A highly organized structure mediating nuclear localization of a Myb2 transcription factor in the protozoan parasite Trichomonas vaginalis.
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.
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chien-Hsin
CH
Chu
Lung-Chun
LC
Chang
Hong-Ming
HM
Hsu
Shu-Yi
SY
Wei
Hsing-Wei
HW
Liu
Yu
Y
Lee
Chung-Chi
CC
Kuo
Dharmu
D
Indra
Chinpan
C
Chen
Shiou-Jeng
SJ
Ong
Jung-Hsiang
JH
Tai
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002467:Cell Nucleus / Q000378:metabolism*; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry; D040461:Gene Components; D049409:Luciferases, Firefly / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D010449:Peptide Mapping; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014246:Trichomonas vaginalis / Q000378:metabolism*
C520248:Myb2 protein, Trichomonas vaginalis; D019913:Nuclear Localization Signals; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D049409:Luciferases, Firefly