Dynamics of ribosome association with the endoplasmic reticulum membrane [Elektronische Ressource] / vorgelegt von Julia Schaletzky

Julia Schaletzky

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

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 9
2.4 Ribosome binding to the rough endoplasmic reticulum 10
2.5 SRP-dependent targeting 12

3 Aim 15

4 Result 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 ribosomplex 52
engaged in translocation
4.10.3 Structure of a bacterial ribosome-channel complex 57
4.10.4 Structure of a bacterial ribosomplex 60
engaged in translocation
I4.10.5 Binding of bacterial ribosomes to bacterial and eukaryotic 66
membranes

5 Discusion 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 CaCl -competent E.coli cells 2
6.1.4 Transformation of CaCl -competent cells 2
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
II6.3 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 Abreviatons 106

9 Curiculm Vitae 109
III1 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. Consisten