Ran-dependent Signal-mediated Nuclear Import Does Not Require GTP Hydrolysis by Ran

Abstract

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.Many nuclear proteins contain a nuclear localization sequence (NLS), 1 the classical type of which consists of either a single or bipartite stretch of primarily basic amino acids. The presence of an NLS on a reporter results in its rapid transport from the cytoplasm through the nuclear pore complex (NPC) and into the nuclear interior. This process can be reconstituted in vitro using cells that have been treated with a low concentration of digitonin, which permeabilizes the plasma membrane to the passage of macromolecules but leaves the nuclear membrane intact (1). Addition of cytosol as a source of import factors, a nucleoside triphosphate (NTP) as a source of energy, and import substrate to these permeabilized cells results in import of the substrate into the nucleus. From this cytosol, four import factors have been identified that together efficiently support nuclear import of a basic NLS-containing substrate in permeabilized cells in the absence of cytosol. The receptor for the classical or basic NLS (karyopherin/importin ␣), together with karyopherin/importin ␤ 1 , targets the import substrate to the NPC (2-8). Thus the addition of these two import factors alone to an in vitro import assay results in binding of import substrate at the nuclear envelope in a characteristic rim pattern. Subsequent passage of import substrate from the NPC into the nuclear interior requires the addition of two other import factors: p10/NTF2 (9, 10) and the small GTPase Ran (11,12). The exact role of the nuclear import factor p10, which preferentially binds GDP-Ran rather than GTP-Ran, has yet to be defined. Ran, however, has been shown to be a key player not only for the nuclear import of a variety of substrates bearing different types of NLSs, but also for the nuclear export of most RNAs and a number of proteins containing different types ...

β-Catenin Shows an Overlapping Sequence Requirement but Distinct Molecular Interactions for Its Bidirectional Passage through Nuclear Pores

“…Although nuclear transport in vivo is an energy-consuming process, a number of in vitro studies have demonstrated that the NPC translocation step of transport does not require any metabolic energy (22)(23)(24)(25)(26). In the importin/exportin pathways, metabolic energy is supplied by the Ran GTPase cycle (27), which operates on both sides of the NPC, to assure the directionality of cargo transport.…”

The Death Effector Domain Protein PEA-15 Prevents Nuclear Entry of ERK2 by Inhibiting Required Interactions

“…Cells plated on coverslips 24 h before permeabilization were washed once with import buffer (20 mM Hepes-KOH, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol) and permeabilized with 70 g/ml digitonin in transport buffer for 5 min on ice. Cells were washed and inverted over 40 ml of import mix, which contained 0.6 -0.8 M GFP-ERK2 (38 -50 g/ml) or 5 g/ml NLS-BSA-TRITC (0.07 M); for NLS-BSA import, cells were incubated with 0.5 M karyopherin-1, 0.25 M karyopherin-2, 2 M Ran, and 0.4 M p10/NTF2 (31). Permeabilized cells were incubated for 15 min or the indicated times and were washed and fixed in 3.7% formaldehyde for 15 min.…”

“…Import Assays-Import assays were performed as described by using REF52 cells grown in medium supplemented with 10% fetal bovine serum and 1% glutamine (31). Cells plated on coverslips 24 h before permeabilization were washed once with import buffer (20 mM Hepes-KOH, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol) and permeabilized with 70 g/ml digitonin in transport buffer for 5 min on ice.…”

“…Elution from the latter was performed with a step salt gradient. Karyopherin-1, karyopherin-2, Ran, p10, and glutathione S-transferase (GST)-NUP153c were purified as described (31)(32)(33). Rhodamine-labeled bovine serum albumin (BSA) was coupled to a peptide containing the nuclear localization sequence (NLS) of the simian virus 40 T antigen to make the control import substrate NLS-BSA (32).…”

Ceramide regulation of nuclear protein import

“…Transport is initiated upon energy-independent NLS recognition by a heterodimeric NLS receptor (17,18) composed of an a subunit (importin-a), which recognizes the NLS (19), and a b subunit (20) (importin-b), which mediates nuclear pore complex docking at the nuclear envelope (21)(22)(23). Translocation of the NLS-receptor assembly through the nuclear pore complex is an energydependent process (24)(25)(26) controlled by a RanGTP/GDP cycle (27)(28)(29). Importin-a is returned to the cytosol by CAS (for Cellular Apoptosis Susceptibility), a nuclear export protein specific for the a subunit (30).…”