CEA, iBEB, Service de Biochimie et Toxicologie Nucléaire/LBSP, Bagnols-sur-Cèze, France.
Cell Cycle. 2013 Feb 1;12(3):463-72. doi: 10.4161/cc.23367. Epub 2013 Jan 16.
GTPases are molecular switches that regulate a wide-range of cellular processes. The GPN-loop GTPase (GPN) is a sub-family of P-loop NTPase that evolved from a single gene copy in archaea to triplicate paralog genes in eukaryotes, each having a non-redundant essential function in cell. In Saccharomyces cerevisiae, yGPN1 and yGPN2 are involved in sister chromatid cohesion mechanism, whereas nothing is known regarding yGPN3 function. Previous high-throughput experiments suggested that GPN paralogs interaction may occur. In this work, GPN|GPN contact was analyzed in details using TAP-Tag approach, yeast two-hybrid assay, in silico energy computation and site-directed mutagenesis of a conserved Glu residue located at the center of the interaction interface. It is demonstrated that this residue is essential for cell viability. A chromatid cohesion assay revealed that, like yGPN1 and yGPN2, yGPN3 also plays a role in sister chromatid cohesion. These results suggest that all three GPN proteins act at the molecular level in sister chromatid cohesion mechanism as a GPN|GPN complex reminiscent of the homodimeric structure of PAB0955, an archaeal member of GPN-loop GTPase.
GTPases 是一类分子开关，调节着广泛的细胞过程。GPN 环 GTPase（GPN）是 P 环 NTPase 的一个亚家族，从古菌中的单个基因拷贝进化而来，在真核生物中则有三个重复的基因，每个基因都具有细胞中不可或缺的功能。在酿酒酵母中，yGPN1 和 yGPN2 参与姐妹染色单体的凝聚机制，而 yGPN3 的功能则未知。先前的高通量实验表明，GPN 基因家族成员之间可能存在相互作用。在这项工作中，我们使用 TAP-Tag 方法、酵母双杂交实验、计算机能量计算和位于相互作用界面中心的保守 Glu 残基的定点突变，详细分析了 GPN|GPN 接触。结果表明，该残基对细胞活力至关重要。染色单体凝聚实验表明，yGPN3 与 yGPN1 和 yGPN2 一样，在姐妹染色单体凝聚中也发挥作用。这些结果表明，所有三种 GPN 蛋白在姐妹染色单体凝聚机制中作为 GPN|GPN 复合物发挥作用，类似于 PAB0955 的同源二聚体结构，PAB0955 是 GPN 环 GTPase 的古菌成员。
