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Investigations of primary active transporters expressed in Xenopus laevis oocytes [Elektronische Ressource] : Wilson disease protein, a P-type ATPase and proteorhodopsin, a light driven proton pump / von Éva Lőrinczi

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159 pages
Investigations of primary active transporters expressed in Xenopus laevis oocytes Wilson Disease Protein, a P-type ATPase and Proteorhodopsin, a light driven proton pump Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften Vorgelegt beim Fachbereich Chemische und pharmazeutische Wissenschaften der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Éva Lőrinczi aus Bukarest Frankfurt am Main 2006 Vom Fachbereich Chemische und pharmazeutische Wissenschaften der Johann Wolfgang Goethe-Universität als Dissertation Angenommen Dekan: Prof. Harald Schwalbe Gutacher: Prof. Ernst Bamberg Prof. Clemens Glaubitz Datum der Disputation: 29.08.2006 2Contents Foreword 7 On the importance of transmembrane proteins for cell homeostasis 7 WILSON DISEASE PROTEIN 9 1. Introduction 11 1.1. History 14 +1.2 Physiological importance of Cu -ATPases 15 1.2.1 Wilson Disease Protein (WNDP) 1.2.2 Menkes Disease Protein (MNKP) 1.3 Structure of WNDP 16 1.4 Functional properties of WNDP 19 1.5 The copper-dependent localisation of WNDP and MNKP 21 1.6 Molecular mechanism of copper-dependent trafficking 23 1.7 Disease mutations that affect the function of WNDP and MNKP 26 1.8 The aim of the work 28 2. Results 29 2.1 Topological model of WNDP 29 2.
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Investigations of primary active transporters
expressed in Xenopus laevis oocytes

Wilson Disease Protein, a P-type ATPase
and
Proteorhodopsin, a light driven proton pump





Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften




Vorgelegt beim Fachbereich
Chemische und pharmazeutische Wissenschaften
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main






von
Éva Lőrinczi
aus Bukarest







Frankfurt am Main
2006


























Vom Fachbereich Chemische und pharmazeutische Wissenschaften der
Johann Wolfgang Goethe-Universität als Dissertation Angenommen























Dekan: Prof. Harald Schwalbe
Gutacher: Prof. Ernst Bamberg
Prof. Clemens Glaubitz
Datum der Disputation: 29.08.2006

2Contents

Foreword 7
On the importance of transmembrane proteins for cell homeostasis 7
WILSON DISEASE PROTEIN 9
1. Introduction 11
1.1. History 14
+1.2 Physiological importance of Cu -ATPases 15
1.2.1 Wilson Disease Protein (WNDP)
1.2.2 Menkes Disease Protein (MNKP)
1.3 Structure of WNDP 16
1.4 Functional properties of WNDP 19
1.5 The copper-dependent localisation of WNDP and MNKP 21
1.6 Molecular mechanism of copper-dependent trafficking 23
1.7 Disease mutations that affect the function of WNDP and MNKP 26
1.8 The aim of the work 28
2. Results 29
2.1 Topological model of WNDP 29
2.2 Expression of the full length WNDP in oocytes 30
2.3 Localisation of WNDP at the cell surface using immunoluminescence 31
2.4 Immunogold labelling of the surface expressed WNDP 33
2.5 Functional characterisation of the HA-tagged WNDP 34
3. Discussion 39
3.1 WNDP is expressed in Xenopus laevis oocytes 40
3.2 The chemiluminescence experiments confirm the suggested topology of WNDP 40
3.3 The effects of sequence modifications on the surface expression of WNDP in oocytes 41
3.3.1 The N-terminal copper binding domains are not necessary for plasma membrane localisation41
3.3.2 The triple-Leu motif might be involved in the retrieval of WNDP from the cell surface 42
3.3.3 Mutations supposed to impair catalytic activity of WNDP not always affect its plasma
membrane localisation 43
4. Conclusions 45
PROTEORHODOPSIN 46
1. Introduction 48
Retinylidene proteins 48
1.1 Bacteriorhodopsin 48
1.1.1 Photocycle, spectral characteristics, structure and photocurrents: a summary 49
1.1.2 Probing the photocycle of BR with blue light 53
1.2 Proteorhodopsin 54
1.2.1 Structure 55
1.2.2. Photocycle and spectral characteristics 57
1.2.3 Photocurrents of Proteorhodopsin 60
31.2.4 Proteorhodopsin variants 62
2.3 The aim of the work 64
2. Results 65
2.1 Action spectrum of PR wild-type 65
2.2 Voltage dependence of the proton transport in the presence or absence of pH 66
gradients 66
2.2.1. In the presence of a pH gradient (asymmetrical pH conditions)
2.2.2. In the absence of a pH gradient (symmetrical pH conditions) 68
2.3. Investigating the effects of the applied electric field 69
2.4 The effect of azide on photocurrents of PR wt 79
2.5 Light intensity dependence of PR wt 80
2.6 Mutants of proteorhodopsin
2.6.1 Mutations of the proton donor E108 82
2.6.2 Mutations of the proton acceptor D97 88
2.6.3 L105Q, the spectral tuning switch 94
2.7 Localisation of bacteriorhodopsin (BR) and proteorhodopsin (PR) at the surface of the oocyte
membrane, investigated by chemiluminescence 95
2.7.1 Functional assessment of HA-tagged retinylidene proteins 97
3. Discussion 99
3.1 Action spectrum of PR wild-type 99
+3.2 Regulation of H translocation by pH and transmembrane potential in PR wt 100
3.3 The photocycles of PR and BR probed by potential changes and laser flash experiments 101
3.4 Light intensity dependence of PR wild-type 106
3.5 Mutants of proteorhodopsin6
3.5.1 Mutations of the proton donor E1087
3.5.2 Mutations of the proton acceptor D979
3.5.3 L105Q, the spectral tuning switch 113
3.6 The effect of azide on the photocurrents of proteorhodopsin4
3.7 Inward pumping by proteorhodopsin8
3.8 Localisation of bacteriorhodopsin (BR) and proteorhodopsin (PR) at the surface of the oocyte
membrane, investigated by chemiluminescence 123
+3.8.1 Whether the HA-tag interferes with the function of an HA-tagged retinal H pump depends on
its position 124
4. Conclusions 125
Materials and Methods 127
On the use of Xenopus laevis oocytes for the heterologous expression of membrane proteins 128
1. Molecular biology 129
1.1 cDNA constructs and cRNA synthesis 129
Wilson Disease Protein (WNDP)9
Proteorhodopsin (PR) and Bacteriorhodopsin (BR)9
1.2 Heterologous expression in Xenopus laevis oocytes 130
2. Surface detection using chemiluminescence (WNDP HA-constructs, PR- and BR-NT-HA) 130
3. Western Blot (WNDP-, PR-, BR- HA-constructs)1
44. Electron microscopy (WNDP constructs) 132
4.1 Postembedding immunogold labelling2
4.2 Freeze-fracture replica labelling3
5. Expression of WNDP constructs in Sf9 cells4
6. Electrophysiology: two-electrode voltage clamp (PR- and BR constructs) 135
Zusammenfassung 138
Bibliography 144
Acknowledgements 156
Glossary 157
Curriculum Vitae 159


5Foreword

On the importance of transmembrane proteins for cell homeostasis

Cell membranes are vital for the life of the cell, and further on for the organisation of tissues,
organs and proper function of an organism. Inside eukaryotic cells they confer specificity to
the membranous organelles (nucleus, endoplasmatic reticulum, Golgi apparatus,
mitochondria, chloroplasts, vacuoles). The fluid mosaic model of cell membranes proposed by
J. Singer and G. Nicolson (1972) is still valid today. In this model membranes are built up by
phospholipids with peripheral and integral proteins (Singer and Nicolson, 1972). The authors
also recognised that the latter protein group is of crucial importance for the structural integrity
of membranes. Since the publication of this model, a tremendous amount of information has
been learned about the variety, structure and function of integral (or transmembrane) proteins.
They confer functional specificity to the different types of membranes and contribute to
communication between cells.
Transport proteins are a subgroup of transmembrane proteins which have a great importance
in the cell homeostasis by facilitating the passage of ions and molecules, for which the lipid
bilayer has a low permeability; by building up electro- and or chemical gradients across
membranes necessary for cell energetics. Transport proteins can be divided into two major
groups: channels (transport their substrate in the direction of its electrochemical gradient and
interact only weakly with it) and carriers or transporters (most of which transport their
substrate against its concentration gradient and undergo a series of conformational changes to
transfer the bound substrate across the membrane = ‘active transport’) (based on The
Molecular Biology of the Cell). Transmembrane ion gradients, necessary for excitability,
energy storage and volume regulation are built up by two broad classes of active transport
systems. Primary active transport utilises a primary energy source, such as light, redox energy
or energy derived from ATP hydrolysis. In secondary active transport, the uphill movement of
an ion species or solute molecule is coupled to the downhill movement of another ion species
(Adam et al., 1995; Bröer and Wagner, 2003; Läuger, 1991).
The present work wishes to contribute with information on two members of the
primary active transporter group, which differ both in structure and function: Wilson Disease
Protein which uses the energy released by ATP hydrolysis to transport copper across cell
membranes, and Proteorhodopsin, which uses the energy of light to build up a proton
gradient across the bacterial cell membrane.












Wilson Disease Protein

The effect of sequence variations on the plasma membrane localisation
in Xenopus laevis oocytes