Mitochondria are membrane-bound organelles of endosymbiotic origin with limited protein-coding capacity. The import of nuclear-encoded proteins and nucleic acids is required and essential for maintaining organelle mass, number, and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNA conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with six monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of the Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure, a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.

中文翻译：

---

拟南芥线粒体 tRNA 输入受体 SAM 结构域的结构分析

线粒体是内共生起源的膜结合细胞器，具有有限的蛋白质编码能力。核编码蛋白质和核酸的输入对于维持细胞器的质量、数量和活性是必需的和必要的。由于植物线粒体不能编码所需的所有必需 tRNA 类型，因此胞质 tRNA 的导入对于细胞器的维持至关重要。最近，两种线粒体外膜蛋白，称为Tric1和Tric2，作为tRNA输入成分，被证明参与胞质tRNA的输入。 Tric1/2 结合 tRNA C 端无菌 Alpha 基序 (SAM) 结构域中的保守残基。在这里，我们报告了 Tric1 SAM 结构域的 X 射线晶体结构。我们确定了 SAM 结构域形成螺旋超结构的能力，每个螺旋圈有 6 个单体以及负责其形成的关键氨基酸残基。我们确定 Tric1 SAM 结构域的寡聚化可能在蛋白质功能中发挥作用，在此位置引入更大侧链的 Gly241 突变会破坏寡聚体并导致 RNA 结合能力丧失。此外，Tric1/2 敲除品系与突变 Tric1 的互补未能恢复有缺陷的植物表型。 SAM 结构域和 Tric1 的 AlphaFold2 结构预测支持环状五聚体或六聚体结构。在六聚体结构的情况下，观察到尺寸足以将 tRNA 转移穿过线粒体膜的孔。我们的结果强调了 Tric1 寡聚化对于蛋白质功能的重要性。