Molecular mechanism of protein import into mitochondria

鸟氨酸转氨甲酰酶（OTC）是一种由32个氨基酸组成的NH_2-端前序合成的前体酶，在线粒体内转运。我们在大肠杆菌中表达了大鼠土霉素的前体和成熟形式，并以变性形式纯化了它，结果表明纯化的土霉素具有良好的生物学活性。在兔网织红细胞裂解液存在下，OTC前体可被转运到分离的线粒体中。当盐酸胍变性的成熟土霉素被稀释并在0 ℃孵育时，它被重新激活至比活性为纯化的成熟酶的18%。出乎意料的是，胍变性的OTC前体在类似条件下被活化至类似值。天然的和再活化的OTC作为修剪剂在蔗糖梯度上沉淀。这些观察结果表明，预序列并不能阻止OTC前体折叠成具有酶活性的三聚体形式。蔗糖梯度离心分析表明，OTC前体，但不是成熟的OTC，与网织红细胞裂解液中存在的蛋白因子形成复合物。该因子被命名为PBF（前序列结合因子）。脲变性土霉素前体在无PBF存在下形成不溶性聚集体，但在有PBF存在下形成可溶性复合物，土霉素前体合成前体的微摩尔浓度抑制土霉素前体-PBF复合物的形成。纯化的PBF显著刺激纯化的OTC前体进入分离的线粒体。因此，PBF与前体蛋白的结合很可能是线粒体蛋白输入的第一步。PBF与前体的前序列部分结合，并可能以运输能力的形式保持它们。

Ornithine, transcarbamylase (OTC) is initially synthesized as a precursor with a transient NH_2-terminal presequence of 32 amino acid residues, then is imported posttranslationally into the mitochondrial matrix. We expressed precursor and mature form of rat OTC in Escherichia coli, purified it in a denatured form, and showed that the purified. OTC precursor could be transported into isolated mitochondria in the presence of rabbit reticulocyte lysate. When guanidine-HCl-denatured mature OTC was diluted and incubated at OﾟC, it was reactivated to a specific activity of 18% of that of the purified mature enzyme. Unexpectedly, guanidine-denatured OTC precursor was activated to a similar value under similar conditions. The native and reactivated OTC sedimented on sucrose gradients as trimmers. These observations indicate that the presequence does not prevent the OTC precursor from folding into an enzymatically active trimetic form. Sucrose gradient centrifugation analysis showed that the OTC precursor, but not mature OTC, formed a complex with a protein factor(s) present in reticulocyte lysate.The cytosolic protein factor was highly purified from reticulocyte lysate by affinity chromatography using OTC precursor-bound Sepharose. This factor was named PBF (presequence binding factor). Urea-denatured OTC precursor formed an insoluble aggregate in the absence of PBF, but formed a soluble complex in its presence.Formation of the OTC precursor-PBF complex was inhibited by micromolar concentrations of the synthetic presequence of the OTC precursor. The purified PBF markedly stimulated the import of purified OTC precursor into the isolated mitochondria. Thus, binding of PBF to precursor proteins may well be the first step of mitochondrial protein import. PBF binds to the presequence portion of the precursors and may hold them in a transport-competent form.