Abstract
Nucleocytoplasmic transport of thousands of molecules is an essential feature of eukaryotic cells. The bulk of the active transport between cytoplasm and nucleus is performed by Ran-dependent nuclear transport receptors known as karyopherins or Importin beta-like proteins. Karyopherins involved in the transport of cargoes into the nucleus are called importins, while karyopherins known as exportins export cargoes out of the nucleus. The directionality of transport is driven by a gradient of the small GTPase Ran. In case of importins, RanGTP binding triggers the release of the cargo, while for exportins, the binding of RanGTP mediates the cargo binding. One of the few bidirectional karyopherins that can both import and export cargoes is Importin13 (Imp13). In addition to several other import cargoes, Imp13 imports Mago-Y14, a core component of the exon-junction-complex (EJC), and Ubc9, the only E2-conjugating enzyme of the sumoylation pathway, into the nucleus. The only export cargo identified to date for Imp13 is the eukaryotic initiation factor 1A (eIF1A), which is essential for translation initiation in the cytoplasm. When I started working on Imp13, detailed structural knowledge on the import factors Importin beta (Imp beta) and Transportin (Tnp) and on the export receptor CAS was available. Only two karyopherins Msn5 and Imp13, were known to be bidirectional transport receptors. In 2009, the structures of the exportins Crm1, ExportinT (Expt) and Exportin5 (Exp5) in complex with RanGTP and their cargoes were published. In the same year, Exportin4 (Exp4) was also identified as a bidirectional transport receptor. However, the molecular understanding of how bidirectional transport receptors such as Imp13 function was limited. How Imp13 can recognize diverse cargoes lacking a typical nuclear localization signal (NLS) or nuclear export signal (NES) was an unresolved question. Furthermore, the means with which RanGTP associates to the same receptor to mediate both cargo release and cargo binding were unclear. During my PhD work I used biochemical assays and x-ray crystallography to elucidate the molecular mechanism underlying Imp13 mediated transport. I first focused on the import branch of the Imp13 cycle. Using biochemical assays, I investigated the nuclear transport mechanism of Mago-Y14 by Imp13. I crystallized and solved the structure of the Imp13-Ubc9 complex at 2.8 Å resolution. Furthermore, I characterized the transport mechanism by in vitro co-precipitation-, competition- and sumoylation assays to further complement information obtained from the structures. I found that Imp13 employs completely different interaction surfaces for binding its import cargoes Mago-Y14 and Ubc9. Ubc9 is bound by the N-terminal arch of Imp13 with the same interaction surface also used for RanGTP binding, while Mago-Y14 is bound in the middle and C-terminal part of Imp13. In in vitro competition assays, both cargoes can displace each other from Imp13 to a similar extent, but cannot bind at the same time. Consistently, the dissociation constants (KD) of Imp13 for both import cargoes is in the same range, between 230 and 370 nM. Furthermore, RanGTP induces the release of both import cargoes by different mechanisms. RanGTP directly competes for the binding surface of Ubc9 on Imp13, whereas it displaces Mago-Y14 by a steric hindrance mechanism. In the second half of my PhD, I focused on the export part of the Imp13 cycle. I crystallized and solved the Imp13-RanGTP-eIF1A complex at 3.6 Å resolution. eIF1A is bound at the inner surface of the C-terminal arch of Imp13 adjacent to RanGTP with a KD of 3 µM. The import cargo Mago-Y14 and the export cargo eIF1A share the same interaction surface on Imp13. The comparison between Imp13 import and export complexes reveals that the direction of the transport is determined by a mutually exclusive binding between import cargo and RanGTP and a concomitant binding with the export cargo. Furthermore, I solved the structure of the cytosolic cargo-free state of Imp13 at 3.0 Å resolution. The structure of free Imp13 reveals an overall open conformation, largely incompatible with the binding of eIF1A in the cytoplasm. In conclusion, Imp13 is a remarkably versatile nuclear transport factor which recognizes the shape of its cargoes by wrapping around them with its inner surface and thereby changing its conformation ranging from an open superhelix to a closed ring. A simple principle in which the size and charge of the cargo determines the direction of transport underlies Imp13's unusual function in nucleocytoplasmic transport.
