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Conformational dynamics of coatomer [Elektronische Ressource] : functional and structural studies / presented by Julian David Langer

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124 pages
I Dissertation submitted to the Combined Faculties for the Natural Sciences a nMda thfeomr atics of the Ruperto-Carola University of Heidelbergm,a nGye r for the degree of Doctor of Natural Sciences presented by Julian David Langer, Diplom-Chemiker born in Heidelberg Oral examination: . II Conformational dynamics of coatomer: functional and structural studies. Referees: Prof. Dr. Felix Wieland Prof. Dr. Irmgard Sinning ITable of contents Table of contents......................................................................................................I Abstract (english).....................................................................................................IV Abstract (german).....................................................................................................V Abbreviations...........................................................................................................................VI List of figures..........................................................................................................VII List of tables............................................................................................................IX 1. Introduction....................................................................................................................11.1.
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I








Dissertation




submitted to the
Combined Faculties for the Natural Sciences a nMda thfeomr atics
of the Ruperto-Carola University of Heidelbergm,a nGye r
for the degree of

Doctor of Natural Sciences






presented by

Julian David Langer, Diplom-Chemiker
born in Heidelberg


Oral examination: . II







Conformational dynamics of coatomer:
functional and structural studies.















Referees: Prof. Dr. Felix Wieland
Prof. Dr. Irmgard Sinning I
Table of contents

Table of contents......................................................................................................I
Abstract (english).....................................................................................................IV
Abstract (german).....................................................................................................V
Abbreviations...........................................................................................................................VI
List of figures..........................................................................................................VII
List of tables............................................................................................................IX
1. Introduction....................................................................................................................1
1.1. The Secretory Pathway................................................................................1
1.2. Vesicular Transport: The three coating system.s...........................................5.......
1.2.1. Clathrin-coated vesicles........................................................................5
1.2.1.1. Structural studies on clathrin-coated vesicle.s.......................................6
1.2.1.2. Conformational changes in adaptor proteins...............................................9
1.2.1.2.1 . The adaptor protein 2 (AP-2) system............................................9
1.2.1.2.2. The adaptor protein 1 (AP-1) system...........................................11
1.2.1.2.3 . Golgi-localizing,γ -adaptin ear homology domain, Arf-binding p.r.o1te2ins.
1.2.2. COPII-coated vesicles........................................................................1..2
1.2.3. COPI-coated vesicles..........................................................................1..4
1.2.3.1 . The COPI budding process.......................................................................15
1.2.3.2. Summary: Comparison of COPI with the other vetiosinc ulsaystems.........20
1.3. Aims of present work..............................................................................23
2. Results.........................................................................................................24
2.1. Conformational dynamics of coatomer.................................................................2..4...
2.1.1. A conformational change γ-inC OP: Screening p24-family members..........2..4.....
2.1.2. Limited proteolysis and screening of other coa tosmuebrunits...........................26
2.1.3. Labeling of coatomer with fluorescent dyes...............................................27
2.1.4. Labeling with amine-reactive dyes.......................................................8......2
2.1.5. Functionality of labeled coatomer.......................................................3..1.....
2.1.6. Specific immobilization of labeled coatomer......................................................34
2.1.7. Surfaces with Cy3-Cy5-labeled coatomer..........................................................39
2.1.8. Inter- or intramolecular FRET?.........................................................4..0....
2.1.9. Assessing the number of attached dyes..................................................41
2.1.10. Approximating the FRET efficiency .E...................................................42app
2.1.11. Interaction of coatomer with cytoplasmic tail nsd omofa iligand proteins...........43
2.1.12. ELISA-like binding assay....................................................................4..6.
2.1.13. Rare events.......................................................................................................48
2.2. Electron Microscopic investigation of COPI vesicle.s................................................49
2.2.1. Preparation of COPI vesicles in vitro..................................................4..9......
2.2.2. Embedding of chemically fixed vesicles into ax .m..a..t.r.i.................................52
2.2.3. Cryo-electron microscopy of COPI vesicles gener atine dvitro..........................53
2.2.4. Using "backed"-Quantifoil..........................................................................5..5.
2.2.5. Direct preparation and imaging of COPI vesicle..s............................................57 II
3. Discussion......................................................................................................64
3.1. Conformational dynamics of coatomer.......................................................6..4...
3.1.1. Data presented in this work..............................................................6..4..
3.1.2. Membrane protein capture and coat lattice form.a..t.io..n..................................66
3.2. Electron microscopy and tomography of COPI vesi.c.l.e.s........................................69
4. Materials and Methods.................................................................................................70
4.1. Materials......................................................................................................70
4.1.1. Reagents...........................................................................................70
4.1.2. Peptides..............................................................................................70
4.1.3. Beads................................................................................................7 1
4.1.4. Molecular weight standards for SDS – PAGE....................................................71
4.1.5. Protease – Inhibitors..........................................................................72
4.1.6. Antibodies..........................................................................................72
4.1.6.1. Primary antibodies.......................................................................7..2.....
4.1.6.2. Secondary antibodies.........................................................................73
4.1.7. Activated fluorophores........................................................................7.3
4.2. Equipment................................................................................................................74
4.2.1. FPLC-Anlagen.....................................................................................74
4.2.2. SMART..............................................................................................74
4.2.3. Spectrophotometer..............................................................................7.4
4.2.4. Single molecule-sensitive confocal setup...................................................74
4.2.5. Electron microscopes...........................................................................7..7
4.3. Methods....................................................................................................................77
4.3.1. SDS – PAGE.......................................................................................77
4.3.1.1 . Stock solutions for SDS - PAGE........................................................77
4.3.1.2. Separation gels..........................................................................7..8....
4.3.1.3 . Stacking gels.............................................................................7..9..
4.3.2. Sample preparations..........................................................................7..9
4.3.2.1. Sample preparation for SDS – PAGE........................................................79
4.3.2.2. Tri-Chloro-acetic acid (TCA) – precipitation........................................79
4.3.2.3. Immunoprecipitation (IP)....................................................................80
4.3.3. Staining of proteins in SDS – gels..................................................8..0.....
4.3.3.1. Coomassie-Staining............................................................................80
4.3.3.2. Silver stain.................................................................................8.1
4.3.4. Western blot analysis.......................................................................................8.1
4.3.4.1. Transfer of proteins separated by SDS-PAGE onto FP-VDmembranes.....81
4.3.4.2. Ponceau-Staining of proteins on PVDF-membranes.............................82
4.3.4.3. Immunochemical detection of proteins on PVDF-memnebsra...................82
4.3.5. Bradford assay..................................................................................83
4.3.6. Isolation of coatomer from rabbit liver cy.to..s.o..l..........................................83
4.3.6.1. Isolation of rabbit liver cytosol....................................................3......8
4.3.6.2. Ammoniumsulfate precipitation of rabbit liver ocly..t.o.s.............................83
4.3.6.3. DEAE anion exchange chromatography of ASP...................................84
4.3.6.4. SourceQ anionic exchange chromatography of the DEpAoEol.................84
4.3.6.5. Concentration of the SourceQ-Pools...................................................84
4.3.6.6. Buffers for coatomer preparation.......................................................85
4.3.7. Labeling coatomer...............................................................................86
4.3.8. Analysis of labeled coatomer subunits.....................................................86
4.3.9. Isolation of rat liver Golgi...................................................................................87
4.3.10. Pull down experiments.......................................................................8..7. III
4.3.11. Membrane binding assay....................................................................8..8....
4.3.12. In vitro COPI vesicle budding assay............................................................8......8
4.3.12.1 . 17h gradient purification..............................................................8......8
4.3.12.2. 1h cushion centrifugation...................................................................89
4.3.12.3 . Sucrose-free preparation of crude vesicles........................................90
4.3.13 . Grid preparation fonr egative staining........................................................90
4.3.14. Grid preparation for cryo electron tomography............................................90
4.3.15. Precipitation assay............................................................................................91
4.3.16. Limited proteolysis............................................................................................91
4.3.17. Antibody purification...........................................................................91
4.3.18. Surface preparation............................................................................92
4.3.19. Calculation of FRET efficiency E...........................................................92appr
4.3.20. ELISA-like binding assay....................................................................9..4.
4.3.21. Electron microscopy and tomography.........................................................95
4.3.21.1 . Aquisition of tilt series...........................................................................9..5...
4.3.21.2. Tomographic reconstruction......................................................................95
4.3.21.3 . Segmentation of tomograms...............................................................96
5. References....................................................................................................97

Acknowledgments.................................................................................................................113 IV
Abstract

In my PhD thesis I have investigated molecularn ismesc hin the biogenesis of membrane
vesicles.
Formation of transport vesicles involves polymtieorniz aof cytoplasmic coat proteins. In COPI
vesicle biogenesis, the heptameric complex coa toims erecruited to donor membranes by
the interaction of multiple coatomer subunits withb udthdein g machinery. Specific binding to
the trunk domain of coatomer's suγb-uCnOitP of the Golgi membrane protein p23 induces a
conformational change in tγh-es ubunit, leading to polymerization of the cionm vpilterxo .
Using a combination of biochemical assays ands aayn baassed on single-molecule, single-
pair fluorescence resonance energy transfer, dw et hafitn this conformational change is only
induced by dimers of the p24-family proteins d p2p32 4,a nand neither by the other p24-
family members nor by cargo proteins. This cotinofnoarml achange takes place in individual
coatomer complexes, independent of each othetrh,e arneda rrangement induced γ-inC OP is
transmitted within the complex toα -istsu bunit.α -COP is one of coatomer's subunits capable
of binding to dibasic cargo motifs, and alsoa nsahlogwys to the Clathrin molecule. We
propose a model in which capture of membranne mpraoctehinery triggers cage formation in
the COPI system.
At the nanometer resolution I started inves tigathtien g structure of the lattice of
conformationally changed coatomer on COPI vesicglesn erated in vitro from purified Golgi
membranes and coating machinery, using cryo ne letcotmroography. Initial data on coated
vesicles and coated buds is presented. V
Zusammenfassung

In meiner Doktorarbeit habe ich die molekularehna niMsemcen der Biogenese von COPI-
Vesikeln untersucht.
Der Transport von Proteinen und Membranen in eeinuekra ryotischen Zelle erfolgt über
vesikuläre Träger. Zur Bildung dieser Vesikel peorilsyimeren Hüllproteine, die sowohl in einer
löslichen, cytosolischen Form als auch in einebr anmgembundenen Form vorliegen. In der
Biogenese eines COPI Vesikels bindet der heptamHeürell komplex Coatomer an die
Donormemrbanen über multiple Interaktionen mit Madsecrh inerie zur Abknospung der
Vesikel. Die Interaktion dγe-r Untereinheit von Coatomerγ -C(OP) mit dem transmembran-
Protein p23 induziert einen Konformationswechsel γ-inCOP, der zur Polymerisation des
Komplexes in vitro führt.
In dieser Arbeit wurden biochemische und biolpishcyhseik aMethoden verwendet, um diesen
Konformationswechsel in Coatomer zu untersuchen. beDia wurde ein Verfahren zur
Untersuchung der Konformation individueller Coatomr-eKomplexe mit Einzelmolekül-
Fluoreszenz-Resonanz-Energie-Transfer etabliert. r Debeschriebene Konformationswechsel
in γ-COP wird nur durch dimere der Proteine p23 u ndin dpu2z4iert, und nicht durch andere
Mitglieder der p24-Familie oder Frachtproteine.f inEdr et in einzelnen Coatomer-Komplexen
statt, und wird in die periphere Untereαi-nhCeOiPt weitergeleitet. α-COP ist eine der beiden
Untereinheiten von Coatomer, die Frachtmoleküle dmibiatsischen Signalsequenzen binden;
zudem zeigtα -COP Analogien zu Clathrin übαe-r solenoide undβ -Propeller-Domänen. In
dieser Arbeit wird ein Modell vorgeschlagen, in diem Bindung von spezifischen
Transmembranproteinen die Polymerisierung der Hürollpteine und die Ausbildung der COPI-
Vesikelhülle induziert.
In einem zweiten Projekt wurde die Struktur votonm eCro ain der COPI-Hülle auf in vitro
generierten COPI Vesikeln durch cryo-Elektronenmoiksrkopie und Tomographie untersucht.
Erste tomographische Rekonstruktionen von Vesikelnu nd COPI-Knospen an
Donormembranen werden gezeigt. VI
Abbreviations

AA (aa) Amino Acids
AP Adaptor protein
APD Avalanche photo diode
APS Ammonium persulfate
Arf ADP-ribosylation factor
ATP Adenosin tri-phosphate
BFA Brefeldin A
BSA Bovine serum albumin
COP Coat protein
DMMA Dimethylmaleic acid anhydride
DMSO Dimethyl sulfoxide
DNA Desoxyribonucleic acid
DTT Dithiothreitol
E Approximated energy transfer efficiency App
EDTA Ethylendiaminetetra-acetic acid
ER Endoplasmic reticulum
ERGIC ER-Golgi intermediate compartment
FRET Fluorescence Resonance Energy Transfer
GAP GTPase activating protein
GDP Guanosine diphosphate
GEF Guanosine nucleotide exchange factor
GST Glutathione-S-transferase
GTP Guanosine tri-phosphate
h hour
Hepes 4-(2-hydroxyethyl)-1-piperazin-ethanc sualfcoidn i
HPLC High pressure liquid chromatography
HRP Horse radish peroxidase
IgG Immunoglobulin class G
IMAC Immobilized metal affinity chromatography
IP Immunoprecipitation
IPTG Isopropyl-1-βth-iDo-galactopyranoside
K Dissociation constant D
kDa kilo-Dalton
kHz kilo-Hertz
MALDI Matrix assisted laser desorption/ioniza tio n
min minute
NP-40 Nonidet® P40 (Nonylphenylpolyethylenoel )g lyc
nt Nucleotides
OD Optical density
PBS Phosphate buffer saline
PBS-T Phosphate buffer saline + Tween 20
PCR Polymerase chain reaction
PMSF Phenylmethulsulfonyl fluoride
PVDF Polyvinyldifluoride
rpm Revolutions per minute
s second
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide geelelctrophoresis
SNARE Soluble N-ethylmaleimide sensitive factatocrh maetnt protein receptor
TCA Trichloroacetic acid
TEMED N, N, N’, N’-Tetramethylethylenediamine
TGN Trans Golgi network
TMB Tetramethylbenzidin
wt wild type VII
List of figures


Figure 1: Schematic representation of the Secretory Pa.t hway
Figure 2: Scheme of the Clathrin triskelion and desCilganthsr ino f lattices.
Figure 3: Structural similarities of coat proteins.
Figure 4: Scheme of the COPI budding process.
Figure 5: Scheme of p24-family members structure anndc ess eqoufe cytoplasmic
domains.
Figure 6: Precipitation of coatomer by p24-family .p roteins
Figure 7: Limited proteolysis of coatomer.
Figure 8: Analysis of coatomer after labeling witnht admiffeoruents of Cy3-NHS esters.
Figure 9: Analysis of coatomer after labeling with erNsH So-f eCsty3 and Cy5.
Figure 10: Functionality of labeled coatomer: Bindien g cytoto ptlhasmic domains of p23
and OST48.
Figure 11: Functionality of labeled coatomer: Membrane bin dainsgsay.
Figure 12: Functionality of labeled coatomer: In avtiitoron fofr mCOP vesicles.
Figure 13: Immobilisation strategies to specifically tethebre ledla coatomer to glass
surfaces.
Figure 14: Low intensity surface scans of glass surafateceds wcitoh a BSA-antibody
solution.
Figure 15: Low intensity surface scans of glass surafateceds wcitoh a BSA-antibody
(CM1) solution and incubated with Cy3-Cy5-laboealetdo mcer.
Figure 16: Low intensity surface scans (20µm x 20µm) osfu rfgalacesss coated with a
BSA-antibody solution and incubated with a 1u:r1e omf ixtCy3-labeled
coatomer and Cy5-labeled coatomer.
Figure 17: Single-pair FRET in coatomer.
Figure 18: Normalized histogram for Cy3-Cy5-labeled coatonmoe rp,e ptide added.
Figure 19: Normalized histogram for Cy3-Cy5-labeled cr,o ataofmteer addition of OST48.
Figure 20: Normalized histogram for Cy3-Cy5-labeled co,a toamfter addition of p23d.
Figure 21: ELISA binding curves to monitor t hea ndK the total number of available D
binding sites of coatomer for cargo proteins pinr estehnece and absence of
dimeric p23.
Figure 22: Fluorescence intensity trace of a Cy3-Cy5- lacboealteodmer that oscillates
between two FRET states.
Figure 23: Images of γGST-PCOPI vesicles, purified by sucrose gradient ubamndit tesd
to negative stain. VIII
Figure 24: Images of γGST-PCOPI vesicles purified either via a 17h-gr(aAd-ieCn)t or
centrifugation on a sucrose cushion).
Figure 25: Images of COPI vesicles (2.5mg of Golgi) prwepitha reGdTγPS, purified by
centrifugation on a sucrose cushion, and subtom itntedg ative stain.
Figure 26: Images of COPI vesicles prepared with GrToP , ine mvbitedded in a polymer
matrix and stained with Ruthenium Red an.d OsO4
Figure 27: Images of COPI vesicles (2.5mg of Golgi)d pwreitpha reGTγPS, purified by
centrifugation on a sucrose cushion, and subcmryiott ede lectron microscopy.
Figure 28: Low (A) and high (B) magnification ImagIe s veosf iclCeOsP, deposited on a
carbon-backed Quantifoil grid, embedded in vi triecoeu. s
Figure 29: Low (A) and high magnification (B) ImagIe s veosf icCleOsP with 6nm gold
dots deposited on carbon-backed Quantifoil, anedd demd bin vitroues ice.
Figure 30: Section of a tomogram of a sample of pPuγrSif-ieCdO PI GTvesicles
deposited on a thin carbon film, embedded uisn ivcietr.e o
Figure 31: TEM image of GTP-COPI vesicles embedded ino uvsi treice.
Figure 32: Section of a tomogram recorded of COPI soanmtapilneisn g cCOPI vesicles
directly applied to a Quantifoil grid after ptiroenp.a ra
Figure 33: Sections of a tomogram recorded of COPI csoanmtapilneisn g COPI vesicles
directly applied to a Quantifoil grid after ptiroenp.a ra
Figure 34: Rendering of coated vesicle boxed in Figsuinreg 3th3e Aumira software
package.
Figure 35: Sections of a tomogram of a sample direedc tlyto aap plQuantifoil grid after
preparation, containing a coated bud.
Figure 36 Rendering of the coated bud boxed in Figiunrge t3he5 Ausmira software
package.
Figure 37: Model for putative role of the conformationagle cihnaα -COP during COPI
vesicle formation.
Figure 38: Structure of NHS-ester-derivative of theh orfeluso rop
Figure 39: Scheme of the custom-built confocal setnu p thuis eds tuidy.
Figure 40: Properties of filters and dichroic mirrorsy ede mpinlo the single-molecule
sensitive setup.
Figure 41: Fluorescence intensity trace of a typyi cadl onsoinrg-l and acceptor-labeled
coatomer complex, and scheme of data extraction.
Figure 42: Schematics of steps involved in calcuElatio. n of App

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