Identification of a Classical Bipartite Nuclear Localization Signal in the Drosophila TEA/ATTS Protein Scalloped

Drosophila melanogaster wing development has been shown to rely on the activity of a complex of two proteins, Scalloped (Sd) and Vestigial (Vg). Within this complex, Sd is known to provide DNA binding though its TEA/ATTS domain, while Vg modulates this binding and provides transcriptional activation through N- and C-terminal activation domains. There is also evidence that Sd is required for the nuclear translocation of Vg. Indeed, a candidate sequence which shows consensus to the bipartite family of nuclear localization signals (NLSs) has been identified within Sd previously, though it is not known if it is functional, or if additional unpredicted signals that mediate nuclear transport exist within the protein. By expressing various enhanced green fluorescent protein (eGFP) tagged constructs within Drosophila S2 cells, we demonstrate that this NLS is indeed functional and necessary for the proper nuclear localization of Sd. Additionally, the region containing the NLS is critical for the wildtype function of ectopically expressed Sd, in the context of wing development. Using site-directed mutagenesis, we have identified a group of five amino acids within this NLS which is critical for its function, as well as another group of two which is of lesser importance. Together with data that suggests that this sequence mediates interactions with Importin-α3, we conclude that the identified NLS is likely a classical bipartite signal. Further dissection of Sd has also revealed that a large portion of the C-terminal domain of the protein is required its proper nuclear localization. Finally, a Leptomycin B (LB) sensitive signal which appears to facilitate nuclear export is identified, raising the possibility that Sd also contains a nuclear export signal (NES)

Identification of a putative bipartite NLS.

<p>(A) A schematic diagram of Sd. Sd contains two known functional domains, the TEA (DNA binding) domain and the Vestigial interacting domain (VID), as shown. At the C-terminus of the TEA domain, there is a 17 amino acid stretch from R145 to R161 which closely matches the consensus classic bipartite NLS sequence ([K/R]₂[X]₁₀[K/R]_>3/5). (B) The region corresponding to the bipartite NLS shows strong identity with a variety of TEAD proteins from both animals and <i>Choanozoa</i> protists. Arrowheads mark the sites of the two N-terminal and five C-terminal residues known to be important for the classical bipartite sequence. ‘X’ marks the 10 intervening amino acids lying between the two termini. A ‘+’ indicates a basic residue (L/R) lies at one of the N- or C-terminal critical sites in the consensus sequence of the aligned TEAD proteins. The dark shading indicates identity with the consensus, while the lighter shading indicates similarity.</p

Quantification of the cellular distribution of the eGFP tagged peptides.

<p>The eGFP fusion constructs from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g002" target="_blank">Figs. 2A–G</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g003" target="_blank">3A–E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g005" target="_blank">Fig. 5D</a> were assayed for the percentage of eGFP signal seen in the nuclei of the expressing cells; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#s4" target="_blank">materials and methods</a>. (S.E.M) is the standard error of the mean. A † denotes a construct with diffuse or nuclear excluded signal (<58% nuclear signal). N is the total number of cells measured from at least two independent transfections. The next four columns represent four arbitrary localization patterns along with the mean nuclear signal each grouping represents. For each peptide, the percentage of cells that fall into one of the four categories is indicated. The means of the experimental constructs TEA-eGFP, NLS-eGFP and NES-eGFP are statistically different from their control (eGFP) at p<0.001. Likewise, NLS-eGFPx2, TEA-eGFPx2 and TEAΔNLS-eGFPx2 are significantly different from eGFPx2, at p<0.0001. Finally the mean of the control eGFP-Sd was significantly different from the four reporter constructs in which the NLS was mutated, at p<0.001. Nuc. = Nuclear. Excl. = Excluded.</p

Sd contains a sequence at amino acids 332–347 which increases the cytoplasmic fraction of a fused eGFP tag in a leptomycin B (LB) sensitive manner.

<p>(A) Schematic of Sd with the putative NES marked. The domains of Sd are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g001" target="_blank">Fig. 1A</a>. Hydrophobic residues are underlined. The open and closed arrowheads mark the boundaries of the region intact in SD Δ344–440 and missing in SD Δ294–440, respectively. (B) Alignment of several TEAD proteins. Dark shading indicates hydrophobic residues L, I, V, M and F, while light shading indicates hydrophobic residues C, W, A or T. (C) Sd amino acids 330–347 were fused N-terminal to eGFP and assayed for spatial distribution. A representative cell showing nuclear exclusion of the fusion protein is shown. See the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g002" target="_blank">Figs. 2A–G</a> for details. (D) Nuclear fraction of eGFP tagged constructs in LB treated and untreated S2 cells. eGFP-tagged isoforms of Sd which contained the NES (NES, Sd ΔNLS, Sd Δ348–440 and Sd Δ392–440), and Sd fragments in which the NES was deleted (Sd Δ301–355 and Sd Δ294–440), were tested for nuclear localization in the presence and absence of Leptomycin B. N is >15 for all conditions. *** indicates a significant difference at P<0.001. Error bars are the standard error of the mean.</p

The intact NLS is necessary for both the proper nuclear translocation of Sd and Importin-α3 binding.

<p>(A–E) Localization of the indicated eGFP reporter tagged proteins in transiently transfected in S2 cells with DAPI stained nuclei and visualized via confocal microscopy. See the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g002" target="_blank">Figs. 2A–G</a> for details. (A) eGFP-SD. When Sd is expressed in S2 cells, reporter activity is predominantly nuclear. (B) eGFP-SD ΔNLS. Deleting amino acids 143–163 of Sd disrupts its localization and leads to diffuse reporter activity throughout both the nucleus and cytoplasm. (C) eGFP-SD mNLS^N+C. Mutation of the six basic amino acids identified as being part of the classical consensus bipartite sequence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g001" target="_blank">Fig. 1</a>) to N causes disruption of localization similar to that seen when the NLS is deleted. (D) eGFP-SD mNLS^N. When the two N-terminal basic amino acids are mutated to N, a lesser disruption of the nuclear signal is observed (compare to A). (E) eGFP-SD mNLS^C. Sd with the four C-terminal basic amino acids mutated to N drives diffuse localization of the eGFP reporter, similar to that seen for SD ΔNLS and SD mNLS^N+C. (compare to panels B and C, respectively). (F) Co-IP of Sd and Imp-α3. Cells expressing 3xFLAG-Sd, 3xFLAG-Sd mNLS^N+C as well as cells mock transfected with water alone were lysed, immunoprecipitated with αFLAG beads and analyzed via western blotting. Detection was with anti-FLAG or anti-Imp-α3. Detection with anti-FLAG ensures expression of the two tagged proteins is approximately equal. The lysate of all cells had a strong Imp-α3 signal. Imp-α3 co-immunoprecipitated strongly with 3xFLAG-Sd, while only weakly with 3xFLAG-Sd mNLS^N+C. The mock transfected cells showed almost no Imp-α3 signal after immunoprecipitation, controlling for the specificity of the anti-FLAG beads.</p

The NLS of Sd is directs an eGFP tag to the nucleus.

<p>(A–G) Localization of the indicated eGFP reporter tagged peptides in transiently transfected in S2 cells with DAPI stained nuclei and visualized via confocal microscopy. A¹–G¹ are the green (eGFP) channels. A²–G² are the blue (DAPI) channels. A³–G³ are the green and blue channels (merge). Hatched lines indicate the boundary of cells, as determined by the extent of the weak cytoplasmic signal. Percentages indicate the percent nuclear signal relative to total signal measured in the given cell. (A) eGFP. When eGFP is expressed alone, diffuse expression is seen throughout the cell, including the nucleus. (B) TEA-eGFP. A fragment of Sd stretching from amino acids 88–178 (which includes the entire TEA/NLS domain) shows almost exclusive reporter activity within the nucleus of the expressing cells. (C) NLS-eGFP. Amino acids 143–163 of Sd (which includes the NLS and two flanking amino acids on either side) drives reporter expression to the nucleus. (D) eGFPx2+HA (referred to hereafter as eGFPx2). eGFPx2 expression is excluded from the nucleus. (E) TEA-eGFPx2. A TEA-eGFPx2 fusion is primarily nuclear. (F) eGFPx2+NLS. This construct is found throughout the cell, but is enriched in the nucleus. (G) TEAΔNLS-eGFPx2. When the NLS is removed from the TEA domain, it is no longer able to direct the tag to the nucleus.</p

The C-terminal domain can act to both repress and facilitate the nuclear localization of Sd.

<p>A series of internal deletions and truncations of Sd were generated, expressed with a fused N-terminal eGFP marker in S2 cells and assayed for cellular distribution. (A) Schematic of the various Sd isoforms generated along with a summary table of the localization experiments. The domains of Sd are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g001" target="_blank">Fig. 1A</a>. ‘mNLS^N+C’ is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g003" target="_blank">Fig. 3C</a>. A † denotes a construct with diffuse or nuclear excluded signal (<58% nuclear signal). N is the total number of cells measured from at least two independent transfections. The next four columns represent four arbitrary localization patterns along with the mean nuclear signal each grouping represents. For each peptide, the percentage of cells that fall into one of the four categories is indicated. (B–E) Representative cells showing 81%, 65%, 47% and 31% nuclear signal (B, C, D and E, respectively). See the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g002" target="_blank">Figs. 2A–G</a> for details.</p

During wing development, 3xFLAG-PMSD and 3xFLAG-SD mNLS^N+C act as dominant negative forms of Sd.

<p>(A and B) Localization of the indicated mRFP reporter tagged proteins in transiently transfected in S2 cells with DAPI stained nuclei and visualized via confocal microscopy. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g002" target="_blank">Figs. 2A–G</a> for details. (A) Sd-mRFP expression. Sd strongly localizes an mRFP tag to the nucleus. (B) PMSD-mRFP expression. Sd tagged with a N-terminal palmitoylation/myristoylation sequence (PMSD) and C-terminal mRFP tag shows strong localization to the cytoplasmic membrane of S2 cells. (C–E) Light micrographs of flies with the indicated genotypes. (C) Wildtype Oregon-R (Ore^R) fly. (D–E) Males containing either <i>UAS</i>-<i>3xFLAG</i>-<i>PMSD</i> or <i>UAS</i>-<i>3xFLAG</i>-<i>SD mNLS^N+C</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021431#pone-0021431-g003" target="_blank">Fig. 3C</a>) inserted on the 2^nd chromosome and balanced over <i>CyO</i> were crossed (two independent lines/insert) to virgin females homozygous for <i>sd</i>-GAL4 and the resultant progeny were scored. Insets are magnified views of the wing tissue. Scale bars are 1 mm (D–E) or 0.1 mm (D and E insets). Arrows indicate the wing, while arrowheads indicate the haltere. (D) Female fly containing <i>UAS</i>-<i>3xFLAG-PMSD</i> under the control of <i>sd</i>-GAL4. Almost no wing or haltere tissue is present. (E) Female fly containing <i>UAS</i>-<i>3xFLAG-mNLS^N+C</i> under the control of <i>sd</i>-GAL4. Again, virtually no wing or haltere tissue is present.</p