Multiple mechanisms promote the inhibition of classical nuclear import upon exposure to severe oxidative stress.

In growing HeLa cells, severe stress elicited by the oxidant hydrogen peroxide inhibits classical nuclear import. Oxidant treatment collapses the nucleocytoplasmic Ran concentration gradient, thereby elevating cytoplasmic GTPase levels. The Ran gradient dissipates in response to a stress-induced depletion of RanGTP and a decreased efficiency of Ran nuclear import. In addition, oxidative stress induces a relocation of the nucleoporin Nup153 as well as the nuclear carrier importin-beta, and docking of the importin-alpha/beta/cargo complex at the nuclear envelope is reduced. Moreover, Ran, importin-beta and Nup153 undergo proteolysis upon oxidative stress. Caspases and the proteasome degrade Ran and importin-beta; however, ubiquitination of these transport factors is not observed. Inhibition of caspases in stressed cells alleviates the mislocalization of importin-beta, but does not restore the Ran concentration gradient or classical import. In summary, inhibition of classical nuclear import by hydrogen peroxide is caused by a combination of multiple mechanisms that target different components of the transport apparatus.