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«The role of a putative arginine finger and guanine nucleotides in homo- and heterodimerization of two GTPases involved in protein import into ...»

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Doctoral Thesis ETH No. 15980

The role of a putative arginine finger and

guanine nucleotides in homo- and heterodimerization

of two GTPases involved in protein import into chloroplasts

A dissertation submitted to the


for the degree of

Doctor of Sciences

presented by


dipl. Natw. ETH, Swiss Federal Institute of Technology Zürich born 16.04.1975 citizen of Hochdorf/LU and Jonschwil/SG accepted on the recommendation of Prof. Dr. N. Amrhein, examiner Prof. Dr. A. Helenius, co-examiner Prof. Dr. F. Kessler, co-examiner Index 1 Index Index 1


6 Zusammenfassung 8 Abbreviations 10 1. Introduction 13 1.1. Chloroplasts, the photosynthetic plastids 13 1.1.1 The evolutionary origin of chloroplasts 14 1.1.2 The coordination of three different genomes in plant cells 15 Nuclear control of chloroplast development 15 Chloroplast control of nuclear gene expression 16 1.1.3. Chloroplast biogenesis 18 The cue (CAB underexpressed) mutants 18 Tight linkage between chloroplast protein import and chloroplast biogenesis 19 1.2. Protein import into chloroplasts 19 1.2.1. General overview 19 1.2.2. The three stages of import characterized by their energy requirement 21 1.2.3. The sites of GTP and ATP consumption during protein import into chloroplasts 22 1.3. The chloroplast translocation machinery 23 1.3.1. Chronology of the research on the chloroplast translocation machinery 23 1.3.2. The Toc-complex 24 Toc159

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Abstract The vast majority of the chloroplast proteins is synthesized in the cytosol with an Nterminal transit peptide and has to be posttranslationally imported into chloroplasts.

This import is facilitated by import machineries at the outer and inner chloroplast membrane, termed the Toc- (translocon at the outer chloroplast membrane) and the Tic- (translocon at the inner chloroplast membrane) complex, respectively. The trimeric Toc-complex at the outer chloroplast membrane consists of Toc34 and Toc159, two integral membrane GTP binding proteins involved in preprotein recognition and binding, and Toc75, forming part of the protein-conducting channel through the outer chloroplast membrane.

The three-dimensional structure of Toc34 from pea, psToc34, has been recently solved, revealing new insights into the function of psToc34. In the crystal, psToc34 formed dimers with bound GDP, resembling a GTPase with its corresponding GAP (GTPase activating protein). It has been hypothesized that one monomer functions as GAP for the other monomer in the pea Toc34 dimer, suggesting that dimerization and GTP hydrolysis are functionally related. Furthermore, the location of Arg133 at the interface of the pea Toc34 dimer suggests that this residue functions as arginine finger. An arginine finger is inserted by many GAPs into the active site of their corresponding GTPase, thereby activating GTP hydrolysis.

To examine the arginine finger hypothesis, we mutated the putative arginine finger (Arg130) of the Arabidopsis ortholog of psToc34, atToc33, to alanine (atToc33 R130A). As Arg130 was found not to be required for GTP hydrolysis, Arg130 does not appear to be an arginine finger. Moreover, GTP hydrolysis by atToc33 wt is slow in comparison to the rate of GTP hydrolysis by activated GTPases, suggesting that one monomer does not function as GAP for the other monomer in the atToc33 dimer.

Instead, Arg130 appears to be essential for atToc33 homodimerization. Moreover, it was shown that Arg130 is involved in heterodimerization of atToc33 and atToc159.

The crystal structure of the psToc34 homodimer suggests that psToc34 homodimerizes preferentially in the GDP bound form. Homodimerization of atToc33, the Arabidopsis ortholog of psToc34, was indeed found to be favoured in presence of GDP.

Abstract 7 Toc159 does not only exist in an integral membrane form, but also in a soluble, cytosolic form. Similar to SRα forming the receptor for SRP54 at the ER membrane, Toc34 appears to function as receptor for soluble Toc159. Furthermore, the interaction between Toc34 and Toc159 is essential for chloroplast biogenesis in vivo and therefore likely plays a crucial role during protein import into chloroplasts.

It is still under debate which guanine nucleotide is bound to the two GTPases when they interact. We provide evidence that the heterodimerization between atToc33 and atToc159 is favoured in presence of GDP, suggesting that GTP hydrolysis is a prerequisite for this interaction.

Zusammenfassung 8


Die grosse Mehrheit der Chloroplasten-Proteine wird im Cytosol mit einem Nterminalen Transitpeptid synthetisiert und anschliessend posttranslational in die Chloroplasten importiert. Dieser Import wird durch Import-Maschinerien in der äusseren und inneren Chloroplasten-Membran bewerkstelligt, die Toc-, respektive Tic-Komplex genannt werden. Der trimere Toc-Komplex besteht aus drei Komponenten: Toc34 und Toc159, zwei GTP-bindende Membran-Proteine, die an Erkennung und Bindung der Vorläuferproteine beteiligt sind, und Toc75, ein Protein das einen Teil des Kanals in der äusseren Chloroplasten-Membran bildet durch den die Vorläuferproteine transportiert werden.

Die kürzlich publizierte Kristall-Struktur von Toc34 aus Erbsen lieferte neue Erkenntnisse bezüglich der Funktion von psToc34. PsToc34 liegt in den Kristallen als Dimer vor, das GDP gebunden hat. Weil dieses psToc34 Dimer einem GTPase/GAP (GTPase aktivierendes Protein)-Komplex sehr ähnlich ist, wurde vermutet, dass im psToc34 Dimer ein Monomer als GAP für das andere Monomer dient. Das impliziert, dass Dimerisierung und GTPase Aktivität miteinander verknüpft sind. Des Weiteren hat die Position eines Arginin-Restes (Arg133) im psToc34 Dimer zur Spekulation geführt, dass dieser Arginin-Rest ein Arginin-Finger sein könnte. Solche ArgininFinger finden sich in vielen GAPs. Sie ragen ins aktive Zentrum der dazugehörigen GTPase und tragen so zur Beschleunigung der GTP-Hydrolyse bei.

Um diese Arginin-Finger Hypothese zu überprüfen, haben wir den vermeintlichen Arginin-Finger (Arg130) von atToc33, dem zu psToc34 orthologen Protein in Arabidopsis, zu einem Alanin mutiert. Es wurde nachgewiesen, dass Arg130 nicht für die GTP-Hydrolyse benötigt wird. Deshalb scheint Arg130 kein Arginin-Finger zu sein. Zusätzlich war die GTP-Hydrolyse von atToc33 wt zu langsam im Vergleich zu anderen aktivierten GTPasen, was gezeigt hat, dass im atToc33 Dimer nicht ein Monomer als GAP für das andere Monomer dient.

Im Gegensatz dazu scheint der mutmassliche Arginin-Finger für die Dimerisierung von atToc33 essentiell zu sein. Ebenso konnte nachgewiesen werden, dass Arg130 an der Heterodimerisierung zwischen atToc33 und atToc159 beteiligt ist.

Zusammenfassung 9 Wie die Kristall-Struktur gezeigt hat, homodimerisiert psToc34 bevorzugt in GDPgebundener Form. AtToc33, das orthologe Arabidopsis Protein zu psToc34, bildet Homodimere ebenfalls bevorzugt in GDP-gebundener Form.

Toc159 kommt nicht nur in Membran-gebundener Form, sondern auch in löslicher Form im Cytosol vor. Es wurde gezeigt, dass Toc34 der Rezeptor für die lösliche Form von Toc159 ist. Eine analoge Situation findet sich an der ER-Membran, wo SRα als Rezeptor für SRP (Signalerkennungs-Partikel) dient. Die Interaktion zwischen Toc34 und Toc159 ist essentiell für die Chloroplastenbiogenese in vivo und scheint deshalb eine sehr wichtige Rolle im Import von Vorläuferproteinen in Chloroplasten zu spielen. Wir zeigen, dass atToc33 und atToc159 bevorzugt in GDPgebundener Form interagieren. Dies weist darauf hin, dass vermutlich die GTPHydrolyse eine Voraussetzung für eine stabile Interaktion zwischen atToc33 und atToc159 ist.

Abbreviations 10 Abbreviations

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atToc…, atTic… indicates the Toc-, Tic-protein of A. thaliana psToc…, psTic… designates the Toc-, Tic-protein of P. sativum Toc…, Tic… refers to Toc- and Tic-proteins of both A. thaliana and P. sativum, unless specified otherwise

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1. Introduction

1.1. Chloroplasts, the photosynthetic plastids The uptake of a cyanobacterium by a eukaryotic cell marked the evolutionary birth of the plant cell. Since this event the relationship between the eukaryotic host and the cyanobacterial endosymbiont has become closer and closer. The endosymbiont lost its autonomy and most of its genome (Martin et al., 1998, 2002), giving rise to a group of organelles, called plastids. Plastids are organelles that are characteristic for plant and algal cells. They are one of the primary features that distinguish plant cells from other eukaryotic cells (Pyke, 1999). In higher plants different types of plastids exist that vary in size, shape, content and function (Thomson and Whatley, 1980).

But they all originate from proplastids, which can be found in dividing cells in meristems. These proplastids develop into plastid types adapted to different functions (Thomson and Whatley, 1980; Surpin and Chory, 1997; Bauer et al., 2001).

Amyloplasts, elaioplasts and proteinoplasts are specialized in the storage of starch, lipids and proteins, respectively. Chromoplasts accumulate carotenoids and provide the red, orange and yellow colors of many flowers, old leaves and fruits.

Chloroplasts, the photosynthetic plastids, are, due to the presence of chlorophyll, characteristic for green plant tissues. Proplastids develop into chloroplasts when leaf cells differentiate in the presence of light or into etioplasts in the absence of light (Chory, 1991, 1993; Surpin and Chory, 1997).

Chloroplasts, like mitochondria, have two envelope membranes separated by an intermembrane space. Within the chloroplast stroma, a third membrane system, the thylakoids, exists. The photosynthetic apparatus is located at the thylakoid membrane (Allen and Forsberg, 2001) which surrounds the thylakoid lumen.

The fact that unique reactions, such as light-induced production of ATP and NADPH and carbon assimilation, take place in chloroplasts, turns chloroplast research into an important topic in plant research.

Introduction 14

1.1.1. The evolutionary origin of chloroplasts

The endosymbiotic theory for the formation of eukaryotic cells containing chloroplasts and mitochondria is now widely accepted (Surpin and Chory, 1997). This theory states that these organelles arose from endosymbiontic events involving a eubacterial ancestor and a eukaryotic host cell (Margulis, 1970). It is thought that cyanobacteria and α-proteobacteria are the eubacterial ancestors of chloroplasts and mitochondria, respectively (Gray, 1999).

Several facts support the prokaryotic origin of chloroplasts and mitochondria. They contain their own genome which consists of several copies of a circular chromosome (Surpin and Chory, 1997). Chloroplasts proliferate by simple fission involving cell division proteins with high sequence identity to bacterial and cyanobacterial proteins (Osteryoung and Vierling, 1995). Chloroplast ribosomes are 70S in size, i.e. of the prokaryotic type, and chloroplast ribosomal-proteins show structural similarities to those of cyanobacteria (Meng et al., 1989).

Furthermore, sequence comparison of genes encoding ribosomal proteins revealed a gene organization in Synechococcus sp. similar to that of chloroplasts of red and brown algal species (Sugita et al., 1997). This sisterhood of green plants and red algae was also affirmed by sequence comparison of marker proteins, underlining that they have the same ancestor and that endosymbiosis was established before the separation of green plants and red algae (Moreira et al., 2000).

A critical step in the transition from the autonomous endosymbiont to the organelle was the reduction of the genome. Many endosymbiont genes were lost and most of those retained were transferred to the nucleus (Douglas, 1998; Martin et al., 1998).

Recent data suggest that around 18% of the coding genes of Arabidopsis were acquired from the cyanobacterial ancestor of chloroplasts (Martin et al., 2002).

Transfer of genes to the nucleus necessitated targeting of cytosolically synthesized gene products to the chloroplast as well as their subsequent import. This resulted in the establishment of an import complex in the outer and inner membrane of chloroplasts, called Toc- and Tic-complex, respectively (discussed in detail later). For various proteins of this import machinery homologues, or at least similar ORF sequences, were detected in the Synechocystis sp. genome (Bölter et al., 1998b;

Reumann et al., 1999; Reumann and Keegstra, 1999; Fulda et al., 2002), supporting again the hypothesis for a cyanobacterial origin of chloroplasts and suggesting that, Introduction 15 at least in part, the import machinery evolved from ancient cyanobacterial proteins.

Moreover, also the Sec-, Tat- and SRP-translocons for protein import into or across the thylakoid membrane, show clear signs of having a cyanobacterial origin (Robinson et al., 2000; Schnell, 2000; Dyall et al., 2004). The present conception is that many transport proteins of the outer membrane are of eukaryotic origin and those in the inner membrane are of mixed origin (Dyall et al., 2004).

1.1.2. The coordination of three different genomes in plant cells

The presence of three different genomes, namely in the nucleus, in mitochondria and in chloroplasts, led to the evolution of various mechanisms to coordinate gene expression in plant and algal cells.

Much of the regulatory traffic between the nucleus and the chloroplast occurs from nucleus to chloroplast (anterograde signaling). But the flow of information is not entirely unidirectional, as signals from chloroplasts influence gene expression in the nucleus (retrograde signaling) (Jarvis, 2001; Rodermel, 2001). Nuclear control of chloroplast development

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