title: NO66, a highly conserved dual location protein in the nucleolus and in a special type of synchronously replicating chromatin.
abstract: It has recently become clear that the nucleolus, the most prominent nuclear subcompartment, harbors diverse functions beyond its classic role in ribosome biogenesis. To gain insight into nucleolar functions, we have purified amplified nucleoli from Xenopus laevis oocytes using a novel approach involving fluorescence-activated cell sorting techniques. The resulting protein fraction was analyzed by mass spectrometry and used for the generation of monoclonal antibodies directed against nucleolar components. Here, we report the identification and molecular characterization of a novel, ubiquitous protein, which in most cell types appears to be a constitutive nucleolar component. Immunolocalization studies have revealed that this protein, termed NO66, is highly conserved during evolution and shows in most cells analyzed a dual localization pattern, i.e., a strong enrichment in the granular part of nucleoli and in distinct nucleoplasmic entities. Colocalizations with proteins Ki-67, HP1alpha, and PCNA, respectively, have further shown that the staining pattern of NO66 overlaps with certain clusters of late replicating chromatin. Biochemical experiments have revealed that protein NO66 cofractionates with large preribosomal particles but is absent from cytoplasmic ribosomes. We propose that in addition to its role in ribosome biogenesis protein NO66 has functions in the replication or remodeling of certain heterochromatic regions.
authors.affiliation: Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Eilbracht
authors.forename: Michaela
authors.initials: M
authors.lastname: Reichenzeller
authors.forename: Michaela
authors.initials: M
authors.lastname: Hergt
authors.forename: Martina
authors.initials: M
authors.lastname: Schnölzer
authors.forename: Hans
authors.initials: H
authors.lastname: Heid
authors.forename: Michael
authors.initials: M
authors.lastname: Stöhr
authors.forename: Werner W
authors.initials: WW
authors.lastname: Franke
authors.forename: Marion S
authors.initials: MS
authors.lastname: Schmidt-Zachmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology*; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D049308:Dioxygenases; D005434:Flow Cytometry; D006367:HeLa Cells; D006570:Heterochromatin / Q000737:chemistry; D056466:Histone Demethylases; D006801:Humans; D019394:Ki-67 Antigen / Q000096:biosynthesis; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D029867:Xenopus Proteins / Q000096:biosynthesis* / Q000502:physiology*; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: C000715594:CBX5 protein, human; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D018076:DNA, Complementary; D006570:Heterochromatin; D019394:Ki-67 Antigen; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen; C492860:RIOX1 protein, Xenopus; D029867:Xenopus Proteins; D000090266:Chromobox Protein Homolog 5; D013395:Sucrose; D012313:RNA; D049308:Dioxygenases; D056466:Histone Demethylases; C492859:RIOX1 protein, human