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 - PubMed (original) (raw)

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

J Bear et al. Mol Cell Biol. 1999 Sep.

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.

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Figures

FIG. 1

FIG. 1

hTAP is conserved between species and contains homologies to other human proteins. (A) Comparison of amino acid sequences of human TAP and the predicted TAP proteins from mouse and rat. Lowercase letters indicate nonconserved residues. Boldface letters indicate the predicted classical NLS. The newly identified aa 1 to 60 of TAP are boxed. (B) Homology of the N-terminal TAP peptide to regions in the hnRNP K and X. laevis hnRNP C. Identical amino acids are shaded in black; similar amino acids are shaded in grey. The box indicates the position of the high-affinity bZip-like RBD identified in hnRNP C. (C) Comparison of hTAP to the predicted TAP-like human protein TAPX2. Identical and similar amino acids are indicated as in panel B. The NLS-NES region is indicated with a hatched bar, the RBD is shown with dotted bar, and the C-terminal portion is indicated with a black bar.

FIG. 1

FIG. 1

hTAP is conserved between species and contains homologies to other human proteins. (A) Comparison of amino acid sequences of human TAP and the predicted TAP proteins from mouse and rat. Lowercase letters indicate nonconserved residues. Boldface letters indicate the predicted classical NLS. The newly identified aa 1 to 60 of TAP are boxed. (B) Homology of the N-terminal TAP peptide to regions in the hnRNP K and X. laevis hnRNP C. Identical amino acids are shaded in black; similar amino acids are shaded in grey. The box indicates the position of the high-affinity bZip-like RBD identified in hnRNP C. (C) Comparison of hTAP to the predicted TAP-like human protein TAPX2. Identical and similar amino acids are indicated as in panel B. The NLS-NES region is indicated with a hatched bar, the RBD is shown with dotted bar, and the C-terminal portion is indicated with a black bar.

FIG. 2

FIG. 2

The genuine hTAP is a 70-kDa protein. Extracts from human 293 cells transfected with the different TAP expression plasmids were separated on a 10% denaturing polyacrylamide gel, blotted onto nitrocellulose, and incubated with an anti-TAP antiserum and then 125I-labeled protein A. The relevant positions of the Rainbow molecular weight markers (Amersham) are indicated on the right. The arrow indicates the endogenous hTAP protein. Lane 1, not transfected (n/t); lanes 2 to 5, transfected with the indicated plasmids.

FIG. 3

FIG. 3

hTAP is a nuclear protein. HLtat cells were transfected with the indicated plasmids, and the tagged proteins were visualized the following day. While the GFP-tagging allows direct visualization in living cells, the HA-tagged proteins were visualized upon staining with anti-HA-antibody and rhodamine-labeled anti-mouse antiserum. The images were obtained by fluorescent microscopy and by use of a CCD camera (A and B; bar = 20 μm) and by confocal microscopy (C to H; bar = 10 μm). Panels: A, GFP; B, GFP–β-Gal; C and D, TAP1-619 GFP; E, HA-tagged TAP1-169; F, TAP1-619 GFP–β-Gal; G, TAP61-619 GFP; H, HA-tagged TAP61-619. Panels C and D show confocal images taken at different planes through the nucleus.

FIG. 4

FIG. 4

Identification of the nuclear localization signal of hTAP. (Top) HLtat cells were transfected with the indicated plasmids, and the TAP hybrid proteins were visualized as described in Fig. 3. All images were obtained by using fluorescent microscopy and a CCD camera. The TAP peptides or the intact hTAP protein were tagged with either GFP–β-Gal (panels A to C, E, and G to J) or GFP (panels D, F, and K). (Bottom) Schematic representation of hTAP indicating the location of the NLS. Different TAP mutants shown in Fig. 4A are represented.

FIG. 5

FIG. 5

Analysis of the C-terminal portion of TAP. (Top) HLtat cells were transfected with the indicated plasmids, and the TAP hybrid proteins were visualized as described in Fig. 3. The TAP peptides were tagged with either GFP (panels A to D and panel I) or GFP–β-Gal (panels E to H). All images were obtained by using fluorescent microscopy and a CCD camera (bar = 20 μm) except panels E to H (bar = 10 μm), for which confocal microscopy was used. Tagging of C-terminal peptides of TAP with GFP (panels A to D) revealed the presence of nuclear retention signal(s), while tagging of the same peptides with GFP–β-Gal (panels E and F) demonstrates that this region lacks an active NLS. (B) Schematic representation of hTAP indicating the locations of the NLS (Fig. 4) and the signal for nuclear retention and rim association. Different TAP mutants shown in Fig. 5A are represented.

FIG. 6

FIG. 6

hTAP is a shuttle protein. Transfected HLtat cells were mixed with an excess of untransfected HLtat cells. The next day, the cells were fused by using PEG, and the GFP fluorescence was detected by using fluorescent microscopy and a CCD camera. (A) Fusion of cells transfected with GFP-tagged TAP1-619 in the absence (top panels) and presence (bottom panels) of the NES(−) Rev M10BL-BFP. (B and C) Fusion of cells transfected with GFP–β-Gal-tagged TAP61-120 and TAP61-110 fixed with 3.7% formaldehyde after 30 and 120 min, respectively. The arrows indicate the donor nuclei.

FIG. 7

FIG. 7

Identification of the NES of hTAP. (A) Different peptides of hTAP, as indicated, were inserted between two moieties of GFP in pTat-GFP (52). HLtat cells were transfected and analyzed as described above. All images were obtained by confocal microscopy. Bar = 10 μm. (B) Schematic representation of hTAP indicating the locations of the NLS (Fig. 4), the signal(s) for nuclear retention and rim association (Fig. 5), and the NES. Different hTAP mutants shown in the fusion assay (Fig. 6) and the export assay (Fig. 7A) are represented.

FIG. 8

FIG. 8

Signals identified on hTAP. Schematic representation of hTAP shows the NLS and NES are distinct elements that are partially overlapping. In addition, residues 100 to 110 were shown to contribute to nuclear import or nuclear retention. The NLS and NES are located next to, but are distinct from, the recently characterized RBD (4). The RBD encompasses the LRR (47). The nuclear retention and rim association signal(s) are located in the C-terminal portion of hTAP.

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