Browsing by Subject "GTPase"

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Die kleine GTPase RagA und der potentielle Pankreastumormarker TCTP, zwei Bindungspartner des Kerntransport-Proteins RanBP3
(2006) Schleicher Lilia; Biesalski, Hans-Konrad
RanBP3 is a component of the Crm1-mediated protein export from the nucleus. It favors the binding of export substrate to the RanGTP-Crm1 complex in the nucleoplasm. Upon translocation through the nuclear pore into the cytoplasm, the export complex is dissociated by RanBP1 and RanGAP. It was the objective of this thesis to advance the understanding of RanBP3 function by identifying novel binding partners. Using Yeast-Two-Hybrid Screening, two new interactors of nuclear protein RanBP3 were found, the small GTPase RagA and the translationally controlled tumour protein (TCTP). As a minimal requirement a short C-terminal part of RagA (residues 275-313) binds to the N-terminal region of RanBP3. Full length recombinant RagA was expressed. To substantiate the two-hybrid interactions, the binding of RagA to RanBP3 was verified by in vitro binding assays using recombinant proteins. RagA competes with exportin Crm1 for binding to RanBP3. Likewise, the nucleotide exchange factor for Ran, RCC1, binds to the RanBP3-Ran complex and inhibits RagA binding. Expression of GFP-fusion proteins established the cellular localisation of RagA in living and fixed cells. It is found predominantly in and at the nucleus. A model of RagA-import and -export is suggested. Residues 100-172 in the C-terminal part of TCTP form the binding site for its interaction with RanBP3. Using Northern Blot analysis, two mRNAs of TCTP were found in all human tissues examined. The TCTP sequence was completed using an ovarian cDNA library and the TCTP was cloned and expressed. By means of quantitative PCR it was shown that TCTP belongs to the small group of very abundant mRNA molecules in the cell. The TCTP-RanBP3 interaction was verified by in vitro binding assays using recombinant proteins. A polyclonal antibody to TCTP was generated. By using a GFP-TCTP fusion protein and anti-TCTP antibody the cellular localisation of TCTP was analysed. In Cos7-cells TCTP is found in and around the nucleus. In human cell lines most of the TCTP is localised at the nuclear membrane. Using specific anti-TCTP antibody it was shown that the TCTP level is increased in pancreatic tumours. A differential diagnosis of pancreatitis and pancreas tumour is feasible. TCTP has the potential to become a pancreatic tumour marker or a prognostic factor for ductal type adenocarcinomas of the exocrine pancreas.