DataSheet1_Molecular insights into the axon guidance molecules Sidestep and Beaten path.pdf

The transmembrane protein Sidestep (Side) functions as a substrate-bound attractant for motor axons in Drosophila. Outgrowing motor axons recognize Side via Beaten path Ia (Beat) and migrate along Side-expressing tissues. Here, we report a structure-function analysis of these guidance molecules using a variety of mutant lines and transgenic constructs. Investigation of Side mutants shows that the exchange of a single amino acid (L241H) in the second immunoglobulin domain disturbs Side function and subcellular localization. Overexpression of Side and Beat deletion constructs in S2 cells and muscles demonstrate that the first Ig domains of both proteins are necessary for their interaction. Furthermore, subcellular distributions of several Beat constructs identify functional domains and suggest a potential posttranslational processing step in ER compartments. In fact, fusing full-length Beat at both the N- and C-terminus with GFP and mCherry, respectively, shows that the N-terminal domain is transported to the plasma membrane and exposed on the cell surface, while the C-terminal domain accumulated in the nucleus. Taken together, these results give insights into the interaction of Side and Beat and imply that Beat might be subject to proteolytic cleavage during maturation.

跨膜蛋白Sidestep（简称Side）在果蝇中作为底物结合型运动轴突吸引性导向因子发挥功能。向外延伸的运动轴突通过Beaten path Ia（简称Beat）识别Side，并沿着表达Side的组织迁移。本研究通过多种突变体品系与转基因构建体，对这类导向分子开展结构-功能分析。对Side突变体的研究显示，其第二个免疫球蛋白（immunoglobulin, Ig）结构域内单个氨基酸（L241H）的替换会破坏Side的功能与亚细胞定位。在S2细胞与肌肉组织中过表达Side与Beat的缺失构建体，结果表明两种蛋白的首个Ig结构域是二者相互作用所必需的结构。此外，对多款Beat构建体的亚细胞分布分析明确了其功能结构域，并提示在内质网（endoplasmic reticulum, ER）区室中存在潜在的翻译后加工步骤。实际上，将全长Beat分别在N端与C端融合绿色荧光蛋白（green fluorescent protein, GFP）与mCherry后，可观测到N端结构域被转运至质膜并暴露于细胞表面，而C端结构域则在细胞核内积累。综上，本研究结果阐明了Side与Beat的相互作用机制，并提示Beat在成熟过程中可能经历蛋白水解切割。