Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan.
Radioisotope Research Center, Yokohama City University School of Medicine, Japan.
FEBS Lett. 2020 Jan;594(2):383-392. doi: 10.1002/1873-3468.13592. Epub 2019 Sep 17.
Parathyroid hormone-related protein (PTHrP) is transported to both the secretory pathway and the nucleus/nucleolus by its dual targeting signals, that is, an N-terminal signal peptide and nuclear targeting signal. Curiously, reporter proteins such as enhanced green fluorescent protein strongly affect the localization of the fusion protein. Here, we report a novel methionine tag for S-labelling added to the C-terminus of its prepro-form, which has no methionine and cysteine residue other than the initiation methionine that enables analyses of the molecular mechanism of its dual localization without the effects of the reporter proteins. Mutational analyses including insertion of a glycosylation site for the tagged PTHrP revealed that the evolutionarily conserved regions in the signal peptide and the pro-region facilitate the redirection of ppPTHrP from the secretory pathway to the nuclear targeting pathway.
甲状旁腺激素相关蛋白（PTHrP）通过其双重靶向信号，即 N 端信号肽和核靶向信号，被运输到分泌途径和核/核仁。奇怪的是，报告蛋白如增强型绿色荧光蛋白强烈影响融合蛋白的定位。在这里，我们报告了一种新的甲硫氨酸标签，用于在其前体形式的 C 端添加 S-标记，该前体形式除起始甲硫氨酸外不含其他甲硫氨酸和半胱氨酸残基，这使得可以在不受报告蛋白影响的情况下分析其双重定位的分子机制。包括插入标记 PTHrP 的糖基化位点的突变分析表明，信号肽和前导区中的进化保守区域有助于将 ppPTHrP 从分泌途径重新导向核靶向途径。
