Oxidative stress mislocalizes and retains transport factor importin-α and nucleoporins Nup153 and Nup88 in nuclei where they generate high molecular mass complexes

Abstract

Nuclear trafficking of proteins requires the cooperation between soluble transport components and nucleoporins. As such, classical nuclear import depends on the dimeric carrier importin-α/β1, and CAS, a member of the importin-β family, which exports importin-α to the cytoplasm. Here we analyzed the effect of oxidative stress elicited by diethyl maleate (DEM) on classical nuclear transport. Under conditions that do not induce death in the majority of cells, DEM has little effect on the nucleocytoplasmic concentration gradient of Ran, but interferes with the nuclear accumulation of several reporter proteins. Moreover, DEM treatment alters the distribution of soluble transport factors and several nucleoporins in growing cells. We identified nuclear retention of importin-α, CAS as well as nucleoporins Nup153 and Nup88 as a mechanism that contributes to the nuclear concentration of these proteins. Both nucleoporins, but not CAS, associate with importin-α in the nuclei of growing cells and in vitro. Importin-α generates high molecular mass complexes in the nucleus that contain Nup153 and Nup88, whereas CAS was not detected. The formation of high molecular mass complexes containing importin-α, Nup153 and Nup88 is increased upon oxidant treatment, suggesting that complex formation contributes to the anchoring of importin-α in nuclei. Taken together, our studies link oxidative stress to the proper localization of soluble transport factors and nucleoporins and to changes in the interactions between these proteins.