Importin (IMP) ! is a nuclear import receptor that recognizes cargo proteinscontaining classic nuclear localization sequences (NLSs) and facilitates their transportinto the nucleus. Although NLS-binding regions are highly conserved, the sixdifferent IMP !s in mammals show distinct NLS-binding specificities. The mainaims of this study were to gain new insight into the structure of the human IMP!protein and in particular to determine the optimal NLS sequences recognized byseveral human IMP!s using a novel NLS peptide library screening approach. Anadditional goal was to compare the NLS-binding specificity and nuclear importefficiency of different IMP !s. Overall, this study has generated new quantitativedata regarding the NLS-binding specificities of IMP!s.IMP! is comprised of two functional domains, a C-terminal domain, the shortN-terminal importin "-binding domain, and a large !-helical central domain(NLS-binding domain) consisting of 10 armadillo repeats. One of the aims of thisproject was to determine the structures of representative members of each of the threedifferent IMP! subfamilies in order to gain insight into their NLS bindingspecificities. However, crystallization of full length h-IMP!s did not provepossible, almost certainly due to the flexible multiple domains of h-IMP!s, whichcan exist in a variety of conformational states, and inhibit the formation of a singlethree-dimensional array in the crystal. In the published mouse IMP! crystalstructure, the large !-helical central domain appears to be largely structured, withthe Arm repeats unchanged in conformation when bound to an NLS. Proteases(endopeptidase Glu-C and chrymotrypsin) were used to perform limited proteolysis toremove the N-terminal and C-terminal domains to leave a stable, central !-helicaldomain. The truncated IMP! derivatives appear to be the most suitable forcrystallization. Micro-crystals of truncated IMP!s were successfully obtainedduring this study. However, more work is clearly required to grow better crystalsthat will enable the structures to be solved.A second aim of this project was to investigate the cargo binding specificity of severalhuman IMP!s. A NLS peptide library screening approach was used to determinethe cargo binding specificity of IMP!s. The optimal NLSs identified for thedifferent IMP!s obtained from screening peptide library 1 [comprising peptides ofthe sequence: G-S-E-F-E-S-P-X-K-X-X-X-X-E-A, where X represents any aminoacids except for C and W; K at residue 9 (P2 position) was fixed] agree well withprevious findings that IMP!-recognized NLSs are highly basic. IMP! binds itsoptimized NLS with high affinity. Binding studies confirmed that different NLSswere functional, and that different IMP!s were able to mediate nuclear import oftheir respective optimal NLS most efficiently. IMP!s are also able to importcargoes containing NLSs which are not optimally recognized; but at a slower rate orto a lesser extent.A second degenerate peptide library, peptide library 2, was constructed without Lysfixed at position 9. Unexpectedly, the NLSs identified using this library werecompletely different from conventional NLSs or the NLSs identified from peptidelibrary 1, with non-basic residues preferred in the P1-P6 positions. Strikingly, Lyswas not preferred at P2 in any of the selected peptides, implying that the majorNLS-binding site on IMP!s may not be the only site for interacting partners. Thatthe peptides identified were functional as NLSs was tested by reconstituting transportin vitro, showing that the T5NLS2 (optimal NLS of h-IMP!5 from screening ofpeptide library 2; G-S-E-F-E-S-P-I-G-K-A-T-E-A) could confer efficient nuclearimport as mediated by h-IMP!5/". This implies that non-basic NLS sequencesmay be able to confer nuclear import via IMP!/".Different IMP!s appear to be specialized in their efficiency to mediate transport ofparticular proteins with high efficiency. Reconstituting nuclear import in vitro usingpurified components confirmed this hypothesis, the results showing that different IMP!s are able to mediate nuclear import of their optimal NLSs most efficiently. Inaddition, different IMP!s are able to mediate the nuclear import of proteins carryingNLSs that bind with lower affinity, import these at a slower rate compared to that bythe optimal IMP!. NLS binding affinity thus appeared to correlate well withnuclear import efficiency.The efficiency of nuclear import by IMP!s, as conferred by several different basicNLSs by IMP!s, was compared. One of the important findings was that differentIMP!s have different nuclear transport efficiencies according to the specific cargoes.T-ag-like NLS-containing proteins are able to be transported into the nucleus veryefficiently by human IMP!1/" and IMP!3/", whereas cargoes containingbipartite NLSs are able to be transported to the nucleus very efficiently by IMP!7/"and IMP!3/". This implies that h-IMP!3 is able to transport cargoes containingeither monopartite or bipartite NLSs efficiently into nucleus, consistent with the factthat it is the most conserved among all IMP!s across evolution.Overall, this study has generated new quantitative data characterizing theNLS-binding specificities of IMP!s. Importantly, this highlights importantpotential differences in the interaction between IMP!s and their target substrates,indicating that distinct IMP!s may have different physiological roles.