A New Role for Nuclear Transport Factor 2 and Ran: Nuclear Import of CapG

Abstract

The small GTPase Ran plays a central role in nucleocytoplasmic transport. Nuclear transport of Ran itself depends on nuclear transport factor 2 (NTF2). Here, we report that NTF2 and Ran control nuclear import of the filamentous actin capping protein CapG. In digitonin‐permeabilized cells, neither GTPγS nor the GTP hydrolysis‐deficient Ran mutant RanQ69L affect transit of CapG to the nucleus in the presence of cytosol. Obstruction of nucleoporins prevents nuclear transport of CapG, and we show that CapG binds to nucleoporin62. In addition, CapG interacts with NTF2, associates with Ran and is furthermore able to bind the NTF2–Ran complex. NTF2–Ran interaction is required for CapG nuclear import. This is corroborated by a NTF2 mutant with reduced affinity for Ran and a Ran mutant that does not bind NTF2, both of which prevent CapG import. Thus, a ubiquitously expressed protein shuttles to the nucleus through direct association with NTF2 and Ran. The role of NTF2 may therefore not be solely confined to sustaining the Ran gradient in cells.

The F-actin filament capping protein CapG is a bona fide nucleolar protein

“…Purification of Myc-CapG-V5-His 6 has been previously described [10]. Human His 6 -RanQ69L and His 6 -Ran wild-type were expressed and purified as described [19].…”

“…In a previous study, we reported that CapG interacts directly with Ran GTPase [10]. Since Ran is predicted to localize in the nucleolus by a proteome study (http://www.lamondlab.com/NOPdb/) and has been reported to accumulate in the nucleolus in in vitro experiments [23], we also tested Ran in our experiments (Fig.…”

“…Its capping activity is activated by calcium and inhibited by membrane polyphosphoinositides but unlike Gelsolin, CapG does not sever actin filaments [8]. CapG also localizes to the nucleus [9] and we recently showed that CapG is imported in the nucleus by the transport receptor NTF2 and Ran GTPase, a major regulator of nucleo-cytoplasmic transport [10]. The nuclear form of CapG has specifically been shown to promote cell invasion [11].…”

“…The nuclear form of CapG has specifically been shown to promote cell invasion [11]. Interestingly, CapG interacts with both the cytoplasmic (GDP-loaded) and nuclear (GTP-loaded) form of Ran [10]. The nucleotide cycle of Ran is responsible for directionality of transport across the nuclear envelope [12].…”

“…To evaluate the energy requirements of transport between the nucleoplasm and the nucleolus, an in vitro system has been conceived analogous to the in vitro import assay used for nucleo-cytoplasmic transport analysis [10,15]. In contrast to the in vitro import assay where digitonin is used to selectively permeabilize the plasma membrane, leaving the nuclear membrane intact, this assay includes CHAPS in the import reaction mix, which additionally results in permeabilization of the nuclear envelope [16].…”