ABSTRACT
RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Guanine Nucleotide Exchange Factors , alpha Karyopherins , Amino Acid Sequence , Animals , Binding Sites , Cell Membrane Permeability , Cytosol/physiology , DNA-Binding Proteins/chemistry , Female , HeLa Cells , Humans , Molecular Sequence Data , Nuclear Proteins/metabolism , Ovary/physiology , Recombinant Fusion Proteins/metabolism , Xenopus Proteins , Xenopus laevis , ran GTP-Binding Protein/metabolismABSTRACT
The nuclear pore complex (NPC) connects the cytoplasm and nucleus through the nuclear envelope and serves as the pipeline for moving material between the two compartments. Macromolecules that move through the NPC range in size from the very small (for example, ions and ATP) to the very large (for example, ribonucleoprotein particle complexes). Unlike translocation across other organelle membranes, proteins do not have to be unfolded to be transported through the NPC, and the NPC also routinely transports large, multicomponent substrates in both directions. This review focuses on current understanding of the different mechanisms by which macromolecules move across the NPC.
Subject(s)
Nuclear Envelope/physiology , Animals , Biological Transport , Diffusion , Humans , Microscopy, Electron , Models, Biological , Nuclear Envelope/ultrastructure , Signal TransductionABSTRACT
Nuclear import of classical nuclear localization sequence-containing proteins involves the assembly of an import complex at the cytoplasmic face of the nuclear pore complex (NPC) followed by movement of this complex through the NPC and release of the import substrate into the nuclear interior. This process has historically been thought to require nucleotide hydrolysis as a source of energy. We found, using hydrolysis-resistant GTP analogs and a mutant Ran unable to hydrolyze GTP, that transport of classical nuclear localization sequence containing substrate through the NPC and release of the substrate into the nucleus did not require hydrolysis of GTP by Ran. In fact, for movement of this type of import substrate into the nuclear interior we did not observe a requirement for hydrolysis of any nucleotide triphosphate. We did, however, find that a pool of free GTP (or its structural equivalent) must be added, probably because the GDP Ran that is added must be converted to GTP Ran during the import process. We found that a requirement for GTP hydrolysis can be restored to an import mixture consisting of recombinant import factors by the addition of RCC1, the Ran guanine nucleotide exchange factor.
