MS-based quantitative analysis of the CRM1 export pathway and spatial proteomics of the Xenopus laevis oocyte
von Samir Karaca
Datum der mündl. Prüfung:2014-10-27
Betreuer:Prof. Dr. Henning Urlaub
Gutachter:Prof. Dr. Henning Urlaub
Gutachter:Prof. Dr. Dirk Görlich
EnglischNuclear 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.
Keywords: Nucleocytoplasmic transport; CRM1 dependent nuclear export; Nucleocytoplasmic partition; Quantitative mass spectrometry; Proteomics