FU Berlin Digitale Dissertation
Luiza Bengtsson : Novel integral membrane proteins of the inner nuclear membrane Characterization of LUMA, Native LAP2β complexes Neue integrale Membranproteine der inneren Kernmembran
|Abstract| |Table of Contents| |More Information|

Abstract The nuclear envelope (NE) is one of the least characterized structures in the eukaryotic cell. Although the dynamics of its alterations during the cell cycle are well described, the detailed study of its functional roles is hampered by the small number of known proteins specifically located to it. This work focuses on the identification of novel components of the inner nuclear membrane (INM) and their characterization. Besides that, interaction partners of LAP2β, a protein known to reside in the INM, were searched for. Through combining subcellular fractionation methods and proteomic tools, a proteomic screen of the NE was performed. Based on the experimental evidence that INM proteins resist extraction with TritonX-100, two novel integral membrane proteins were classified as INM proteins. These proteins are KIAA0810 and LUMA. Their localization to the INM was confirmed by indirect immunofluorescence of transiently transfected cells overexpressing these proteins. LUMA, a protein of an apparent molecular weight of 45kDa, displays no similarity to any known protein and contains no homology domains. The hydropathy profile for LUMA suggests four membrane spanning segments with a large loop between the first two. By overexpressing deletion mutants in COS-7 cells, the topology of LUMA could be determined and the INM targeting domain identified. Both, N- and C-terminus of LUMA point towards the ER lumen, the large loop is oriented to the nucleoplasmic side. The smallest part of LUMA which is sufficient for INM localization are the amino acids 201-345, comprising the C-terminal part of the nucleoplasmic loop and the second transmembrane segment. Overexpression of full length LUMA induces a pronounced phenotype in the cells - the NE shows a swollen appearance and has vesicle like structures attached to it. Additionally, the nuclear chromatin appears less dense. A knock-down of LUMA leads to the formation of vesicle-like structures containing chromatin outside the nucleus and an altered chromatin structure inside. A chromosome binding assay showed that LUMA binds chromosomes and causes their decondensation. These observations indicate a role for LUMA in the regulation of gene expression and/or the cell cycle. They also show that LUMA is crucial for the integrity of the NE structure. To understand the functions of NE, a detailed understanding of the interplay between its proteins is necessary. To investigate that, native complexes of LAP2β were purified and characterized. Applying different techniques, after solubilization of the membranes with n-dodecyl-beta-maltoside, high molecular weight complexes containing LAP&2beta; could be isolated. Besides LAP2β, LAP2ε was present in these complexes, but no lamins. Further experiments are needed for closer characterization of the isolated complexes.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title 0 TITLE PAGE, TABLE OF CONTENTS 1 INTRODUCTION 8 1.1 Functional organization of the cell nucleus 8 1.2 The nuclear envelope 11 1.2.1 The integral proteins of the inner nuclear membrane 14 1.2.2 The lamina 21 1.3 Questions and approaches 23 2 RESULTS AND DISCUSSION 24 2.1 The nuclear envelope proteome 24 2.1.1 Fractionation of nuclear envelopes and the protein content of the fractions 24 2.1.2 Discovery of two novel proteins of the inner nuclear membrane 27 2.1.3 Localization of KIAA0810 and LUMA in COS-7 cells 27 2.1.4 Bioinformatic characterization of LUMA and KIAA0810; KIAA0668 - a third novel protein of the inner nuclear membrane? 29 2.1.4.1 LUMA 30 2.1.4.2 KIAA0810 32 2.1.4.3 KIAA0668 36 2.1.5 Localization of KIAA0668 in COS-7 cells and its biochemical characterization 37 2.2 Functional characterization of LUMA 40 2.2.1 Overexpression of LUMA in COS-7 cells - comparison with other INM proteins 40 2.2.2 Localization of LUMA deletion mutants - inner nuclear membrane targeting domain and topology of LUMA 43 2.2.3 Downregulation of LUMA in 3T3 cells 47 2.2.3.1 Messenger primer walking 48 2.2.3.2 Transfection of anti-sense oligonucleotides into 3T3 cells 49 2.2.4 Chromatin binding assay 51 2.2.5 Concluding remarks on LUMA 53 2.3 Functional characterization of LAP 2b - the native LAP 2b complexes 54 2.3.1 Solubilization of nuclear envelopes 54 2.3.2 Gel filtration 55 2.3.2.1 FPLC 55 2.3.2.2 SMART high performance liquid chromatography 57 2.3.3 Glycerol gradient centrifugation 58 2.3.4 Blue Native Gel Electrophoresis 59 3 SUMMARY 62 4 ZUSAMMENFASSUNG 64 5 METHODS 66 5.1 Molecular Biology 66 5.1.1 RNA isolation 66 5.1.2 cDNA synthesis 67 5.1.3 PCR 68 5.1.4 Vector construction 70 5.1.4.1 Restriction digest 70 5.1.4.2 Dephosphorylation 71 5.1.4.3 Agarose gel electrophoresis 71 5.1.4.4 Purification of DNA fragments 71 5.1.4.5 Ligation 72 5.1.4.6 TOPO cloning 72 5.1.5 Preparation of competent E.coli 73 5.1.6 Transformation into E.coli 74 5.1.7 Analytical and preparative plasmid preparation 74 5.1.7.1 Analytical plasmid preparation 74 5.1.7.2 Preparative plasmid preparation 75 5.1.8 Sequence analysis of the DNA 75 5.1.9 Protein expression in E.coli 76 5.1.10 In vitro transcription 77 5.1.11 Messenger primer walking 78 5.2 Cell Biology 79 5.2.1 Cell culture (N2a, COS-7, 3T3, CHO) 79 5.2.2 Transfection of mammalian cells 79 5.2.3 Immunofluorescence staining of cells 80 5.2.4 Fluoroscence microscopy 80 5.2.5 Preparation of metaphase chromosomes from CHO cells 80 5.2.6 Preparation of nuclei from N2a cells 81 5.2.7 Preparation of nuclear envelopes from N2a nuclei 82 5.3 Biochemistry 83 5.3.1 Protein concentration assay (Bradford) 83 5.3.2 Solubilization of nuclear envelopes 83 5.3.2.1 TX-100 and urea/carbonate 83 5.3.2.2 n-Dodecyl-b-maltoside, Tween 80 83 5.3.3 SDS PAGE 84 5.3.4 Western Blot 85 5.3.4.1 Semi dry Blot 85 5.3.4.2 Immunodetection 85 5.3.5 2D-BAC/SDS-PAGE 86 5.3.6 Tryptic in gel digest 87 5.3.7 MALDI-TOF mass spectrometry 89 5.3.7.1 Sample preparation 89 5.3.7.2 ZipTip purification 90 5.3.7.3 Identification of proteins 90 5.3.8 TCA protein precipitation 90 5.3.9 Gel filtration 91 5.3.10 Glycerol gradient centrifugation 91 5.3.11 Cross-linking GluDH 92 5.3.12 Blue Native Electrophoresis 92 5.3.12.1 2D BNE/SDS PAGE 93 5.3.13 Purification of proteins expressed in E.coli 93 5.3.14 Chromosome binding assay 94 5.4 Bioinformatics 95 6 LIST OF ABBREVIATIONS 96 7 REFERENCES 98 8 PUBLICATIONS 112 9 CURRICULUM VITAE 113 10 ACKNOWLEDGMENTS 114

More Information:
Online available:	http://www.diss.fu-berlin.de/2002/100/indexe.html
Language of PhDThesis:	english
Keywords:	nuclear envelope, LUMA, proteome, gene expression
DNB-Sachgruppe:	32 Biologie
Date of disputation:	11-Jun-2002
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Prof. Dr. Ferdinand Hucho
Second Referee:	PD Dr. Shiao Li Oei
Third Referee:	Prof. Dr. Jürgen-H. Fuhrhop
Contact (Author):	l.bengtsson@web.de
Contact (Advisor):	hucho@chemie.fu-berlin.de
Date created:	17-Jun-2002
Date available:	18-Jun-2002

 

---

|| DARWIN|| Digitale Dissertationen || Dissertation|| German Version|| FU Berlin|| Seitenanfang ||

---

	Fragen und Kommentare an: darwin@inf.fu-berlin.de
© Freie Universität Berlin 1999