transfer
it to the reaction centers where the energy drives electron transport (Jansson, 1999; Koziol,
2007). LHCI and LHCII are each composed of several pigment binding proteins called Lhca
and Lhcb, respectively. Lhca and Lhcb belong to a multi-gene family encoding proteins with
one to four transmembrane helices and several conserved chlorophyll and xanthophyll binding
sites (Pichersky, 1996; Koziol, 2007). They likely evolved through gene duplication of high-
light inducible proteins of cyanobacteria which function in acclimation to light stress
(Dolganov, 1995; Jansson, 1999; Montané, 2000; Koziol, 2007). LHCI is tightly associated
with the PSI core complex. It exhibits low temperature fluorescence emission shifted toward
longer wavelengths as compared to LHCII.

Despite its central role in photosynthesis, the exact composition of LHCI and our
understanding of the role of the single Lhca proteins and their function in adaptation of the
photosynthetic capacity to varying environmental conditions is far from complete. For
example, genome sequence information has provided valuable data on the number and
structure of lhca genes, however, their corresponding proteins have not always been
identified. This is partly due to the difficulty of separating the different Lhca. Quantitative
determinations of Lhca have been hampered by the lack of methods for absolute
quantification of proteins in complexes.

In the present study, two eukaryotic organisms, the green alga Chlamydomonas reinhardtii
(Chlamydomonas) and Lycopersicon esculentum (Solanum lycopersicum, tomato) as a land
plant, were studied with respect to composition of their LHCI and the association of LHCI
with PSI. A detailed study of Chlamydomonas LHCI was aimed at determining its qualitative
composition and the stoichiometry of its Lhca with respect to the PSI core complex.

The LHCI-PSI complex has a remarkable ability to adjust itself to changing environmental
conditions. Under iron deficiency, PSI levels are drastically reduced, and LHCI is remodelled
and becomes energetically uncoupled from PSI (Moseley, 2002a). In this thesis, the
1 remodelling of the LHCI-PSI complex occuring under iron deficiency was investigated in
detail with special focus on the mechanism of the uncoupling of LHCI from PSI.

With the advances in mass spectrometry (MS) techniques, proteomics have become an
important tool in the analysis of protein complexes. Two-dimensional gel electrophoresis (2-
DE) and one-dimensional gel electrophoresis (1-DE) have been applied throughout this study
to identify and quantify single Lhca obtained from purified complexes or thylakoid
preparations. As a method for protein quantification, stable isotope labelling was established
for use in Chlamydomonas and successfully applied in the determination of the Lhca-PSI
stoichiometry.
2 2. Background

2.1 Photosystem I is a light-driven plastocyanin or cytochrome c – ferredoxin 6
oxidoreductase

Light-dependent electron flow from H O to NADPH in the photosynthetic apparatus is carried 2
out by the concerted action of four multiprotein complexes of the thylakoid membrane; PSII,
the cytochrome b f complex, PSI-LHCI and the adenosine triphosphate (ATP) synthase 6
(Dekker, 2005; Nelson, 2006). PSII initiates the process by the light-driven removal of
electrons from H O in the lumenal compartment of the thylakoids and transferring them to a 2
pool of mobile lipophilic electron carriers referred to as the plastoquinone pool. Plastoquinol
does not deliver electrons directly to PSI, but instead delivers them to the cytochrome b f 6
complex which transfers them to either of the soluble lumenal electron carriers plastocyanin
or cytochrome c which can donate electrons to PSI (Cramer, 2006). Plastocyanin serves as an 6
electron donor for land plants, algae and most photosynthetic prokaryotes. In contrast to land
plants (Weigel, 2003), green algae and cyanobacteria can also use cytochrome c as an 6
electron donor to PSI. Thus the cytochrome b f complex mediates electron flow between the 6
two photosystems and also plays a key role in processes such as state transitions and cyclic
electron flow that tune PSII and P