Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Arch Insect Biochem Physiol. 2021 Mar;106(3):e21768. doi: 10.1002/arch.21768. Epub 2021 Feb 28.
Bombyx mori Masculinizer protein (BmMasc) is essential for both masculinization and dosage compensation in B. mori. We previously identified a bipartite nuclear localization signal (NLS) of BmMasc and two essential residues (lysine at 274 [K274] and arginine at 275 [R275]) implicated in its function. Sequence comparison showed the presence of putative NLSs in lepidopteran Masc proteins, but their functional properties and critical residues are unknown. Here we characterized a putative NLS of Ostrinia furnacalis Masc (OfMasc) using B. mori ovary-derived BmN-4 cell line. Deletion and alanine scanning mutagenesis revealed that a putative NLS is required for nuclear localization of OfMasc. However, mutations at both K227 and R228, which correspond to K274 and R275 of BmMasc, respectively, do not greatly abolish the NLS activity. Additional mutagenesis analysis revealed that triple mutations at K227, R228, and K240 almost completely inhibited OfMasc nuclear localization. These results suggest that lepidopteran Masc proteins possess a common functional NLS, but the critical residues for its activity are different. Moreover, we examined the masculinizing activity of OfMasc derivatives and found that nuclear localization is not required for the masculinizing activity of OfMasc. The results from our studies indicate that lepidopteran Masc proteins function in the cytoplasm to drive masculinizing cascade.
家蚕雄性化蛋白（BmMasc）对于家蚕的雄性化和剂量补偿都是必不可少的。我们之前鉴定了 BmMasc 的二分核定位信号（NLS）和两个与功能相关的必需残基（274 位的赖氨酸[K274]和 275 位的精氨酸[R275]）。序列比较表明鳞翅目 Masc 蛋白存在潜在的 NLS，但它们的功能特性和关键残基尚不清楚。在这里，我们使用家蚕卵巢衍生的 BmN-4 细胞系对野桑蚕 Masc（OfMasc）的一个潜在 NLS 进行了特征描述。缺失和丙氨酸扫描诱变表明，一个潜在的 NLS 对于 OfMasc 的核定位是必需的。然而，分别对应于 BmMasc 的 K274 和 R275 的 K227 和 R228 突变并没有极大地消除 NLS 活性。进一步的突变分析表明，K227、R228 和 K240 的三重突变几乎完全抑制了 OfMasc 的核定位。这些结果表明，鳞翅目 Masc 蛋白具有共同的功能 NLS，但对于其活性的关键残基不同。此外，我们检查了 OfMasc 衍生物的雄性化活性，发现核定位对于 OfMasc 的雄性化活性不是必需的。我们的研究结果表明，鳞翅目 Masc 蛋白在细胞质中发挥作用，驱动雄性化级联反应。
