title: Analysis of nucleolar protein fibrillarin mobility and functional state in living HeLa cells.
abstract: Fibrillarin is an evolutionarily-conserved and obligatory protein component of eukaryotic cell nucleoli involved in pre-rRNA processing and methylation. In vertebrates the fibrillarin molecule contains two cysteine residues (Cys99 and Cys268) whose sulfhydryl groups are able to establish intramolecular -S-S- bridges. However, the functional state of fibrillarin with reduced or oxidized thiol groups is still practically unstudied. Besides, there are no data in the literature concerning existence of the -S-S- fibrillarin form in human cells. To answer these questions, we used plasmids encoding native human fibrillarin and its mutant form devoid of cysteine residues (fibrillarinC99/268S) fused with EGFP for temporary transfection of HeLa cells. The mobile fraction localizing the enzymatically active protein molecules and the fluorescence half-recovery time characterizing the rate of enzymatic reactions were determined by the FRAP technique using a confocal laser scanning microscope. Measurements were carried out at 37 and 27°C. The results show that the fibrillarin pool in HeLa cells includes two protein forms, with reduced SH groups and with oxidized SH groups forming intramolecular -S-S- bridges between Cys99 and Cys268. However, the absence of Cys99 and Cys268 has no effect on intracellular localization of fibrillarin and its main dynamic parameters. The human fibrillarin form without disulfide bridges is included into the mobile protein fraction and is consistent with its functionally active state.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia. v.barygina@mail.ru
authors.forename: V V
authors.initials: VV
authors.lastname: Barygina
authors.forename: V P
authors.initials: VP
authors.lastname: Veiko
authors.forename: O V
authors.initials: OV
authors.lastname: Zatsepina
mesh_terms: D000595:Amino Acid Sequence; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; C047458:fibrillarin; D003545:Cysteine