State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361102, China.
National Institute of Biological Sciences, Beijing 102206, China.
G3 (Bethesda). 2021 Oct 19;11(11). doi: 10.1093/g3journal/jkab290.
Using genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Single-domain camelid antibodies generated against GFP have been engineered as nanobodies or GFP-binding proteins (GBPs) that can bind GFP as well as some GFP variants with high affinity and selectivity. In this study, we have used GBP-mCherry fusion protein as a tool to perturb the natural functions of a few kinetochore proteins in the fission yeast Schizosaccharomyces pombe. We found that cells simultaneously expressing GBP-mCherry and the GFP-tagged inner kinetochore protein Cnp1 are sensitive to high temperature and microtubule drug thiabendazole (TBZ). In addition, kinetochore-targeted GBP-mCherry by a few major kinetochore proteins with GFP tags causes defects in faithful chromosome segregation. Thus, this setting compromises the functions of kinetochores and renders cells to behave like conditional mutants. Our study highlights the potential of using GBP as a general tool to perturb the function of some GFP-tagged proteins in vivo with the objective of understanding their functional relevance to certain physiological processes, not only in yeasts, but also potentially in other model systems.
利用基因突变在体内研究蛋白质功能是现代生物学的一个核心范例。针对 GFP 产生的单域骆驼科抗体已被设计成纳米体或 GFP 结合蛋白（GBP），它们可以高亲和力和选择性地结合 GFP 以及一些 GFP 变体。在这项研究中，我们使用 GBP-mCherry 融合蛋白作为工具，干扰裂殖酵母 Schizosaccharomyces pombe 中几个着丝粒蛋白的自然功能。我们发现，同时表达 GBP-mCherry 和 GFP 标记的内着丝粒蛋白 Cnp1 的细胞对高温和微管药物噻苯达唑（TBZ）敏感。此外，由几个带有 GFP 标签的主要着丝粒蛋白靶向的着丝粒靶向 GBP-mCherry 导致忠实染色体分离的缺陷。因此，这种设置会影响着丝粒的功能，使细胞表现得像条件突变体。我们的研究强调了使用 GBP 作为一种通用工具来体内干扰某些 GFP 标记蛋白功能的潜力，目的是了解它们对某些生理过程的功能相关性，不仅在酵母中，而且在其他模型系统中也有可能。
