Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein.
NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P(270)KKRKAP(276)) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.
Kutty
R Krishnan
RK
Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. kuttyk@nei.nih.gov
Chen
Shanyi
S
Samuel
William
W
Vijayasarathy
Camasamudram
C
Duncan
Todd
T
Tsai
Jen-Yue
JY
Fariss
Robert N
RN
Carper
Deborah
D
Jaworski
Cynthia
C
Wiggert
Barbara
B
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D019556:COS Cells; D002452:Cell Count; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
D000906:Antibodies; D003598:Cytoskeletal Proteins; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; C428450:RAI14 protein, human; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D006639:Histidine