Structural and Biochemical Studies of Cotranslational Protein Transport across the Endoplasmic Reticulum

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1 Structural and Biochemical Studies of Cotranslational Protein Transport across the Endoplasmic Reticulum Inaugural-Dissertation zur Erlangung des akademischen Grades Doktor der Naturwissenschaften - Dr. rer. Nat.- am Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Diplom-Biochemiker Jürgen Helmers Berlin 2003

2 Die vorliegende Arbeit wurde im Zeitraum Januar 1997 bis August 2003 im Labor von Prof. Günter Blobel an der Rockefeller University in New York, NY, USA angefertigt. Gutachter 1: Gutachter 2: Günter Blobel, M.D., Ph.D. Prof. Dr. Dr. Manfred Schweiger Eingereicht am: Tag der mündlichen Prüfung:

3 for Anja and Leoni

4 I Table of Contents 1 Introduction Protein Transport across the Membrane of the Endoplasmic Reticulum The Signal Hypothesis Cotranslational Protein Transport Posttranslational Protein Transport Components of the Protein Targeting and Translocation Process The Ribosome Eukaryotic Protein Biosynthesis The Signal Recognition Particle The Signal Recognition Particle Receptor The Protein Conducting Channel Guanine Nucleotide Binding Proteins Molecular Switches Guanine Nucleotide Exchange Factors The GTPase Cycle during Cotranslational Protein Targeting Cotranslational Protein Transport is dependent on GTP Signal Sequence Transfer to the PCC Three Dimensional Electron Microscopy of Macromolecular Assemblies Electron Microscopy Negative stain Cryo Electron Microscopy Three Dimensional Reconstruction using Single Particle Analysis Image Formation in the Electron Microscope The Contrast Transfer Function Contrast Transfer Function (CTF) Correction Single Particle Analysis Three Dimensional Reconstruction of Single Particle Specimens using Reference Projections Material and Methods Materials...27

5 II Bacterial Strains Yeast strains Plasmids E. coli Media Yeast media Rich media Minimal Media (Drop Out Plates) Solutions Solutions for Gel Electrophoresis Buffers for the Manipulation of Yeast Chemicals Enzymes, Kits, and Miscellaneous Materials Detergents Lipids Protease Inhibitors Antibodies Nucleic Acids Equipment Computer Software Molecular Biological and Biochemical Methods Molecular Biology Polymerase Chain Reaction (PCR) Desalting of PCR Products Plasmid Preparation Restriction Digestion Gel Purification of DNA Ligation Transformation Colony PCR Purification of Recombinant Proteins Overproduction of Recombinant Proteins Protein Purification using a 6xHIS Tag...42

6 III Protein Purification using a Glutathione Affinity Tag Loading of purified SRβ with mantgdp SRβ Nucleotide Exchange Assay Yeast genetics Protein A-Tagging of Yeast Proteins Generation of Double Stranded DNA for Recombination Introduction of DNA into Yeast Cells Verification of Proper Gene Integration Purification of the Signal Recognition Particle (SRP) Purification of the Heptameric- and the Sec61p complex Purification of Non-Translating Ribosomes Purification of Programmed Ribosome Nascent Chain Complexes Reconstitution of RNC-Sec61 Complexes Cryo Electron Microscopy Preparation of Holey EM Grids Preparation of Cryo EM Grids Data Collection in the Cryo Electron Microscope Scanning of Micrographs Three Dimensional Reconstruction Methods Micrograph Selection Particle Selection Initial Alignment Particle Quality Control Calculation of an Initial 3D-Structure Structure Refinement Results Architecture of the Non-Translating 80S Ribosome Purification of Non-Translating Ribosomes Preparation of Cryo Electron Microscopy Grids of Non-Translating Ribosomes Image Reconstruction of Non-Translating Ribosomes Multireference Alignment Calculation of an Initial 3D-Model...65

7 IV Iterative Refinement of the Initial 3D-Model Movement of Expansion Segment Architecture of the Protein-Conducting Channel in Association with the Translating 80S Ribosome Purification of Ribosome Nascent Chain Complexes Reconstitution of the Sec61p Ribosome Nascent Chain Complex Reconstruction and Overall Structure of the RNC-Channel Complex Presence of trna in the Programmed Ribosome The Ribosome-Channel Connection Structure and Function of the Protein-Conducting Channel The GTPase Cycle of the SRP Receptor β-subunit Sbh1p and Sbh2p share Sequence Homology with the Sec7 domain Sbh1p and Sbh2p function as the GEFs for SRβ Sbh1p functions as the GEF for SRβ in the Trimeric Sec61p but not in the Heptameric Complex Discussion Movement of Expansion Segment The Protein Conduction Channel Associated with the Translating 80S Ribosome D-Reconstruction of the RNC-Sec61p Complex The Connection between the Ribosome and the Sec61p Complex Structure and Function of the Protein Conducting Channel Binary Model for Cotranslational Protein Translocation and Membrane Protein Integration The GTPase Cycle of the SRP Receptor β-subunit References Abbreviations List of Publications Acknowledgments Summary Zusammenfassung...127