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Dynamics of Ribosome Association with the Endoplasmic Reticulum Membrane

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Dynamics of Ribosome Association with the Endoplasmic Reticulum Membrane Dynamics of Ribosome Association with the Endoplasmic Reticulum Membrane Dissertation zur Erlangung des Doktorgrades der Fakultaet fuer Biologie der Ludwig-Maximilians-Universitaet Muenchen vorgelegt von Dipl.-Biochem. Julia Schaletzky Mai 2006 Die vorliegende Arbeit wurde an der Harvard Medical School in Boston, MA (USA) unter der Anleitung von Prof. Tom Rapoport durchgefuehrt. Die dreidimensionale Struktur der von mir isolierten Komplexe wurde in Kollaboration mit Prof. Chris Akey (Boston University, USA) unter Anleitung von Dr. Jean-François Ménétret durch Cryo-Elektronenmikroskopie ermittelt. Die vorliegende Arbeit wurde zur Beurteilung eingereicht am 01.06.2006. Gutachter: Prof. Dr. J. Soll PD Dr. E. Schleiff Prof. Dr. M. Hayashi Prof. Dr. K. Jung Rigorosum: 20.09.2006 Ehrenwoertliche Versicherung Hiermit versichere ich, dass ich die vorliegende Arbeit selbstaendig verfasst und keine anderen als die von mir angegebenen Quellen und Hilfsmittel verwendet habe. Ferner erklaere ich, dass ich anderweitig nicht versucht habe, eine Dissertation einzureichen oder mich einer Doktorpruefung zu unterziehen. Die vorliegende Arbeit ist nicht als Ganzes oder in Teilen einer weiteren Pruefungskommission vorgelegt worden. Boston, 12.05.06 ........................................ (Julia Schaletzky) ACKNOWLEDGEMENTS I would like to thank Prof. Tom Rapoport for his continuing support and advice during the supervision of my project, and for being a great teacher and mentor. I would like to express my gratitude to Prof. Juergen Soll for generously agreeing to be my advisor and to lead my PhD committee. Prof. Stefan Jentsch deserves thanks for agreeing to examine my PhD thesis and for his constant support and help during the pursuit of my PhD. I am also grateful to Prof. Walter Neupert for advice and help. Parts of this thesis are the results of a fruitful collaboration with the laboratory of Prof. Chris Akey, especially with Dr. Jean-François Ménétret. I would like to thank both of them for their enthusiasm, effort and constant discussion and for giving me the opportunity to learn electron cryo-microscopy. I am grateful to Prof. Reid Gilmore, Dr. Andrea Neuhof, Dr. Sven Heinrich, Dr. William Clemons, Dr. Jochen Zimmer, Dr. Michael Rape and dog H3 for plasmids, purified ribosomes and proteins. Dr. Andrea Neuhof deserves thanks for starting the SRP-dependent targeting project. I would like to thank all former and present members of the Rapoport Lab for discussion, advice, encouragement, practical help and for keeping the spirits high. I thank Lorna Fargo and Carol Sawyer for being always helpful and making sure things are running smoothly in the lab. I would like to thank Sue Wood and Vivian Holmes for handling my visa issues so that I could work on my PhD without becoming an illegal alien. I would like to thank the Boehringer Ingelheim Fonds for continuous nonbureaucratic support, especially Dr. Hermann Froehlich, Claudia Walther and Monika Beutelspacher. I would like to thank my family and friends for support and encouragement. Great thanks go to Michael for never-ending discussions, advice, infectious enthusiasm and patience during late nights in the lab. TABLE OF CONTENTS 1 Abstract / Zusammenfassung 1 2 Introduction 5 2.1 Different ways of protein translocation 5 2.2 Protein translocation by Sec61 7 2.3 The oligomeric state of Sec61 9 2.4 Ribosome binding to the rough endoplasmic reticulum 10 2.5 SRP-dependent targeting 12 3 Aim 15 4 Results 16 4.1 Characterization of Ribosome binding to the rough ER membrane 16 4.2 Characterization of SRP-dependent targeting under competition conditions 19 4.3 Characterization of the new binding sites used by the SRP-system 25 4.4 A free channel population observed after membrane saturation 28 4.5 Importance of the free channel population for SRP-dependent targeting 31 4.6 Differences between free and bound Sec61 34 4.7 Biochemical analysis of the ribosome-channel junction 39 4.8 Working model of SRP-dependent targeting 44 4.9 Conditions for dissociation of prebound ribosomes 47 4.10 Structural characterization of ribosome-channel complexes by 49 electron cryo-microscopy 4.10.1 Structure of a eukaryotic ribosome-channel complex 49 4.10.2 Structure of a eukaryotic ribosome-channel complex 52 engaged in translocation 4.10.3 Structure of a bacterial ribosome-channel complex 57 4.10.4 Structure of a bacterial ribosome-channel complex 60 engaged in translocation I 4.10.5 Binding of bacterial ribosomes to bacterial and eukaryotic 66 membranes 5 Discussion 69 5.1 Why is SRP needed for targeting? 69 5.2 The mechanism of SRP-dependent targeting 70 5.3 Evolutional conservation in prokaryotes 72 5.4 Differences in structures determined from membranes and in detergent 74 5.5 The nature of the pore 75 5.6 Ribosome dissociation from the ER membrane 76 5.7 Different oligomeric states of Sec61 in the ER membrane 77 6 Materials and Methods 79 6.1 Molecular Biology 79 6.1.1 Bacterial strains 6.1.2 Bacterial growth 6.1.3 Preparation of CaCl2-competent E.coli cells 6.1.4 Transformation of CaCl2-competent cells 6.1.5 Plasmid purification 6.1.6 DNA/RNA precipitation 6.1.7 Cloning 6.1.7.1 Polymerase chain reaction (PCR) 6.1.7.2 TA-overhang cloning 6.1.7.3 Restriction digest 6.1.7.4 Ligation 6.1.7.5 Agarose gelelectrophoresis 6.2 Purification of microsomes, proteins and reconstitution 81 6.2.1 Preparation of microsomes 6.2.2 Purification of c.f. Sec61, SRP and SRP-receptor 6.2.3 Overexpression and purification of E.c. SecYEG 6.2.4 Reconstitution of proteoliposomes II 6.3 Analytical Methods 84 6.3.1 Polyacrylamide-Gelelectrophoresis and staining 6.3.2 Autoradiography 6.3.3 Immunoblotting and Antibodies 6.3.4 Scintillation counting 6.3.5 Concentration determination 6.4 In vitro transcription / translation 86 6.4.1 In vitro transcription 6.4.2 In vitro translation 6.4.2.1 Reticulocyte lysate system 6.4.2.2 Wheat germ extract system 6.4.2.3 E.coli S30 extract system 6.4.2.4 Isolation of RNCs 6.4.2.5 Labeling of endogenous RNCs in RMs 6.5 Targeting reactions and ribosome binding experiments 88 6.6 Radiolabeling of ribosomes and dissociation experiments 88 6.7 Solubilization of membranes and sucrose gradient centrifugation 89 6.8 Crosslinking and antibody-binding experiments 89 6.9 Co-immunoprecipitation 90 6.10 Electron Cryo-Microscopy of Ribosome-Channel Complexes 91 6.10.1 Preparation of 70S-SecYEG and 80S-Sec61 complexes 6.10.2 Preparation of eukaryotic RNC-Sec61 complexes 6.10.3 Preparation of prokaryotic RNC-SecYEG complexes 6.10.4 Electron Cryo-Microscopy 6.10.5 Three-Dimensional Image Processing and Analysis 7 References 95 8 Abbreviations 106 9 Curriculum Vitae 109 III 1 ABSTRACT Protein translocation across the endoplasmic reticulum (ER) membrane is fundamental for protein sorting and secretion in all kingdoms. Translocation occurs through a protein-conducting channel in the ER membrane, which is formed by Sec61. Targeting of ribosome-nascent chain complexes (RNCs) to Sec61 is mediated by the signal recognition particle (SRP) and its cognate receptor (SR). However, Sec61 has a high affinity for nontranslating ribosomes and it is largely occupied in vivo. We addressed how RNC-SRP complexes can efficiently associate with the rER membrane, although most Sec61 seems to be occupied. We found that the spontaneous dissociation of ribosomes from the ER membrane is extremely slow. Surprisingly, membrane binding of RNC-SRP complexes does not require or cause the dissociation of prebound ribosomes. Instead, RNC-SRP complexes use a Sec61 population for translocation that cannot be bound by nontranslating ribosomes or RNCs alone. Complex formation between RNC-SRP and SR at the ER membrane facilitates the interaction between the RNC and the free Sec61. We have used biochemical and structural approaches to investigate why the free Sec61 fails to be bound by nontranslating ribosomes. Our data suggests that Sec61 is present in two interconvertible forms in the membrane, that are in an equilibrium with each other and provide high-affinity and low-affinity binding sites for ribosomes. The former are quickly occupied, while the latter remain free. The high-affinity binding sites are formed by tetrameric rings of Sec61, which provide several connections that each can break and reform, but together prevent the ribosome from detachment. In contrast, the low-affinity binding sites may correspond to lower oligomeric states of Sec61. Consistent with that, we could capture monomers or dimers of Sec61 bound to eukaryotic ribosomes by electron cryo-microscopy. A ribosome-bound Sec61 dimer was only observed in presence of a nascent chain, which could indicate that Sec61 oligomerization is induced by translocation of a nascent chain. We obtained similar results for bacterial ribosome-channel complexes. However, the low-affinity binding site seems to be more abundant in bacterial membranes, which indicates that the prokaryotic equilibrium between low- and high-affinity binding sites is adjusted differently from eukaryotes. 1 We propose a model, in which Sec61 exists in different oligomeric states in the ER membrane. Tetrameric Sec61 provides a high-affinity binding site for ribosomes and is readily occupied in vivo. Monomeric or dimeric Sec61 is accessible for targeting by the SRP pathway, which facilitates the interaction between RNC and free Sec61. After transfer of the nascent chain into the channel and dissociation of SR and SRP, more Sec61 molecules could be recruited to stabilize the interaction with the translating ribosome. A dynamic oligomerization equilibrium of Sec61 is thus the core of a mechanism that would guarantee efficient protein translocation in vivo. 2 ZUSAMMENFASSUNG Die Translokation sekretorischer Proteine in das Lumen des endoplasmatischen Retikulums (ER) ist evolutionaer konserviert und ueberlebenswichtig in Eukaryoten und Prokaryoten.
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