Chen Chi-Chih, Huang Yu-Ru, Chan Yuen Ting, Lin Hung-Yun, Lin Han-Jia, Hsiao Chwan-Deng, Ko Tzu-Ping, Lin Tai-Wen, Lan Ya-Hsuan, Lin Hsuan-Ya, Chang Hsin-Yang
Department of Life Sciences and Institute of Genome Sciences, National Yang Ming Chiao Tung University, Beitou Dist, No. 155, Sec. 2, Linong St, Taipei City, 112304, Taiwan.
Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung, Taiwan.
BMC Biol. 2024 Jun 13;22(1):136. doi: 10.1186/s12915-024-01933-x.
Most tail-anchored (TA) membrane proteins are delivered to the endoplasmic reticulum through a conserved posttranslational pathway. Although core mechanisms underlying the targeting and insertion of TA proteins are well established in eukaryotes, their role in mediating TA protein biogenesis in plants remains unclear. We reported the crystal structures of algal arsenite transporter 1 (ArsA1), which possesses an approximately 80-kDa monomeric architecture and carries chloroplast-localized TA proteins. However, the mechanistic basis of ArsA2, a Get3 (guided entry of TA proteins 3) homolog in plants, for TA recognition remains unknown.
Here, for the first time, we present the crystal structures of the diatom Pt-Get3a that forms a distinct ellipsoid-shaped tetramer in the open (nucleotide-bound) state through crystal packing. Pulldown assay results revealed that only tetrameric Pt-Get3a can bind to TA proteins. The lack of the conserved zinc-coordination CXXC motif in Pt-Get3a potentially leads to the spontaneous formation of a distinct parallelogram-shaped dimeric conformation in solution, suggesting a new dimer state for subsequent tetramerization upon TA targeting. Pt-Get3a nonspecifically binds to different subsets of TA substrates due to the lower hydrophobicity of its α-helical subdomain, which is implicated in TA recognition.
Our study provides new insights into the mechanisms underlying TA protein shielding by tetrameric Get3 during targeting to the diatom's cell membrane.
大多数尾锚定（TA）膜蛋白通过保守的翻译后途径被转运到内质网。尽管真核生物中TA蛋白靶向和插入的核心机制已得到充分确立，但其在介导植物中TA蛋白生物合成中的作用仍不清楚。我们报道了藻类亚砷酸盐转运蛋白1（ArsA1）的晶体结构，它具有约80 kDa的单体结构，并携带叶绿体定位的TA蛋白。然而，植物中Get3（TA蛋白引导进入因子3）同源物ArsA2识别TA的机制基础仍不清楚。
在此，我们首次展示了硅藻Pt-Get3a的晶体结构，它在开放（核苷酸结合）状态下通过晶体堆积形成独特的椭圆形四聚体。下拉实验结果表明，只有四聚体Pt-Get3a能与TA蛋白结合。Pt-Get3a中缺乏保守的锌配位CXXC基序，可能导致其在溶液中自发形成独特的平行四边形二聚体构象，这表明存在一种新的二聚体状态，在TA靶向时随后会形成四聚体。由于其α-螺旋亚结构域的疏水性较低，Pt-Get3a能非特异性地结合不同子集的TA底物，该亚结构域与TA识别有关。
我们的研究为四聚体Get3在靶向硅藻细胞膜过程中保护TA蛋白的机制提供了新的见解。
