| dc.contributor.advisor | Urlaub, Henning Prof. Dr. | |
| dc.contributor.author | Karaca, Samir | |
| dc.date.accessioned | 2015-09-22T08:21:52Z | |
| dc.date.available | 2015-09-22T08:21:52Z | |
| dc.date.issued | 2015-09-22 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0023-9622-6 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-5274 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | MS-based quantitative analysis of the CRM1 export pathway and spatial proteomics of the Xenopus laevis oocyte | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Urlaub, Henning Prof. Dr. | |
| dc.date.examination | 2014-10-27 | |
| dc.description.abstracteng | Nuclear export is a vital cellular process, which supplies the cytoplasm with essential biological
macromolecules, such as matured tRNAs and ribosomal subunits. This process is carried out by a
set of proteins called exportins. In higher eukaryotes, up to now eight RanGTPase-dependent
exportins have been identified. Among these exportins, CRM1 mediates one of the major nuclear
export pathways with the broadest range of cargoes. So far, more than 100 structurally and
functionally diverse CRM1 cargoes have been described. The CRM1-cargo interaction occurs
through recognition of short peptide sequences, which are called nuclear export signal (NES).
Although, this consensus sequence is commonly present in the primary structure of many
proteins, it might be inaccessible to CRM1. This poses challenges for in silico identification of
CRM1 cargoes. Therefore, this study focused on experimental identification of CRM1 cargoes with
two orthogonal approaches. First, a novel MS-based approach was established to capture in vivo
localization changes between the nucleus and the cytosol upon inhibition of the CRM1 export
pathway with Leptomycin B treatment in HeLa cells. This led to identification of many known and
novel CRM1 cargoes (totally ~140). Some of these were verified by microscopic analysis. Second,
the CRM1 affinity chromatography was employed to selectively enrich RanGTP dependent
interaction partners from X. laevis oocyte extract, which resulted in identification of large number
of CRM1 binders (~640). These two approaches provided a comprehensive catalog of candidate
CRM1 cargoes, which most of them belong to cytoplasmic activities, such as translation,
intracellular membrane trafficking and cytoskeleton based processes. Complementary to CRM1
cargo identification, nucleocytoplasmic distribution of X. laevis proteome was investigated and
this resulted in quantitative mapping of ~6300 proteins. This offered valuable insights into degree
of compartmentation of a eukaryotic cell, and the spatial distribution of distinct molecular
activities, such as RNA metabolism, protein production and degradation. The nucleocytoplasmic
distribution of candidate CRM1 cargoes revealed that ~17% exclusively cytosolic proteins were
RanGTP dependent CRM1 binders. This observation highlights that the permeability barrier of the
nuclear pore complex is alone insufficient to keep cytosolic proteins out of the nucleus and CRM1 safeguards the
nucleus by counteracting leakage of the many cytosolic proteins. | de |
| dc.contributor.coReferee | Görlich, Dirk Prof. Dr. | |
| dc.subject.eng | Nucleocytoplasmic transport | de |
| dc.subject.eng | CRM1 dependent nuclear export | de |
| dc.subject.eng | Nucleocytoplasmic partition | de |
| dc.subject.eng | Quantitative mass spectrometry | de |
| dc.subject.eng | Proteomics | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0023-9622-6-5 | |
| dc.affiliation.institute | Biologische Fakultät für Biologie und Psychologie | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 835366839 | |