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. 2012 Dec;11(12):1441-50.
doi: 10.1128/EC.00190-12. Epub 2012 Oct 5.

Iron-inducible nuclear translocation of a Myb3 transcription factor in the protozoan parasite Trichomonas vaginalis

Hong-Ming Hsu  1 , Yu Lee, Dharmu Indra, Shu-Yi Wei, Hsing-Wei Liu, Lung-Chun Chang, Chinpan Chen, Shiou-Jeng Ong, Jung-Hsiang Tai
Affiliations

Iron-inducible nuclear translocation of a Myb3 transcription factor in the protozoan parasite Trichomonas vaginalis

Hong-Ming Hsu et al. Eukaryot Cell. 2012 Dec.

Abstract

In Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

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Figures

Fig 1
Fig 1
Overexpression of HA-Myb3 in T. vaginalis. (A) In pAP65-2.1-ha-myb3, an ap65-2.1 promoter drives expression of the HA-tagged myb3 gene and the β-tubulin (TUB) promoter drives the neo gene. Restriction sites for constructing the plasmid are indicated. Cells for the experiments were grown in normal medium overnight. (B) Cells overexpressing HA-Myb3 (top) or the control (bottom) were examined by an immunofluorescence assay using the anti-HA antibody and FITC-conjugated mouse IgG. The nucleus was stained with DAPI, and the cell morphology was recorded by phase-contrast microscopy. (C) Cell lysates from the control (lanes 1 and 3) and cells overexpressing HA-Myb3 (lanes 2 and 4) were examined by Western blotting using anti-HA (lanes 1 and 2), anti-Myb3 (lanes 3 and 4, top), and anti-α-tubulin (lanes 3 and 4, bottom) antibodies. (D) Cell lysates (TL) from the control and cells overexpressing HA-Myb3 were fractionated into cytosolic (C) and nuclear (N) fractions for Western blotting using anti-Myb3, anti-Myb1, anti-Myb2, anti-H3K9-Ac, and anti-CyPA1 antibodies. In panels C and D, molecular weight markers (left) and the detected proteins (right) are indicated.
Fig 2
Fig 2
The polybasic sequence and Myb3's nuclear localization. (A) Relative locations of Myb3′ R1R2R3 structural domains (helices), a β-hairpin (two arrows), and a polybasic (PB) sequence are depicted. The PB sequence was mutated to produce a mutant protein, KKRK170–173AAAA. Cells overexpressing HA-Myb3, KKRK170–173AAAA, or the control were grown overnight in normal medium. (B) Cells were examined by an immunofluorescence assay using a mouse anti-HA antibody and FITC-conjugated mouse IgG. The nucleus was stained with DAPI, and the cell morphology was recorded by phase-contrast microscopy. (C) Cell lysates (TL) were fractionated into cytosolic (C) and nuclear (N) fractions for Western blotting using anti-HA (top), anti-H3K9-Ac (middle), and anti-CypA1 (bottom) antibodies. Molecular weight markers (left) and the proteins detected (right) are indicated.
Fig 3
Fig 3
Deletion mapping to define Myb3's NLS. (A) The relative locations of the R1, R2, and R3 domains and a polybasic (PB) sequence in Myb3 are indicated by numbers. Regions spanning various fragments of Myb3 were each fused with an N-terminal HA tag and a C-terminal TetR to produce fusion proteins, the names of which are shown on the left to identify the fusion protein, while the nuclear or cytoplasmic localization of a protein was examined in panel B (indicated by N or C, respectively). The cells were grown in normal medium overnight. (B) Cells overexpressing various proteins or the control, as indicated on the left, were examined by an immunofluorescence assay using a mouse anti-HA antibody and FITC-conjugated mouse IgG. Nuclei were stained with DAPI, and the cell morphology was recorded by phase-contrast microscopy. The relative intensity of the FITC signal in the nucleus (N) versus the entire cell (N+C) for each protein is shown as a histogram at the bottom. The error bars indicate standard deviations. (C) Cell lysates (TL) were fractionated into cytosolic (C) and nuclear (N) fractions for Western blotting using anti-HA (top), anti-H3K9-Ac (middle), and anti-CyPA1 (bottom) antibodies. Signal intensities in samples from nuclear fractions were quantified and are shown as a histogram at the bottom. Molecular weight markers (left) and the detected proteins (right) are indicated. Fusion proteins are indicated by arrowheads and HA-Myb3 by an arrow.
Fig 4
Fig 4
Roles of the NLS C-terminal tail in the nuclear translocation of Myb3. The sequence spanning aa 157 to 165 of HA-Myb3 was mutated to produce NSN157–159AAA, HKE160–162AAA, or ILL163–165AAA. Cells were grown overnight in normal medium. (A) Cells were examined by an immunofluorescence assay using a mouse anti-HA antibody and FITC-conjugated mouse IgG. The nuclei were stained with DAPI, and the cell morphology was recorded by phase-contrast microscopy. (B) Cell lysates (TL) were fractionated into cytosolic (C) and nuclear (N) fractions for Western blotting using anti-HA (top), anti-H3K9-Ac (middle), and anti-CyPA1 (bottom) antibodies. Molecular weight markers (left) and the detected proteins (right) are indicated.
Fig 5
Fig 5
Effects of a conserved isoleucine, I79, on nuclear importation, DNA-binding activity, and the structural integrity of Myb3. I79 in HA-Myb3 was mutated to alanine (I79A) or proline (I79P). Cells were grown overnight in normal medium. (A) Cells were examined by an immunofluorescence assay using a mouse anti-HA antibody and FITC-conjugated mouse IgG. Nuclei were stained with DAPI, and the cell morphology was recorded by phase-contrast microscopy. (B) Cell lysates (TL) were fractionated into cytosolic (C) and nuclear (N) fractions for Western blotting using anti-HA (top), anti-H3K9-Ac (middle), and anti-CyPA1 (bottom) antibodies. Molecular weight markers (left) and the detected proteins (right) are indicated. (C) I79 in the recombinant protein rMyb3(48–180) (lane 2) was mutated to alanine or proline to produce rI79A(48–180) (lane 3) or rI79P(48–180) (lane 4), respectively. An electrophoretic mobility shift assay was performed with coincubation of the recombinant proteins with a γ-32P-labeled MRE-1 probe (lane 1). (D) The secondary structures of the recombinant proteins rMyb3(48–180), rI79A(48–180), and rI79P(48–180) were monitored by far-UV CD spectra. (E) The ternary folding of rMyb3(48–180), rI79A(48–180), and rI79P(48–180) was examined by fluorescence spectroscopy. All fluorescence emission spectra were measured at 300 to 400 nm and 25°C. Protein samples were dissolved in PBS buffer (pH 7.4) with 1 mM dithiothreitol (DTT). rMyb3(48–180) was also denatured with 6 M guanidine hydrochloride.
Fig 6
Fig 6
Iron-inducible nuclear translocation of Myb3. Cells were cultivated overnight in growth medium depleted of iron. (A) Cells overexpressing HA-Myb3 and 4HA-Myb2 were replete with iron and were fixed at the indicated time intervals. (B) Cells overexpressing HA-Myb3 were pretreated with methanol or LMB for 30 min prior to iron repletion and fixed at the indicated time intervals. In panels A and B, an immunofluorescence assay was performed using a mouse anti-HA antibody and FITC-conjugated mouse IgG. Images of DAPI-stained nuclei and cell morphology are shown in Fig. S2 in the supplemental material. Signal intensities were quantified and are shown as a histogram at the bottom in panel A and on the right in panel B. (C) Cell lysates (TL) from nontransfected cells were fractionated into cytosolic (C) and nuclear (N) fractions for Western blotting using anti-Myb3, anti-Myb1, anti-Myb2, anti-H3K9-Ac, and anti-CypA1 antibodies. Molecular weight markers (left) and the detected proteins (right) are indicated.
Fig 7
Fig 7
Sequence elements in the C-terminal tail of Myb3's NLS for iron-inducible nuclear translocation. Cells overexpressing HA-Myb3 and its derived proteins (48–180-TetR or 48–167-TetR [A]; NSN157–159AAA, HKE160–162AAA, or ILL163–165AAA [B]; KKRK170–173AAAA [C]) were grown in normal or iron-depleted medium overnight, and the latter were also supplied with iron and sampled at the indicated time intervals for an immunofluorescence assay using a mouse anti-HA antibody and FITC-conjugated mouse IgG. Images of DAPI-stained nuclei and cell morphology are shown in Fig. S3 in the supplemental material. Each bar in the histograms of the micrographs shows the average signal intensity from the measurements of 300 cells in five microscopic fields.
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