Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide.
Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide.
复制标题
DOI：
10.1083/jcb.105.6.2631
复制
发表时间：
1987-12
期刊：
影响因子：
--
通讯作者：
Rosenberg LE
中科院分区：
文献类型：
--
作者：
Sztul ES;Hendrick JP;Kraus JP;Wall D;Kalousek F;Rosenberg LE
线粒体基质酶鸟氨酸转氨甲基酶(OTC)是以胞质多聚核糖体为前体(POTC)合成的，NH2末端延伸32个氨基酸。我们在这里报道了在体外合成的大鼠POTC被分离的大鼠肝脏线粒体分两个临时分离的步骤内化和切割。第一步，依赖于完整的线粒体膜电位，POTC被转移到线粒体中，并被基质酶切割成一种称为IOTC的产物，其大小介于POTC和成熟OTC之间。该产品位于胰酶保护的线粒体位置。在体内注射体外合成的POTC的青蛙卵母细胞中也产生了相同的中等大小的OTC。POTC的蛋白质降解过程涉及从前体的NH2末端去除24个氨基酸，并利用与第23位的关键精氨酸残基相距两个残基的裂解位点。在第二步中，也是在基质酶的催化下，IOTC通过去除前导序列的其余8个残基转化为成熟的OTC。为了确定这些事件所需的OTC先导肽中的关键区域，我们合成了具有先导改变的OTC前体。用酸性氨基酸残基(天冬氨酸)或“螺旋断裂”氨基酸残基(甘氨酸)替代前导序列的精氨酸23可以抑制IOTC和OTC的形成，但不影响转位。这些突变的前体在先导序列的16和17残基之间的另一个神秘的切割位点上被切割。有趣的是，这种切割发生在距离第15位精氨酸两个残基的位置。数据表明，POTC向成熟OTC的转化是通过形成第三个离散物种进行的：中等大小的OTC。数据进一步表明，在大鼠POTC领导人中，易位所需的基本要素不同于正确切割到IOTC或成熟OTC所需的要素。
The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.