Identification of the transmembrane protein 147 (TMEM147) as a novel component of the Nicalin-NOMO membrane protein complex [Elektronische Ressource] / Ulf Dettmer

Ulf Dettmer

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 38
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München








Identification of the transmembrane protein 147
(TMEM147) as a novel component of the Nicalin-NOMO
membrane protein complex













Ulf Dettmer
aus
Nürnberg
2010
Erklärung


Diese Dissertation wurde im Sinne von §13 Abs. 3 der Promotionsordnung vom 29. Januar
1998 von Frau PD Dr. Konstanze Winklhofer betreut und vor der Fakultät für Chemie und
Pharmazie vertreten.













Ehrenwörtliche Versicherung


Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, am 10. Juni 2010






……………………………………..
(Ulf Dettmer)










Dissertation eingereicht am: 10. Juni 2010
1. Gutachter: PD Dr. Konstanze Winklhofer
2. Gutachter: Prof. Dr. Christian Haass
Mündliche Prüfung am: 26. Juli 2010

2

Meinen Eltern, meiner Schwester Silke und meiner Freundin Alexandra gewidmet.































One step inside doesn’t mean you understand. (title of a song by The Notwist)
3 Table of contents
Table of contents

Summary ............................................................................................................................... 6
Zusammenfassung ................................................................................................................ 7
1 Introduction ..................................................... 8
1.1 γ-Secretase ...................................................................................... 8
1.1.1 Components of γ-secretase ............................................................................... 10
1.1.2 Assembly and transport of γ-secretase .............................................................. 12
1.2 The Nicalin-NOMO complex .................................................. 13
1.2.1 Components of the Nicalin-NOMO complex ...... 16
1.2.2 Assembly of the Nicalin-NOMO complex............................ 18
1.3 Goals of this thesis ................................................................................................ 19

2. Results......................................................................................................................... 20
2.1 Transmembrane protein 147 co-purifies with the Nicalin-NOMO complex ............. 20
2.2 TMEM147 co-localizes with Nicalin and NOMO in the ER ..................................... 24
2.3 The TMEM147 topology is similar to APH-1 .......................................................... 28
2.4 TMEM147, Nicalin, and NOMO stabilize each other .............. 30
2.5 Nicalin controls complex levels by stabilizing excess molecules ............................ 35
2.6 TMEM147 and NOMO differ in their Nicalin binding requirements ......................... 37
2.7 The interaction of TMEM147 with Nicalin/NOMO is evolutionary conserved ......... 40
2.8 TMEM147, like Nicalin and NOMO, is ubiquitously expressed ............................... 43
2.9 Towards the Nicalin interactome using SILAC ....................................................... 46

3. Discussion ................................................................................................................... 51
3.1 A novel core component of the Nicalin-NOMO complex ........................................ 51
3.2 Nicalin-NOMO complex and γ-secretase assembly occur stepwise ....................... 53
3.3 Nicalin is the master regulator of the Nicalin-NOMO complex ................................ 55
3.4 Analysis of the Nicalin interactome ........................................................................ 58
3.5 The Nicalin-NOMO complex beyond Nodal signaling ............................................ 61
3.6 Outlook .................................................................................................................. 63

4. Materials and methods ................................................................................................. 64
4.1 Equipment ............................................................................ 64
4.2 Chemicals ...................................................... 66
4.3 DNA techniques .................... 67
4.3.1 Plasmids......................................................................................................... 67
4.3.2 Oligonucleotides ............................................................. 68
4.3.3 RNA isolation and cDNA synthesis ................................................................. 68
4.3.4 PCR ........................................................ 69
4.3.6 Agarose gel electrophoresis ........................................... 69
4.3.9 Restriction enzyme treatment ................. 70
4.3.10 DNA ligation ................................................................................................... 70
4.3.11 Transformation of competent bacteria ............................................................ 70
4.3.12 DNA isolation from bacteria ............................................................................ 71
4.4 Cell culture ................................. 72
4.4.1 Cell lines................................................................................. 72
4.4.2 Cell cultivation ................................................................................................ 73
4.4.4 Cell transfection.............................................................................................. 73
4.4.5 Cell transduction, lentiviral ....................................................... 74
4.4.6 Cell cryoconservation ..................................................... 75

4 Table of contents
4.5 Protein analysis .................................................................................................... 76
4.5.1 Antibodies ............... 76
4.5.2 Total protein lysates ........................ 77
4.5.3 Membrane protein lysates .............................................................................. 77
4.5.4 Deglycosylation .............................................................................................. 78
4.5.5 Immunoprecipitation (IP) ........................ 78
4.5.6 Density gradient centrifugation ............... 79
4.5.7 SDS-PAGE ...................................... 80
4.5.8 Transfer of proteins to PVDF membranes ...................................................... 81
4.5.9 Immunological detection of proteins ............................................................... 81
4.6 Immunocytochemistry .................................................................... 81
4.7 Mass spectrometry ................................................................ 82
4.8 Zebrafish techniques .............................. 82
4.9 Bioinformatical methods ........ 84

5. List of references ......................................................................................................... 85
6. List of abbreviations ..................................................................... 94
7. List of companies .......................... 97
8. Curriculum vitae ........................................................................................................... 98
9. Publications and meetings ........................................................................................... 99
10. Acknowledgments...................... 100


5 Summary
Summary

Nicastrin is one out of four components of γ-secretase, a membrane protein complex with a
key role in the pathogenesis of Alzheimer’s Disease. Its closest relative is Nicalin (Nicastrin-
like protein) which, together with NOMO (Nodal modulator), is part of a novel, evolutionary
conserved membrane protein complex which is involved in the regulation of the Nodal
signaling pathway in developing zebrafish embryos. Since its native size (200-220 kDa)
could not be satisfyingly explained by the molecular weights of monomeric Nicalin (60 kDa)
and NOMO (130 kDa), the possible existence of additional components, especially in the
low-molecular-weight range, was examined. Using an optimized procedure of the Nicalin
affinity purification approach which had led to the isolation of NOMO, a ~22-kDa protein
could be specifically enriched from membrane protein lysates of cultured human cells and
identified by mass spectrometry as transmembrane protein 147 (TMEM147). TMEM147 is a
novel, highly conserved polytopic membrane protein with a putative topology similar to the γ-
secretase component APH-1. Its specific interaction with Nicalin was confirmed by
immunoprecipitation/immunoblotting experiments and shown to be r