Post-translational insertion of a small tail-anchored protein into the membrane of the endoplasmic reticulum [Elektronische Ressource] / presented by Milan Spasic

Milan Spasic

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2005
• Nombre de lectures: 47
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

INAUGURAL - DISSERTATION
Submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
Rupert-Karl-Universität Heidelberg
for the degree of
Doctor of Natural Sciences
Presented by
Master of Science Milan Spasic
from Belgrade, Serbia
2005
Date of oral examination:POST-TRANSLATIONAL INSERTION OF A SMALL
TAIL-ANCHORED PROTEIN INTO THE MEMBRANE
OF THE ENDOPLASMIC RETICULUM
Referees:
Professor Dr. Bernhard Dobberstein
Professor Dr. Irmgard SinningAcknowledgements
This work has been accomplished in the lab of professor Bernhard Dobberstein in ZMBH,
Heidelberg. I am grateful to Bernhard for his support, suggestions and patience...and most of all for
sharing with me his overwhelming enthusiasm and joy of science. I take this opportunity also to
acknowledge professor Irmgard Sinning for being my second referee.
I would like to thank all the members of the lab for a friendship and support. Oliver Schlenker
from Irmi Sinning's group in BZH, Heidelberg, helped me a lot during experimental work. Thanks Oli
for fruitful discussions, too (especially during meetings in Manchester pubs). My special thanks go to
Ute and Gerry, Mr. Anshuman, Christoph and Sabina. I was lucky to have you around, my friends. It
was a great time...
This work was not funded, but was heavily supported by members of the Heidelberg Serbian-
Croatian-Bosnian community.
Finally, I would like to dedicate this work and efforts put into it to two beautiful angels that are
always watching over me. I love you.
...volim vas...Aleksandru i Maji1. ABSTRACT..........................................................................................................1
2. INTRODUCTION ..................................................................................................3
2.1. Structure and topology of membrane proteins ........................................................................ 3
2.1.1. Integral membrane proteins ................................................................................................... 3
2.1.1.1. Single-spanning integral membrane proteins.................................................................... 4
2.1.1.2. Multi-spanning integral membrane proteins..................................................................... 5
2.1.2. Peripheral membrane proteins ............................................................................................... 5
2.2. Biosynthesis of membrane proteins...........................................................................................6
2.2.1. Topological determinants for insertion of proteins into the bacterial or ER membranes ...... 7
2.2.2. Protein targeting and insertion into the bacterial plasma membrane ..................................... 8
2.2.2.1. Cytosolic factors that mediate targeting to the bacterial membrane................................. 9
2.2.2.2. Components of the bacterial membrane required for protein insertion ...........................10
2.2.2.3. Complexity of membrane protein biogenesis in bacteria.................................................11
2.2.3. Protein targeting and insertion into the endoplasmic reticulum............................................12
2.2.3.1. Cotranslational targeting to the ER..................................................................................13
2.2.3.2. Post-translational Sec-dependent targeting to the yeast ER.............................................15
2.2.3.3. Post-translational targeting and insertion of tail-anchored proteins ................................17
2.3. Analysis of the post-translational targeting and insertion of a tail-anchored ER membrane
protein.........................................................................................................................................19
2.3.1. Ribosome Associated Membrane Protein 4 (RAMP4).........................................................20
3. MATERIALS AND METHODS ...........................................................................22
3.1. Materials .....................................................................................................................................22
3.1.1. Chemicals..............................................................................................................................22
3.1.2. Buffers, solutions and media.................................................................................................22
3.1.3. Bacterial strains and mammalian cell lines...........................................................................24
3.1.4. Enzymes..............24
3.1.5. Oligonucleotides ...................................................................................................................24
3.1.6. DNA standards for electrophoresis.......................................................................................25
3.1.7. Plasmids................................................................................................................................25
3.1.8. Antibodies.............................................................................................................................26
3.1.8.1. Primary antibodies ...........................................................................................................26
3.1.8.2. Secondary antibodies .......................................................................................................26
3.1.9. Protein standards for electrophoresis....................................................................................26
3.1.10. Kits........................................................................................................................................26
3.1.11. Computer programs ..............................................................................................................27
3.2. Methods...................27
3.2.1. DNA manipulation techniques..............................................................................................27
3.2.1.1. Constructions of plasmids..27
3.2.2. In vitro transcription and purification of mRNA ..................................................................29
3.2.3. Protein synthesis ...................................................................................................................30
3.2.3.1. In vitro translation in the rabbit reticulocytes lysate........................................................30
3.2.3.2.translation in wheat germ....................................................................................31
3.2.4. Depletion of nucleotides .......................................................................................................32
3.2.5. Protein precipitation..............................................................................................................32
3.2.5.1. Ammonium sulfate precipitation .....................................................................................32
3.2.5.2. TCA precipitation ............................................................................................................33
3.2.6. Protein electrophoresis..........................................................................................................333.2.6.1. Coomassie staining ..........................................................................................................33
3.2.6.2. Silver staining ..................................................................................................................33
3.2.7. Western blotting....................................................................................................................34
3.2.8. Denaturing immunoprecipitation..........................................................................................34
3.2.9. Preparation and coupling of antibodies to CNBr-sepharose.................................................35
3.2.9.1. Purification of anti-opsin antibodies................................................................................35
3.2.9.2. Coupling of anti-opsin antibodies to CNBr-sepharose ....................................................35
3.2.10. Affinity purification of RAMP4op .......................................................................................36
3.2.11. Gel filtration chromatography of proteins from the HeLa cytosol .......................................36
3.2.12. Preparation of rough microsomal membranes ......................................................................37
3.2.12.1.Preparation of dog pancreas rough microsomal membranes (RM) .................................37
3.2.12.2.Preparation of puromycine-high salt washed membranes (PKRM) ................................38
3.2.12.3.Preparation of trypsin-treated PKRM (PKRM-T) ...........................................................39
3.2.12.4.Preparation of NEM-treated PKRM (PKRM-NEM) .......................................................40
3.2.13. Sucrose density gradient centrifugation................................................................................40
3.2.14. HeLa cells manipulation .......................................................................................................41
3.2.14.1.Transfection .....................................................................................................................41
3.2.14.2.Cell lysis ........................................................................................................................