Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor [published erratum appears in J Cell Biol 1994 Jan;124(1-2):217]

Abstract

Abstract. We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin-permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISAbased assay that allows rapid quantitative analysis. GTP~,S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro nuclear import. Transport inhibition by GTP3,S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography.Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient nuclear import. Biochemical complementation with bacterially expressed protein demonstrated that this essential GTP3,S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate nuclear protein import with these other nuclear activities.

Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

“…In addition to importin family proteins [42,43], the small G-protein, Ran, is also essential for protein import into the nucleus [44,45]. Moreover, asymmetric distribution of Ran, RCC1, RanGAP1 and RanBP1 [46][47][48] results in a steep RanGTP gradient across the nuclear envelope; High RanGTP concentration in the nucleus, whereas high RanGDP concentration in the cytoplasm [30].…”

Essential role of active nuclear transport in apoptosis

“…The GTP hydrolysis of Ran-GTP appears to be required in active nuclear transport, because the microinjection of excess amounts of the Ran-binding unhydrolysable GTP analogue, GTPgS (Ran-GTPgS), inhibits active nuclear transport, probably through the functional inhibition of endogenous Ran (Melchior et al 1993). Anti-PTAC58 antibody inhibits the function of PTAC58 in active nuclear transport (Imamoto et al 1995).…”

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

“…Because RIP1 is not an essential gene and its deletion is not reported to affect endogenous RNA export (Stutz et al, 1995), other factors are probably involved in NES recognition and nuclear export. Export defects have been characterized with mutations in Gsplp (the yeast homologue of a small Ras-like GTP-binding protein required for nuclear import, Ran/TC4; Belhumeur et al, 1993;Kadowaki et al, 1993;Melchior et al, 1993;Moore and Blobel, 1993;Schlenstedt et al, 1995) and in Prp2Op (the yeast homologue of RCC1, a guanine nucleotide exchange protein for Ran/TC4; Aebi et al, 1990;Bischoff and Ponstingl, 1991a,b;Amberg et al, 1993;Kadowaki et al, 1993;Cheng et al, 1995). This has implied a requirement for GTP hydrolysis in RNA export.…”