Department of Biology, Faculty of Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395, Japan.
Division of Organelle Homeostasis, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Biochim Biophys Acta Mol Cell Res. 2019 Mar;1866(3):441-449. doi: 10.1016/j.bbamcr.2018.10.006. Epub 2018 Oct 5.
Proteins harboring peroxisome-targeting signal type-2 (PTS2) are recognized in the cytosol by mobile PTS2 receptor Pex7p and associate with a longer isoform Pex5pL of the PTS1 receptor. Trimeric PTS2 protein-Pex7p-Pex5pL complexes are translocated to peroxisomes in mammalian cells. However, it remains unclear whether Pex5pL and Pex7p are sufficient cytosolic components in transporting of PTS2 proteins to peroxisomes. Here, we construct a semi-intact cell import system to define the cytosolic components required for the peroxisomal PTS2 protein import and show that the PTS2 pre-import complexes comprising Pex7p, Pex5p, and Hsc70 isolated from the cytosol of pex14 Chinese hamster ovary cell mutant ZP161 is import-competent. PTS2 reporter proteins are transported to peroxisomes by recombinant Pex7p and Pex5pL in semi-intact cells devoid of the cytosol. Furthermore, PTS2 proteins are translocated to peroxisomes in the presence of a non-hydrolyzable ATP analogue, adenylyl imidodiphosphate, and N-ethylmaleimide, suggesting that ATP-dependent chaperones including Hsc70 are dispensable for PTS2 protein import. Taken together, we suggest that Pex7p and Pex5pL are the minimal cytosolic factors in the transport of PTS2 proteins to peroxisomes.
含有过氧化物酶体靶向信号类型 2（PTS2）的蛋白质在细胞质中被可移动的 PTS2 受体 Pex7p 识别，并与 PTS1 受体的较长同工型 Pex5pL 结合。三聚体 PTS2 蛋白-Pex7p-Pex5pL 复合物在哺乳动物细胞中被转运到过氧化物酶体中。然而，Pex5pL 和 Pex7p 是否足以作为细胞质成分将 PTS2 蛋白转运到过氧化物酶体中仍不清楚。在这里，我们构建了一个半完整细胞导入系统，以确定将 PTS2 蛋白导入过氧化物酶体所需的细胞质成分，并表明从 pex14 中国仓鼠卵巢细胞突变体 ZP161 的细胞质中分离出的包含 Pex7p、Pex5p 和 Hsc70 的 PTS2 前导入复合物是具有导入能力的。PTS2 报告蛋白可通过重组 Pex7p 和 Pex5pL 在缺乏细胞质的半完整细胞中被转运到过氧化物酶体中。此外，在非水解型 ATP 类似物腺苷酰亚胺二磷酸和 N-乙基马来酰亚胺的存在下，PTS2 蛋白被转运到过氧化物酶体中，这表明包括 Hsc70 在内的 ATP 依赖性伴侣对于 PTS2 蛋白的导入是可有可无的。总之，我们认为 Pex7p 和 Pex5pL 是 PTS2 蛋白转运到过氧化物酶体的最小细胞质因子。
