A nuclear localization signal binding protein in the nucleolus.
We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine-labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.
Meier
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UT
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York 10021.
Blobel
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D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000378:metabolism; D051381:Rats; D011915:Rats, Inbred BUF; D013539:Simian virus 40 / Q000235:genetics; D014981:Xenopus
D000952:Antigens, Polyomavirus Transforming; D009687:Nuclear Proteins; D021382:Protein Sorting Signals