Identification of Novel Import and Export Signals of Human TAP, the Protein That Binds to the Constitutive Transport Element of the Type D Retrovirus mRNAs

Abstract

The nuclear export of the unspliced type D retrovirus mRNA depends on the cis-acting constitutive transport RNA element (CTE) that has been shown to interact with the human TAP (hTAP) protein promoting the export of the CTE-containing mRNAs. We report here that hTAP is a 619-amino-acid protein extending the previously identified protein by another 60 residues at the N terminus and that hTAP shares high homology with the predicted rat and mouse TAP proteins. We found that hTAP is a nuclear protein that accumulates in the nuclear rim and the nucleoplasm. We further demonstrated that hTAP is able to shuttle between the nucleus and the cytoplasm. Identification of the signals responsible for nuclear import (NLS) and export (NES) revealed that they are distinct but partially overlapping. NLS and NES of hTAP are active transferable signals that do not share similarities with known elements. The C-terminal portion contributes further to hTAP's nuclear retention and contains a signal(s) for nuclear rim association. Taken together, our data show that hTAP is a dynamic protein capable of bidirectional trafficking across the nuclear envelope. These data further support hTAP's role as an export factor of the CTE-containing mRNAs.Posttranscriptional regulation is an essential regulatory step of many retroviruses and is necessary for virus production. This key regulatory step mediates the export of the unspliced, fulllength viral RNA, which requires the interaction of viral and/or cellular factors. This controlled export of the viral RNA to the cytoplasm ensures the availability of the genomic RNA for packaging into the progeny virions and the production of the Gag/Pol polyproteins. Among the best-studied export systems are those used by the simian type D retroviruses (SRV/D) and the lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) (for reviews see references 6, 14, and 28).SRV/D expression is controlled by the essential cis-acting constitutive transport element (CTE) (5,12,54,63). The SRV/D CTE (11, 55) and a related CTE-like element in a murine intracisternal A-particle retroelement (54) fold into an extended RNA stem-loop structure containing two conserved internal loops and an AAGA bulge. These loops and the bulge, the spacing of the loops within the RNA element, as well as the overall secondary structure of the element, have been shown to be essential features for CTE function (11,55). Recently, we showed that the human TAP protein (hTAP) binds specifically to these internal loops and promotes nucleocytoplasmic transport of the CTE-containing intron lariat from the Xenopus oocyte nucleus (22). TAP had previously been identified as a factor binding to Tip, a herpesvirus saimiri protein responsible for cell transformation (60). The role of hTAP interaction with Tip is still unclear.Whereas the SRV/D retroviruses have been proposed to utilize the cellular hTAP protein to export their unspliced mRNA (22), HIV-1 uses the viral Rev protein to promote the transport of the Rev responsive element (RRE)-containing...

Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

“…Further evidence for the existence of alternative NPCbinding sites of Tap comes from a study in which GFP-␤-Gal-Tap 61-120 , missing Tap-UBA,Tap-NBR2, and the two NESs was shown to shuttle (Bear et al, 1999). In addition, a separate study found that the truncated Tap 540 -619 mutant, missing the Tap-NBR2 and Nxt1-binding domain, and the two NESs, could also shuttle through the NPC (Schmitt and Gerace, 2001).…”

“…We reported previously that deletion of one of these hydrophobic surfaces, the Tap-UBA domain, obliterated binding to p62 in vitro . A single point mutation within Tap-UBA (S585P) has also been shown to disrupt the association of Tap with the nuclear envelope (Bear et al, 1999) and was thus incorporated into the present study. The second NPC-binding hydrophobic interface, Tap-NBR2, resides within the NTF2-like domain.…”

Karyopherin β2B participates in mRNA export from the nucleus

“…TAP-GFP and GFP-TAP constructs were generated by cloning full-length TAP into the EcoRI͞BamHI sites of pEGFP-N1 or pEGFP-C1 plasmids (CLONTECH), respectively. TAPmutNES-GFP construct was made by substituting a segment excised by the PpuM1 enzyme with a segment generated by PCR with a reverse primer that contained R3A mutations of residues 97, 98, 100, and 105 (38). GFP-TAPmutC construct was made by substituting a BspE1-BglII segment of TAP with a segment generated by PCR using a reverse primer coding for the mutation S5853P (38).…”

“…TAPmutNES-GFP construct was made by substituting a segment excised by the PpuM1 enzyme with a segment generated by PCR with a reverse primer that contained R3A mutations of residues 97, 98, 100, and 105 (38). GFP-TAPmutC construct was made by substituting a BspE1-BglII segment of TAP with a segment generated by PCR using a reverse primer coding for the mutation S5853P (38). HeLa cells were transiently transfected with 0.4 g of plasmid DNA by using Lipofectamine Plus (Life Technologies, Grand Island, NY).…”

A variant of nuclear localization signal of bipartite-type is required for the nuclear translocation of hypoxia inducible factors (1α, 2α and 3α)

“…EGFP-b-Gal tag (about 190 kDa) is too big to di use into the nucleus passively, ensuring identi®cation of a functional NLS which actively targets the fused EGFP-b-Gal to the nucleus (Bear et al, 1999). EGFP-b-Gal tag alone exclusively localized to the cytoplasm (data not shown).…”