Fig. 1. Nucleostemin containes two distinct nucleolus-targeting regions with different nucleolar retention time. (A) Diagram of nucleostemin (NS) protein structure and mutant constructs used to determine the nucleolus-targeting domains of nucleostemin. An SV40 nuclear localization signal (NLS, black circle) was introduced in mutants that lack an endogenous NLS (black boxes). Numbers indicate the aa positions. B, basic; C, coiled-coil; G, GTP-binding; I, intermediate; A, acidic. (B) Subcellular distributions of mutant proteins in U2OS cells were revealed by a C-terminal GFP-tag; counterstaining with anti-B23 antibody is shown in the right upper quadrants in all panels on a 50% scale. Both the B- and the G-domains (nlsG) displayed a nucleolar distribution pattern. Mutation of the conserved GTP-binding residue G256, yielding nlsG(256), did not affect the nucleolar localization of the G-domain alone. In the presence of the I-domain, such a mutation [nlsGI(256)] abolished its nucleolar localization. A cytoplasmic hydrolase protein (Hd3) was tagged with the SV40 NLS to demonstrate that this sequence was not sufficient to confer nucleolar localization. Bar, 10 µm. (C1) FRAP recovery times (x-axis, in seconds) of the B-domain (trace 1), the full-length nucleostemin (trace 2) and the GI-domain (trace 3) were determined in CHO cells transiently transfected with the GFP-fusion constructs. The y-axis represents the percentage of fluorescence intensity in the bleached area relative to the prebleached intensity. (C2) The FRAP recovery time of the GI-domain (nlsGI) and the A-domain deletion mutant (NSdA) was measured as described in the Materials and Methods. (C3) Statistical analyses at 5, 10, 20 and 30 seconds (mean ± standard error of mean (s.e.m.), n=20).