Functional studies of nuclear envelope-associated proteins in Saccharomyces cerevisiae Ida Olsson Stockholm University © Ida Olsson, Stockholm 2008 ISBN 978-91-7155-666-0, pp 1-58 Typesetting: Intellecta Docusys Printed in Sweden by Universitetsservice US-AB, Stockholm 2008 Distributor: Department of Biochemistry and Biophysics, Stockholm University To Carl with love ABSTRACT Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co- ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins. The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharo- myces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nu- clear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and poten- tially in nuclear transport and RNA processing. The integral membrane pro- tein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its abil- ity to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling path- ways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics. In conclusion, the presented results aid in a deeper understanding of func- tions related to the nuclear envelope in revealing a novel link between argin- ine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein. LIST OF PUBLICATIONS The thesis is based on the following publications, which will be referred to by their Roman numerals. I Olsson I., Berrez J.-M,. Leipus A., Östlund C. and Mutvei A. (2007) The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100. Exp. Cell Res. 313:1778-89 II Olsson I. and Mutvei A. (2008) The arginine methyltrans- ferase Rmt2 specifically associates with FG-nucleoporins – implications for a function in nuclear transport. Manuscript. III Leipus A., Olsson I., Berrez J. -M., Hultenby K., Östlund C. and Mutvei A. (2008) Cwh43 is an evolutionary con- served polytopic inner nuclear membrane protein. Submitted to J. Cell Biol.. IV Georgiev A., Leipus A., Olsson I., Berrez J.-M. and Mutvei A. (2008) Characterization of MYR1, a dosage suppressor of YPT6 and RIC1 deficient mutants. Curr. Genet. 53:235- 247 Papers I and IV were reprinted with permissions from the publishers. Additional publications Lebens M., Sun J. B., Sadeghi H., Bäckström M., Olsson I., Mielcarek N., Li B. L., Capron A., Czerkinsky C. and Holmgren J. (2003) A mucosally administered recombinant fusion protein vaccine against schistosomiasis protecting against immunopathology and infection. Vaccine 21 (5-6) 514-20 TABLE OF CONTENTS 1. INTRODUCTION ....................................................................................11 1.1 General introduction...........................................................................11 1.2 Yeast as a model organism.................................................................12 1.3 Functional organization of the nucleus ..............................................13 1.3.1 The nuclear envelope..................................................................13 1.3.2 The nuclear envelope proteome..................................................15 1.3.3 The Nuclear Pore Complex ........................................................17 1.3.4 Nucleocytoplasmic transport ......................................................19 1.3.5 Targeting of proteins to the nuclear envelope ............................20 1.3.6 Chromatin organization and DNA repair....................................21 1.3.7 Function of the nuclear envelope in chromatin organization and gene regulation ....................................................................................22 1.3.8 Role of the nuclear envelope in diseases ....................................22 1.3.9 The nucleolus..............................................................................23 1.4 PKC signalling ...................................................................................24 1.4.1 Cwh43.........................................................................................24 1.5 Post-translational modifications.........................................................25 1.5.1 Arginine methylation..................................................................25 1.5.2 Cellular functions of arginine methylation .................................28 1.5.2 Rmt2 ...........................................................................................30 1.6 Vesicular trafficking...........................................................................31 1.6.1 Rab/Ypt proteins.........................................................................32 1.6.2 Ypt6 ............................................................................................32 2. THE PRESENT STUDY ..........................................................................34 2.1 Aims of the study ...............................................................................34 2.2 Results and discussion........................................................................35 2.2.1 Screen for nuclear membrane associated proteins......................35 2.2.2 The arginine methyltransferase Rmt2 interacts with nucleo-porins (papers I and II) ...................................................................................35 2.2.3 Cwh43 is an integral membrane protein of the inner nuclear membrane (paper III)...........................................................................38 2.2.4 Myr1is a novel membrane associated protein (paper IV)...........40 3. CONCLUDING REMARKS....................................................................43 4. FUTURE PERSPECTIVES......................................................................44 5. SAMMANFATTNING.............................................................................46 6. ACKNOWLEDGEMENTS......................................................................47 7. REFERENCES .........................................................................................49 LIST OF ABBREVIATIONS AdoMet S-adenosyl-L-methionine BMP bone morphogenic protein CWH43 calcoflour white-hypersensitive protein ER endoplasmic reticulum EM electron microscopy GAL galactose GAMT guanidinoacetate methyltransferase GAP GTPase activating protein GEF guanine nucleotide exchange factor GFP green fluoresent protein GTP guanosine 5´- triphosphate INM inner nuclear membrane Kap karyopherin LAP lamina-associated peptide LBR lamin B-receptor MAP mitogen-activated protein MEGA10 decanoyl-N-methylglucamide MYR1 multicopy suppressor of ypt6Δ and ric1Δ NE nuclear envelope NES nuclear export signal NET nuclear envelope transmembrane protein NLS nuclear import signal NPC nuclear pore complex NR nucleoplasmic reticulum ONM outer nuclear membrane ORF open reading frame PKC protein kinase C PM plasma membrane PNS perinuclear space PRMT protein arginine methyltransferase RMT2 protein arginine methyltransferase Smad small mothers against decapentaplegic SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptor SPB spindle pole body TAP tandem affinity purification TGFβ transforming growth factor-β YPT6 yeast protein two 1. INTRODUCTION 1.1 General introduction Proteins, from the Greek word πρώτα ("prota") meaning "of primary impor- tance", are essential components of living cells. They are large organic com- pounds build by long stretches of amino acids joined by polypeptide bonds. The information specifying the sequence of the amino acids is encoded by a gene. Proteins function as catalysts of chemical reactions, provide structural support to cells and organs, and are involved in immune response, transport and a variety of other cellular processes. To understand how cells work and communicate it is of fundamental importance to understand the function of proteins. To achieve this, biochemical and molecular biological tools are used to evaluate the chemical properties, structures, cellular localizations and binding partners of proteins. Genetic tools are used to analyse the cellular pathways in which the proteins work. An important feature of eukaryotic cells is the presence of membrane-bound compartments. The major compartment is the nucleus that encloses the ge- nome. This thesis describes the functions of three proteins associated with the nucleus and the cellular membranes in the budding yeast Saccharomyces cerevisiae. To elucidate the functions