Nuclear-cytoplasmic trafficking of NTF2, the nuclear import receptor for the RanGTPase, is subjected to regulation.

NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s)

The ins and outs of nuclear re-export of retrogradely transported tRNAs in Saccharomyces cerevisiae

In Saccharomyces cerevisiae intron-containing pre-tRNAs are exported from the nucleus to the cytoplasm for removal of the introns, and the spliced tRNAs are returned to the nucleus for reasons that are not understood. The re-imported spliced tRNAs are then subjected to aminoacylation in the nucleolus to ensure that they are functional prior to re-export to the cytoplasm. Previous studies have shown that re-imported spliced tRNAs and mature tRNAs made entirely in the nucleus from intronless precursors are retained in the nucleus of S. cerevisiae in response to glucose, amino acid, nitrogen or inorganic phosphate deprivation. Contrary to these studies, we recently reported that starvation of S. cerevisiae of amino acids or nitrogen results in nuclear accumulation of re-imported spliced tRNAs, but not tRNAs made from intronless precursors. This finding suggests that separate pathways are used for nuclear export of retrogradely transported spliced tRNAs and tRNAs made from intronless pre-tRNAs. In addition, the data support the conclusion that the nuclear re-export pathway for retrogradely transported spliced tRNAs, but not the pathway responsible for nuclear export of tRNAs derived from intronless precursors is regulated during amino acid or nitrogen starvation. This regulation appears to occur at a step after the re-imported spliced tRNAs have undergone aminoacylation quality assurance and, in part, involves the TORC1 signalling pathway. Moreover, it was established that Utp9p is an intranuclear component that only facilitates nuclear re-export of retrogradely transported spliced tRNAs by the β-karyopherin Msn5p. Utp9p acts in concert with Utp8p, a key player in nuclear tRNA export in S. cerevisiae, to translocate aminoacylated re-imported spliced tRNAs from the nucleolus to Msn5p and assist with formation of the Msn5p-tRNA-Gsp1p-GTP export complex. This pathway, however, is not the only one responsible for nuclear re-export of retrogradely transported spliced tRNAs

Tween-80 or deoxycholate treatment does not affect nuclear tRNA export.

<p>HeLa cells were treated with (A) 150 µM Tween-80 or (B) 150 µM deoxycholate for 4 h in serum-free DMEM. The cells were fixed and FISH was used to monitor the distribution of tRNA^Lys. The cells were stained with DAPI to visualize the nucleus. Scale bar represents 10 µm.</p

Tween-20 treatment causes Ran to accumulate in the cytoplasm of HeLa cells and a block in nuclear export of proteins but not nuclear import of proteins.

<p>(A) Tween-20 causes cytoplasmic retention of Ran in HeLa cells. HeLa cells were incubated in fresh serum-free DMEM (Untreated) or in serum-free DMEM containing 150 µM Tween-20 for 4 h. After the 4 h incubation, the cells were left in Tween-20 containing medium (Tween-20), washed and placed in fresh serum-free media (Wash), or had the serum-free DMEM media containing Tween-20 supplemented with 10% FBS (Serum Add) and incubated at 37°C for 1 h. The distribution of Ran was monitored by immunofluorescence microscopy as described in materials and methods. (B) Tween-20 causes nuclear accumulation of Importin-α. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 150 µM Tween-20 in serum-free DMEM for 4 h (Tween-20). Following the 4 h incubation, the cells were washed and incubated in serum-free DMEM for 1 h. The distribution of Importin-α was monitored by immunofluorescence microscopy. (C) Tween-20 causes nuclear accumulation of an NES-EGFP. HeLa cells were transfected with NES-EGFP and allowed to express for 24 h. Post-transfection cells were washed and placed in fresh serum-free DMEM with (Tween-20) or without (Untreated) 150 µM Tween-20 for 4 h. The distribution of NES-EGFP was monitored by direct fluorescent microscopy. (D) Tween-20 does not block nuclear import of the NLS containing Histone H2A. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 150 µM Tween-20 for 4 h. The distribution of Histone H2A was monitored by immunofluorescence microscopy. (E) Tween-20 does not affect TNF-α stimulated nuclear import of NF-κB. HeLa cells were incubated in serum-free DMEM without (Untreated) or with 10 ng/ml TNF-α for 30 min, or with 150 µM Tween-20 for 4 h and then for 30 min with 10 ng/ml TNF-α. The distribution of NF-κB was monitored by immunofluorescence microscopy. The cells were DAPI stained to visualize the nucleus. Scale bar represents 10 µm.</p

Tween-20 causes an increase in the level of protein phosphorylation at tyrosine and threonine residues, but does not appear to cause modification of NTF2.

<p>(A) HeLa cells were incubated in serum-free DMEM without (Untreated) or with 150 µM Tween-20 (Tween-20) for 4 h. The cells were washed and lysed in the presence of sodium fluoride and sodium orthovanadate (Lanes 1 and 2, -AP) or lysed in the absence of phosphatase inhibitors (Lanes 3 and 4, +AP). The cells lysed without phosphatase inhibitors were incubated with alkaline phosphatase for 30 min at 37°C. Following the incubation, 40 µg of each lysate was separated on 10% gels by SDS-PAGE followed by Western blot analysis to monitor the levels of protein phosphorylation at threonine (first row), tyrosine (second row) and serine (third row) residues or actin level (fourth row). Arrows indicate bands of increased phosphorylation. (B) The lysate (40 µg) prepared as in (A) was separated by SDS-PAGE on a 15% gel followed by Western blot analysis to monitor the mobility of NTF2. The relative mobility of NTF2 was monitored in lysate prepared from Untreated (lanes 1 and 3) and Tween-20 (lanes 2 and 4) treated cells. (C) Lysates treated as in (A) were subjected to chromatography using Phostag™-agarose. The resins were washed and bound proteins were eluted using sodium phosphate containing buffer. Total cell lysate (20 µg) (lanes 1and 3, left) and eluted proteins were subjected to Western blot analysis. NTF2 was detected using α-NTF2 (left) and phospho-Thr proteins (lanes 1 and 2, right) were detected with anti-phospho-Thr antibodies.</p