Subcellular Distribution of the Human Putative Nucleolar GTPase GNL1 is Regulated by a Novel Arginine/Lysine-Rich Domain and a GTP Binding Domain in a Cell Cycle-Dependent Manner

GNL1, a putative nucleolar GTPase, belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. Members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization with respect to cell growth remain unknown. To understand the nuclear transport mechanism of GNL1, we have identified a novel arginine/lysine-rich nucleolar localization signal in the NH2-terminus that is shown to translocate GNL1 and a heterologous protein to the nucleus/nucleolus in a pathway that is independent of importin-a and importin-beta. In addition, the present investigation provided evidence that GNL1 localized to the nucleus and the nucleolus only in G2 stage, in contrast to its cytoplasmic localization in the Cl and S phases of the cell cycle. Using heterokaryon assay, we have demonstrated that GNL1 shuttles between the nucleus and the cytoplasm and that the motif between amino acids 201 and 225 is essential for its export from the nucleus by a signal-mediated CRM1-independent pathway. Alanine-scanning mutagenesis of conserved residues within G-domains suggests that the G2 motif is critical for guanine nucleotide triphosphate (GTP) binding of GNL1 and further showed that nucleolar retention of GNU is regulated by a GTP-gating-mediated mechanism. Expression of wild-type GNL1 promotes G2/M transition, in contrast to the G-domain mutant (G2m), which fails to localize to the nucleolus. These data suggest that nucleolar translocation during G2 phase may be critical for faster M-phase transition during cell proliferation. Replacement of conserved residues within the G5 motif alters the stability of GNU without changing GTP binding activity. Finally, our data suggest that ongoing transcription is essential for the efficient localization of GNL1 to the nucleolus. Overall, the results reported here demonstrate that multiple mechanisms are involved in the translocation of GNL1 to the nucleolus in a cell cycle-dependent manner to regulate cell growth and proliferation. (C) 2012 Elsevier Ltd. All rights reserved.