Fig. 7. Nucleolar distribution and the nucleostemin-interaction of RSL1D1 are controlled by separate domains. (A) RSL1D1 contains an L1 domain (aa 150-254), a coiled-coil domain (C) and three putative NLS (black boxes). Myc-tagged truncated RSL1D1 mutants were generated to map nucleostemin-interacting and nucleolus-targeting regions. (B) Affinity binding assays show that GST fusions of both the BC- and GI-domains bind the (aa 317-452) part of RSL1D1 that does not contain the L1- or C-domain. (C-L) Anti-Myc and anti-nucleostemin double-labeled immunofluorescence demonstrate that the 150-316 aa region of RSL1D1 is localized in the nucleolus (C). The N-terminal 1-149 aa region is cytoplasmic by itself (D), and becomes partially nucleolar when tagged with an SV40 NLS (E). Distribution of the C-terminal 317-452 aa region is diffuse in the nucleus (F), with some cells showing more signals in the nucleolus than in the nucleoplasm (G). Within the aa 150-316 segment, the L1 domain (150-254) by itself is primarily cytoplasmic (H), but becomes mostly nucleolar when fused to an SV40 NLS (I,J, nlsL1). The coiled-coil domain (255-316) is diffusely localized in the nucleus (K). The nucleostemin signal is diminished or absent from the nucleolus of many cells expressing nlsL1 (J). Bars, 10 µm.