Rao M R K Subba, Kumari Gita, Balasundaram D, Sankaranarayanan R, Mahalingam S
Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, ECIL Road, Nacharam, Hyderabad 500076, India.
J Mol Biol. 2006 Dec 8;364(4):637-54. doi: 10.1016/j.jmb.2006.09.007. Epub 2006 Sep 8.
A variety of G-proteins and GTPases are known to be involved in nucleolar function. We describe here a new evolutionarily conserved putative human GTPase, guanine nucleotide binding protein-like 3-like (GNL3L). Genes encoding proteins related to GNL3L are present in bacteria and yeast to metazoa and suggests its critical role in development. Conserved domain search analysis revealed that the GNL3L contains a circularly permuted G-motif described by a G5-G4-G1-G2-G3 pattern similar to the HSR1/MMR1 GTP-binding protein subfamily. Highly conserved and critical residues were identified from a three-dimensional structural model obtained for GNL3L using the crystal structure of an Ylqf GTPase from Bacillus subtilis. We demonstrate here that GNL3L is transported into the nucleolus by a novel lysine-rich nucleolar localization signal (NoLS) residing within 1-50 amino acid residues. NoLS identified here is necessary and sufficient to target the heterologous proteins to the nucleolus. We show for the first time that the lysine-rich targeting signal interacts with the nuclear transport receptor, importin-beta and transports GNL3L into the nucleolus. Interestingly, depletion of intracellular GTP blocks GNL3L accumulation into the nucleolar compartment. Furthermore, mutations within the G-domains alter the GTP binding ability of GNL3L and abrogate wild-type nucleolar retention even in the presence of functional NoLS, suggesting that the efficient nucleolar retention of GNL3L involves activities of both basic NoLS and GTP-binding domains. Collectively, these data suggest that GNL3L is composed of distinct modules, each of which plays a specific role in molecular interactions for its nucleolar retention and subsequent function(s) within the nucleolus.
已知多种G蛋白和GTP酶参与核仁功能。我们在此描述一种新的进化上保守的假定人类GTP酶，鸟嘌呤核苷酸结合蛋白样3样（GNL3L）。编码与GNL3L相关蛋白质的基因存在于从细菌、酵母到后生动物中，这表明其在发育过程中起关键作用。保守结构域搜索分析表明，GNL3L包含一个由G5 - G4 - G1 - G2 - G3模式描述的环状排列的G基序，类似于HSR1/MMR1 GTP结合蛋白亚家族。利用枯草芽孢杆菌Ylqf GTP酶的晶体结构为GNL3L获得的三维结构模型鉴定出了高度保守且关键的残基。我们在此证明，GNL3L通过位于1 - 50个氨基酸残基内的一种新的富含赖氨酸的核仁定位信号（NoLS）被转运到核仁中。此处鉴定出的NoLS对于将异源蛋白质靶向核仁是必要且充分的。我们首次表明，富含赖氨酸的靶向信号与核转运受体importin-β相互作用，并将GNL3L转运到核仁中。有趣的是，细胞内GTP的消耗会阻止GNL3L在核仁区室中的积累。此外，G结构域内的突变会改变GNL3L的GTP结合能力，即使在存在功能性NoLS的情况下也会消除野生型核仁保留，这表明GNL3L有效的核仁保留涉及碱性NoLS和GTP结合结构域的活性。总体而言，这些数据表明GNL3L由不同的模块组成，每个模块在其核仁保留和随后在核仁内的功能的分子相互作用中发挥特定作用。
