| dc.description.abstracteng | The nucleus is the most prominent feature of every eukaryotic cell. Since the nucleus contains virtually all the genetic information it separates the transcription and translation. This separation requires a bidirectional process divided into import and export of macromolecules through the nuclear pore complex (NPC) the nuclear transport. This process is largely mediated by receptors of the karyopherin-β superfamily. The driving force behind the nuclear transport is a gradient of the small GTPase Ran across the nuclear envelope. This protein and its gradient simultaneously form an important part in the organization of mitotic spindle and the reconstruction of the nuclear envelope in mitosis. Ran appears in a GTP- and a GDP-bound form. The GTPase switches between these two conformations due to the action of regulatory factors (in both processes the same) and thus directs and regulates the nuclear transport, as well as the organization of the mitotic spindle and the reconstruction of the nuclear envelope. Recently it was discovered that the small GTPase Ran is subject to posttranslational acetylation on multiple lysine residues (Choudhary et al., 2009b).
In this work the small GTPase Ran with acetylated lysine (K37, K38, K60, K71, K99 and K159) was expressed, purified and analyzed functionally as well as structurally in complex with the export receptor Crm1. It could be shown that acetylation of the GTPase at only one lysine has no effect on the binding of the GTPase to the transport receptors Crm1 and Impβ. However, it was found that acetylation of a single lysine effects the RanGAP1 induced GTP hydrolysis of GTPase in complex with Crm1 and Imp7 and on the GTPase alone. The acetylation at specific lysine's still showed GTP hydrolysis even at high concentrations of Crm1/Imp7. In combination with Impβ this could not be observed. These proteins are involved in both the nuclear transport as well as mitosis. On the GTPase alone the acetylation shows, depending on the acetylated lysine's, an accelerating or decelerating effect. Structurally the acetylated mutants (K71Ac and K99Ac) show an additional interaction between the Asp91Ran and the Arg887Crm1, as well as the occurrence of K71Ran in a loop in the switch 2 region with no further significant difference to the already solved structure Crm1:RanGTP:SPN1 (Monecke et . al, 2009; PDB Id 3GJX).
The results presented in this work allow an impression of the function of the acetylated small GTPase Ran, probably in the modulation and fine tuning of the RanGTP-dependent processes in nuclear transport and mitosis. | de |