Different domains cooperate to target the human ribosomal L7a protein to the nucleus and to the nucleoli

The human ribosomal protein L7a is a component of the major ribosomal subunit, We transiently expressed in HeLa cells L7a-beta-galactosidase fusion proteins and studied their subcellular localization by indirect immunofluorescence staining with anti-p-galactosidase antibodies, We have identified three distinct domains responsible for the nuclear targeting of the protein: domain I, amino acids 23-51; domain II, amino acids 52-100; domain III, amino acids 101-220, each of which contains at least one nuclear localization signal (NLS), Through subcellular localization analysis of deletion mutants of L7a-beta-galactosidase chimeras, we demonstrate that domain II plays a special role because it is necessary, although not sufficient, to target the chimeric beta-galactosidase to the nucleoli, In fact, we demonstrate that the nucleolar targeting process requires the presence of domain II plus an additional basic domain that can be represented by an NLS or a basic stretch of amino acids without NLS activity. Thus, when multiple NLS are present, each NLS exerts distinct functions, Domain II drives nucleolar accumulation of a reporter protein with the cooperative action of a short basic amino acid sequence, suggesting a mechanism requiring protein-protein or protein-nucleic acid interactions.