Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Coyoacán, Cd.Mx., Mexico.
Department of Molecular, Cellular, and Developmental Biology, University of California at Santa Barbara, Santa Barbara, California, USA.
J Biol Chem. 2022 Jun;298(6):101984. doi: 10.1016/j.jbc.2022.101984. Epub 2022 Apr 26.
A major challenge in eukaryotic cells is the proper distribution of nuclear-encoded proteins to the correct organelles. For a subset of mitochondrial proteins, a signal sequence at the N terminus (matrix-targeting sequence [MTS]) is recognized by protein complexes to ensure their proper translocation into the organelle. However, the early steps of mitochondrial protein targeting remain undeciphered. The cytosolic chaperone nascent polypeptide-associated complex (NAC), which in yeast is represented as the two different heterodimers αβ-NAC and αβ'-NAC, has been proposed to be involved during the early steps of mitochondrial protein targeting. We have previously described that the mitochondrial outer membrane protein Sam37 interacts with αβ'-NAC and together promote the import of specific mitochondrial precursor proteins. In this work, we aimed to detect the region in the MTS of mitochondrial precursors relevant for their recognition by αβ'-NAC during their sorting to the mitochondria. We used targeting signals of different mitochondrial proteins (αβ'-NAC-dependent Oxa1 and αβ'-NAC-independent Mdm38) and fused them to GFP to study their intracellular localization by biochemical and microscopy methods, and in addition followed their import kinetics in vivo. Our results reveal the presence of a positively charged amino acid cluster in the MTS of select mitochondrial precursors, such as Oxa1 and Fum1, which are crucial for their recognition by αβ'-NAC. Furthermore, we explored the presence of this cluster at the N terminus of the mitochondrial proteome and propose a set of precursors whose proper localization depends on both αβ'-NAC and Sam37.
真核细胞面临的一个主要挑战是将核编码蛋白正确分配到细胞器中。对于一部分线粒体蛋白，其 N 端的信号序列（基质靶向序列 [MTS]）被蛋白复合物识别，以确保它们正确转运到细胞器中。然而，线粒体蛋白靶向的早期步骤仍未被破解。细胞质伴侣新生多肽相关复合物（NAC），在酵母中代表两种不同的异二聚体αβ-NAC 和 αβ'-NAC，被提出参与线粒体蛋白靶向的早期步骤。我们之前曾描述过线粒体外膜蛋白 Sam37 与 αβ'-NAC 相互作用，并共同促进特定线粒体前体蛋白的输入。在这项工作中，我们旨在检测线粒体前体蛋白 MTS 中的区域，这些区域对于它们在被分拣到线粒体时被 αβ'-NAC 识别是相关的。我们使用不同线粒体蛋白的靶向信号（αβ'-NAC 依赖性的 Oxa1 和 αβ'-NAC 非依赖性的 Mdm38）并将其与 GFP 融合，通过生化和显微镜方法研究它们的细胞内定位，并进一步在体内跟踪它们的导入动力学。我们的结果揭示了一些选择的线粒体前体蛋白（如 Oxa1 和 Fum1）的 MTS 中存在带正电荷的氨基酸簇，这对于它们被 αβ'-NAC 识别是至关重要的。此外，我们还探讨了这个簇在整个线粒体蛋白质组的 N 端的存在情况，并提出了一组前体蛋白，它们的正确定位既依赖于 αβ'-NAC 也依赖于 Sam37。
