Identification and Characterization of Importin 13 Substrates
Imke Baade
Doctoral thesis
Date of Examination:
2017-09-07
Date of issue:
2018-05-09
Advisor:
Kehlenbach, Ralph H. Prof. Dr.
Referee:
Kehlenbach, Ralph H. Prof. Dr.
Referee:
Krebber, Heike Prof. Dr.
Persistent Address: http://hdl.handle.net/11858/00-1735-0000-002E-E3DE-A
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Abstract
English
Nuclear pore complexes embedded in the nuclear envelope regulate the bidirectional transport of macromolecules between the nucleus and the cytoplasm. Small molecules can rapidly move through the permeability barrier of the nuclear pore complex, whereas larger macromolecules typically require nuclear transport receptors to facilitate their diffusion. With more than 20 different transport receptors identified, only three have been reported to mediate both nuclear import and export, namely human exportin 4, yeast Msn5 and human importin 13. The latter was characterized as a bidirectional transport receptor in 2001 and since then several importin 13 import cargoes have been identified. For a long time, however, the translation initiation factor eIF1A remained the only established export cargo. For a better understanding of the physiological significance of nuclear transport receptors and their diverse transport competencies, more cargoes need to be identified. The central aim of this study was to expand the range of known importin 13 substrates using an importin 13 overexpression screen and a quantitative proteomics approach based on stable isotope labeling with amino acids in cell culture (SILAC). This approach should allow for the identification of proteins that bind to importin 13 in pull-down experiments under conditions that promote the formation of either import or export complexes. In the overexpression screen, DBC-1, DMAP1, DDX43 and DDX59 were found to be redistributed to the cytoplasm upon importin 13 coexpression, identifying them as possible importin 13 substrates. Interestingly, transport of these proteins has previously been shown to be Nup358-dependent, suggesting a functional link between importin 13 and Nup358, possibly by Nup358 serving as an assembly or disassembly platform for importin 13 transport complexes. Detailed analysis of DBC 1 showed that its coiled-coil domain is required for interaction with importin 13. In the SILAC based screen, more than 200 proteins were identified as potential importin 13 substrates, greatly expanding the repertoire of known cargoes for this transport receptor. Using importin 13 overexpression experiments, RTCA, FEN1, APEX1, SRP14, NSUN2, HNRNPD, XRCC5, BTF3, EIF2D, XRCC6 and SET were validated as potential importin 13 export cargoes, while ERI1 and NELFCD were identified as potential importin 13 import cargoes. In addition, importin 13 seems to function as an exportin for M9 signal sequence containing proteins. Ultimately, the larger spectrum of importin 13 cargoes should give new insights into the physiological significance of importin 13, its bidirectional transport competence, its unique mechanisms of cargo recognition and cargo release and, eventually, the identification of one or possibly even several conserved nuclear localization signals in cargo proteins.
Keywords: nucleocytoplasmic transport; transport receptors; importin 13
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GGNB - Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften [821]
GGNB - Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences