Yon Wesley J, Ha Taekjip, Zheng Yixian, Pedersen Ross T A
Department of Embryology, Carnegie Institution for Science, Baltimore, MD, USA.
Cell, Molecular, Developmental Biology, and Biophysics Program, Johns Hopkins University, Baltimore, MD, USA.
bioRxiv. 2024 Jul 15:2023.05.09.539990. doi: 10.1101/2023.05.09.539990.
α- and β-tubulin form heterodimers, with GTPase activity, that assemble into microtubules. Like other GTPases, the nucleotide-bound state of tubulin heterodimers controls whether the molecules are in a biologically active or inactive state. While α-tubulin in the heterodimer is constitutively bound to GTP, β-tubulin can be bound to either GDP (GDP-tubulin) or GTP (GTP-tubulin). GTP-tubulin hydrolyzes its GTP to GDP following assembly into a microtubule and, upon disassembly, must exchange its bound GDP for GTP to participate in subsequent microtubule polymerization. Tubulin dimers have been shown to exhibit rapid intrinsic nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.
α-微管蛋白和β-微管蛋白形成具有GTP酶活性的异二聚体，这些异二聚体组装成微管。与其他GTP酶一样，微管蛋白异二聚体的核苷酸结合状态控制着分子是处于生物活性状态还是无活性状态。虽然异二聚体中的α-微管蛋白始终与GTP结合，但β-微管蛋白可以与GDP（GDP-微管蛋白）或GTP（GTP-微管蛋白）结合。GTP-微管蛋白在组装成微管后将其GTP水解为GDP，并且在解聚时，必须将其结合的GDP交换为GTP以参与随后的微管聚合。已证明微管蛋白二聚体在体外表现出快速的内在核苷酸交换，导致人们普遍认为细胞中可能不需要微管蛋白鸟嘌呤核苷酸交换因子（GEF）。在这里，我们使用定量结合测定来表明纺锤体组装因子BuGZ与GDP-微管蛋白紧密结合，与GTP-微管蛋白结合较松，与微管结合较弱。我们进一步使用核苷酸交换测定表明BuGZ促进GTP掺入微管蛋白。微管蛋白GEF的发现提示了一种可能有助于细胞中微管快速组装动态的机制。
