Molecular interactions of nuclear transport factor 2

• Main Author: Clarkson, W. D.
• Published: University of Cambridge 1997
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597746

In this Thesis, I describe a molecular dissection of the interaction of NTF2, with Ran and several nuclear pore proteins. To undertake these studies I first cloned and sequenced rat NTF2 cDNA, expressed the protein in bacteria, and purified it to homogeneity. NTF2 was then coupled to Sepharose beads, and used to characterise in detail the molecular interactions of NTF2 with other proteins. NTF2 specifically bound both Ran-GDP and also various repeat-containing nucleoporins, including mammalian p62, and yeast Nsp1p. These interactions were verified by a variety of alternative techniques including the yeast two hybrid screen. Competition experiments indicated that NTF2 has separate binding sites for Ran-GDP and nucleoporins. In addition, I used protein engineering to construct a range of targeted NTF2 mutants based on the known structure of NTF2 to identify the regions of NTF2 involved in these interactions. Although none of the engineered mutant proteins disturbed the nucleoporin binding site on NTF2, several mutants failed to bind Ran-GDP. Using the three dimensional crystal structure of wild-type and two of NTF2 mutants, the key features of the Ran-GDP binding site on NTF2 could be identified. Furthermore, the NTF2 mutants which were unable to bind Ran, were also unable to stimulate nuclear protein import <I>in vitro</I>, and additionally one mutant was found not to be viable in place of the wild type <I>NTF2</I> gene in the yeast <I>Saccharomyces cerevisiae</I>. Taken together, these findings indicate that the NTF2-Ran interaction is essential for efficient nuclear protein import. Finally, the implications of these and other results for understanding NTF2 function during nuclear protein import are discussed.