Role of amphipathic transmembrane domains of single-spanning membrane proteins as determinants of their oligomeric state and subcellular localization.

分子动力学模拟暗示了在介导p24蛋白的TMD-TMD相互作用中的不寻常的极性界面（Contreras-Ernst等人，2012年）。其他报道暗示了TMD的类似极性面参与介导定位到高尔基体和蛋白质-蛋白质相互作用（Machamer等人，1993; Dawson等人，2002年，2003年）。生物信息学分析的初步数据表明，两亲TMD设计是常见的单跨膜蛋白和丰富的早期分泌途径。相对于不同的模型，试图解释特定的本地化酶的高尔基体，我计划调查是否是一个至关重要的决定因素，在实现这一功能的两亲性的TMD。该假设可能会联合收割机所有现有的模型，因为ER定位将通过ER驻留膜蛋白的细胞质修复信号实现，而同样具有两亲性TMD的质膜驻留者将由于其疏水长度增加而被排除在Golgienzyme寡聚体之外（TMD分配模型）。在高尔基体内，高尔基体酶的亲缘识别将通过寡聚体之间基于TMD的相互作用来实现。为了研究两亲性TMD在同源和异源寡聚化中的贡献，将采用基于FRET的蛋白脂质体系统和基于BIrA的体内生物素化测定。两亲性TMD的各自的候选人在其亚细胞定位的作用将通过双色，高分辨率STED纳米显微镜解决。

Molecular dynamics simulations hinted at an unusual polar interface in mediating TMD-TMD interactions for p24 proteins (Contreras-Ernst et al., 2012). Other reports hint at the involvement of similar polar faces of TMDs in mediating localization to the Golgi and protein-protein interactions (Machamer et al., 1993; Dawson et al., 2002, 2003). Preliminary data that arose from bioinformatical analysis suggests that amphipathic TMD design is common for single-spanning membrane proteins and enriched in the early secretory pathway. With respect to the different models that attempt to explain specific localization of enzymes to the Golgi, I am planning to investigate whether the amphipathicity of TMDs is a crucial determinant in achieving this function. This hypothesis might combine all existing models, as ER-localization would be achieved via cytoplasmic retrieval signals of ER resident membrane proteins, while plasma membrane residents that also possess amphipathic TMDs would, due to their increased hydrophobic length, be excluded from Golgienzyme oligomers (TMD-partitioning model). Within the Golgi, kin recognition of Golgi enzymes would then be achieved by TMD-based interactions among oligomers. To study the contribution of amphipathic TMDs in homo- and heterooligomerization, a FRET-based proteoliposomal system and an in vivo BIrA-based biotinylation assay will be employed. The role of the amphipathic TMD of the respective candidate in its subcellular localization will be addressed by two-color, high-resolution STED nanoscopy.