MLF1-interacting protein is mainly localized in nucleolus through N-terminal bipartite nuclear localization signal.
BACKGROUND
The myelodysplasia/myeloid leukemia factor 1-interacting protein (MLF1LP, also called KLIP1 and CENP-50) is reported to localize in both the nucleus and the cytoplasm. To investigate the functions of MLF1IP, its subnuclear localization was studied.


METHODS
MLF1IP was tagged with green fluorescent protein (EGFP). Fibrillarin was tagged with red fluorescent protein (DsRed). EGFP-tagged MLF1IP deletion vectors were also constructed. Plasmid-constructs were transfected into human cervical adenocarcinoma HeLa cells or monkey kidney fibroblast COS-7 cells, and the localization was studied by either confocal fluorescence microscopy or fluorescence microscopy.


RESULTS
Ectopically expressed MLF1IP was localized mainly in the nucleolus. In some cells, small dot-like particles of MLF1IP fluorescence were observed in the nucleoplasm. Co-staining of fibrillarin disclosed that MLF1IP was co-localized with fibrillarin in the nucleolus. Deletion mutants of MLF1IP revealed that the N-terminal bipartite nuclear localization signal (NLS) was responsible for nucleolar targeting.


CONCLUSIONS
MLF1IP was localized mainly in the nucleolus through the N-terminal bipartite NLS and partly in the nucleoplasm featuring small dot-like particles. These findings suggest that MLF1IP may have multi-functions and its different localizations may contribute to carcinogenesis.
Suzuki
Hideaki
H
Department of General Medicine, Institute of DNA Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan.
Arakawa
Yasuhiro
Y
Ito
Masaki
M
Saito
Shinobu
S
Takeda
Nobuakira
N
Yamada
Hisashi
H
Horiguchi-Yamada
Junko
J
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins; D002466:Cell Nucleolus / Q000737:chemistry* / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006367:HeLa Cells; D006657:Histones; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
C485917:CENPU protein, human; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; C047458:fibrillarin; D049452:Green Fluorescent Proteins