Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, Barcelona, Spain.
Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028, Barcelona, Spain.
BMC Plant Biol. 2021 Mar 17;21(1):141. doi: 10.1186/s12870-021-02898-7.
Sterols are structural and functional components of eukaryotic cell membranes. Plants produce a complex mixture of sterols, among which β-sitosterol, stigmasterol, campesterol, and cholesterol in some Solanaceae, are the most abundant species. Many reports have shown that the stigmasterol to β-sitosterol ratio changes during plant development and in response to stresses, suggesting that it may play a role in the regulation of these processes. In tomato (Solanum lycopersicum), changes in the stigmasterol to β-sitosterol ratio correlate with the induction of the only gene encoding sterol C22-desaturase (C22DES), the enzyme specifically involved in the conversion of β-sitosterol to stigmasterol. However, despite the biological interest of this enzyme, there is still a lack of knowledge about several relevant aspects related to its structure and function.
In this study we report the subcellular localization of tomato C22DES in the endoplasmic reticulum (ER) based on confocal fluorescence microscopy and cell fractionation analyses. Modeling studies have also revealed that C22DES consists of two well-differentiated domains: a single N-terminal transmembrane-helix domain (TMH) anchored in the ER-membrane and a globular (or catalytic) domain that is oriented towards the cytosol. Although TMH is sufficient for the targeting and retention of the enzyme in the ER, the globular domain may also interact and be retained in the ER in the absence of the N-terminal transmembrane domain. The observation that a truncated version of C22DES lacking the TMH is enzymatically inactive revealed that the N-terminal membrane domain is essential for enzyme activity. The in silico analysis of the TMH region of plant C22DES revealed several structural features that could be involved in substrate recognition and binding.
Overall, this study contributes to expand the current knowledge on the structure and function of plant C22DES and to unveil novel aspects related to plant sterol metabolism.
甾醇是真核细胞膜的结构和功能组成部分。植物产生复杂的甾醇混合物，其中β-谷甾醇、豆甾醇、菜油甾醇和胆固醇是一些茄科植物中最丰富的物种。许多报道表明，甾醇中豆甾醇与β-谷甾醇的比例在植物发育过程中以及对胁迫的反应中发生变化，这表明它可能在这些过程的调节中发挥作用。在番茄（Solanum lycopersicum）中，豆甾醇与β-谷甾醇的比例变化与唯一编码甾醇 C22-脱饱和酶（C22DES）的基因的诱导相关，该酶专门参与β-谷甾醇向豆甾醇的转化。然而，尽管该酶具有生物学意义，但仍缺乏有关其结构和功能的几个相关方面的知识。
本研究基于共聚焦荧光显微镜和细胞分级分析，报道了番茄 C22DES 在内质网（ER）中的亚细胞定位。建模研究还表明，C22DES 由两个截然不同的结构域组成：一个单一的 N 端跨膜螺旋结构域（TMH）锚定在 ER 膜上，一个朝向细胞质的球状（或催化）结构域。尽管 TMH 足以将酶靶向并保留在 ER 中，但在没有 N 端跨膜结构域的情况下，球状结构域也可能相互作用并保留在 ER 中。观察到缺乏 TMH 的 C22DES 的截断版本无酶活性，这表明 N 端膜结构域对于酶活性至关重要。对植物 C22DES 的 TMH 区域的计算机分析揭示了几个可能参与底物识别和结合的结构特征。
总的来说，这项研究有助于扩展对植物 C22DES 的结构和功能的现有认识，并揭示与植物甾醇代谢相关的新方面。
