A Highly Organized Structure Mediating Nuclear Localization of a Myb2 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

Abstract

Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis. The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.

Regulation of Nuclear Translocation of the Myb1 Transcription Factor by TvCyclophilin 1 in the Protozoan Parasite Trichomonas vaginalis

“…In this regard, the transfected cell line overexpressing HA-Myb1(P107A) may provide a tool to study the downstream target genes of Myb1. The genome of T. vaginalis encodes Ͼ400 Myb-like proteins among which Myb2 and Myb3 exploit highly ordered helical structures spanning the DBD for nuclear translocation (24,25). In addition, Myb1 may use a similar entity for nuclear translocation.…”

“…Similar to c-Myb, the DBDs in Myb1, Myb2, and Myb3 are composed of six helices arranged in two bundles with two antiparallel ␤-sheets unique to Myb3 forming a small hairpin following the helices (21)(22)(23). Intriguingly, the DBDs of Myb2 and Myb3 were also shown to mediate their own nuclear translocation (24,25). A similar structural entity in Myb1 may also mediate its own nuclear translocation.…”

Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

“…The insert restricted by SacI/BglII was cloned into SacI/ BamHI-restricted pET6H (11) to produce pET6H/Myb3(48ϳ180). Sitedirected mutagenesis using a two-step PCR as described above and primer pairs listed in Table S1 in the supplemental material was performed to mutate the I79 isoleucine to a proline or alanine to produce pET6H/ Myb3-I79P(48ϳ180) or pET6H/Myb3-I79A(48ϳ180), respectively.…”

“…A 3= DNA fragment encoding a bacterial tetracycline repressor (TetR) was amplified from p48-143-tetR, which encodes a Myb2-tetR fusion protein (11), using the primer pair myb3(180)tetR-5= and tetR-sac2-3=. The 5= and 3= DNA fragments thus generated were mixed, denatured, and annealed for a second-round PCR using the primer pair ap65-2.1-sac2-5= and tetR-sac2-3=.…”

“…CRM1-mediated nuclear protein export can be inhibited by the fungal metabolite leptomycin B (LMB) (22). The classical NLS (cNLS) comprises a loose consensus polybasic sequence, K(K/R)X(K/R) (23), of the simian virus 40 T antigen that allows the nuclear import of nonnuclear proteins in many heterologous systems but does not function in T. vaginalis (11). Instead, a novel NLS comprising an ordered structure embedded in the R2R3 domain of Myb2 was defined in the parasite (11) (see Fig.…”