Targeting Sequences Conferring Bidirectional Transport by Importin 13

2017-04-20T05:09:29Z (GMT) by Rebecca Davies
Importin 13 (IMP13) is expressed at high levels in many tissues, including the fetal lung, brain, testis and cornea, and has been associated with disease states including X-linked mental retardation and childhood asthma. IMP13 is a member of the karyopherin β family of nuclear transport receptors that are responsible for mediating the transport of proteins between the cytoplasm and the nucleus of eukaryotic cells. <br><br> A wide range of IMP13 nuclear import cargoes have been identified, a large proportion of which are transcription factors, including NF-Y subunits (NF-YB/NF-YC), Pax6, Pax3, Crx and the glucocorticoid receptor (GR). IMP13 is unique among Importin family members in that it has the distinctive ability to transport cargoes both into and out of the nucleus, however, only one IMP13 nuclear export cargo has so far been reported.<br> <br> Previous work in our laboratory (unpublished) has suggested that the most well-characterised nuclear import cargo of IMP13, the E2 ubiquitin conjugating-like enzyme, Ubc9, may also be a nuclear export cargo for IMP13. This thesis maps an IMP13-recognised nuclear localisation signal (NLS) within the N-terminus of Ubc9, comprising of a basic stretch of amino acid residues similar to those recognised by other IMPβ family members. Using the DILIMOT (Discovery of Linear Motifs) consensus site prediction server, additional residues that also contribute to IMP13-mediated nuclear import are identified, suggesting that IMP13 is able to mediate Ubc9 nuclear import in both a conventional and non-conventional manner. This thesis further demonstrates that IMP13 forms a trimeric complex with Ubc9 and RanGTP <i>in vitro</i>, indicative of a conventional exportin (EXP)-nuclear export cargo-RanGTP complex, suggesting that IMP13 can indeed mediate Ubc9 nuclear export.<br> <br> Importantly, this thesis also identifies retinoic acid receptor gamma transcript variant 2 (RARγ2), which belongs to the same family of ligand activated transcriptional regulators as the GR, as a new binding partner for IMP13. It is shown here for the first time that IMP13 can mediate RARγ2 nuclear export via residues within the RARγ2 C-terminal domain, and while two NLSs were also mapped within RARγ2, neither appear to be recognised by IMP13. Surprisingly, IMP13 was also shown to mediate nuclear export of the GR, which was originally identified as a nuclear import cargo of IMP13, demonstrating that IMP13 is able to mediate GR bidirectional transport and highlighting this novel function of IMP13.<br> <br> This thesis discusses the mechanisms behind IMP13 bidirectional transport, examines the potential role it plays during development and disease and has expanded the IMP13 interactome by the identification of a novel binding partner, and establishing a new nuclear export function for an already existing nuclear import cargo. These findings, in conjunction with the identification of two potential modes of nuclear import (conventional and non-conventional), highlight the unique nature of IMP13-mediated nuclear transport. Given the link between IMP13 and disease, and that IMP13 is essential to development, certainly warrants further investigation to gain a better understanding of the mechanisms outlining this unique and important nuclear transport pathway.