C-terminal domain of the mitotic apparatus protein p62 targets the protein to the nucleolus during interphase.
Mitotic apparatuses from sea urchin embryos contain a protein (p62), previously shown to be required for mitotic progression. This protein localizes to the mitotic apparatus during cell division in urchin embryos and mammalian tissue culture cells. We show here by immunofluorescence that p62 is localized to the nucleus of mammalian cells during interphase and is highly concentrated in nucleoli. In addition, a fusion protein composed of full-length p62 and green fluorescent protein also localizes to nucleoli when expressed in COS-7 cells in culture. Analysis of the primary sequence of p62 reveals three distinct domains of the protein based on amino acid charge distribution: the acidic N-terminal domain, the basic C-terminal domain, and the central, M-domain, which contains alternating subdomains of clusters of acidic and basic residues. To identify the domain important for nucleolar localization during interphase, specific domains of p62 alone, or in combination with each other or with beta-galactosidase were fused to green fluorescent protein. Following confirmation of the fusion constructs by sequence analysis, the constructs were expressed in mammalian cells, expression was confirmed by immunoblotting, and the fusion proteins were localized via fluorescence microscopy. The data demonstrate that the C-terminal domain of p62 is both necessary and sufficient for the nuclear localization and nucleolar binding of p62 that is observed during interphase.
Warner
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AK
Department of Biological Sciences, 6044 Gilman Laboratory, Dartmouth College, Hanover, New Hampshire 03755, USA.
Sloboda
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RD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007399:Interphase; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D016415:Sequence Alignment; D001616:beta-Galactosidase / Q000235:genetics
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C104815:mitotic apparatus-associated protein p62, sea urchin; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase