Subcellular localization of RPS19. (A) The use of an anti-RPS19 antibody raised in chicken shows primary localization of RPS19 in the nucleus and particularly to the nucleoli in Cos-7 cells (arrows). A weaker and diffuse cytoplasmic staining is also observed. The left panel represents 4′6-diamidino-2-phenylindole (DAPI) staining for the nucleus; the middle panel, FITC staining of RPS19; and the right panel, the merged picture of DAPI and FITC staining. (B) Nucleolar localization of RPS19 is confirmed after transfection of Cos-7 cells with the recombinant GFP–wild-type RPS19 (left panel). RPS19 colocalizes with the major nucleolar protein nucleolin (middle panel) after transfection of Cos-7 cells with GFP–wild-type RPS19 and the red fluorescent DsRed1-nucleolin fusion protein (right panel). (C) Nucleolar localization of RPS19 (arrows) is also observed in live cells after transfection of Cos-7 cells with GFP-RPS19, ruling out an artifactual effect of cell fixation on RPS19 subcellular distribution. (D) Deletion of the last 3 COOH-terminal amino acids does not impair RPS19 localization compared with wild type (left panel). In contrast, deletion of the last 25 COOH-terminal amino acids dramatically alters RPS19 nucleolar import (middle panel). Furthermore, deletion of the first 14 NH₂ amino acids also impaired RPS19 nucleolar import (right panel). (E) The 22-amino-acid-long NoS identified in the last 25 amino acids fails to import pyruvate kinase into the nucleoli. (F) Addition of the first NoS to the previous pyruvate kinase construct also fails to be imported into the nucleoli of Cos-7 cells, confirming that although these 2 NoS are necessary, they are not sufficient for nucleolar import. Original magnifications: × 63 (A,B,D,E,F); × 40 (C).