Powell S K, Orci L, Craik C S, Moore H P
Department of Physiology-Anatomy, University of California, Berkeley 94720.
J Cell Biol. 1988 Jun;106(6):1843-51. doi: 10.1083/jcb.106.6.1843.
In neuronal and endocrine cells, peptide hormones are selectively segregated into storage granules, while other proteins are exported continuously without storage. Sorting of hormones by cellular machinery involves the recognition of specific structural domains on prohormone molecules. Since the propeptide of insulin is known to play an important role in its three-dimensional structure, it is reasonable to speculate that targeting of proinsulin to storage granules would require a functional connecting peptide. To test this hypothesis, we constructed two mutations in human proinsulin with different predicted structures. In one mutation, Ins delta C, the entire C peptide was deleted, resulting in an altered insulin in which the B and the A chains are joined contiguously. In the other mutation, Ins/IGF, the C peptide of proinsulin was replaced with the unrelated 12-amino acid connecting peptide of human insulin-like growth factor-I; this substitution should permit correct folding of the B and A chains to form a tertiary structure similar to that of proinsulin. By several biochemical and morphological criteria, we found that Ins/IGF is efficiently targeted to storage granules, suggesting that the C peptide of proinsulin does not contain necessary sorting information. Unexpectedly, Ins delta C, which presumably cannot fold properly, is also targeted to granules at a high efficiency. These results imply that either the targeting machinery can tolerate changes in the tertiary structure of transported proteins, or that the B and A chains of insulin can form a relatively intact three-dimensional structure even in the absence of C peptide.
在神经元和内分泌细胞中，肽类激素被选择性地分隔到储存颗粒中，而其他蛋白质则持续输出而不储存。细胞机制对激素的分选涉及对激素原分子上特定结构域的识别。由于已知胰岛素原肽在其三维结构中起重要作用，因此推测将胰岛素原靶向储存颗粒需要一个功能性连接肽是合理的。为了验证这一假设，我们构建了两种具有不同预测结构的人胰岛素原突变体。在一种突变体Ins delta C中，整个C肽被删除，导致胰岛素发生改变，其中B链和A链连续连接。在另一种突变体Ins/IGF中，胰岛素原的C肽被人胰岛素样生长因子-I的不相关的12个氨基酸的连接肽取代；这种取代应允许B链和A链正确折叠以形成类似于胰岛素原的三级结构。通过几种生化和形态学标准，我们发现Ins/IGF能有效地靶向储存颗粒，这表明胰岛素原的C肽不包含必要的分选信息。出乎意料的是，可能无法正确折叠的Ins delta C也能高效地靶向颗粒。这些结果表明，要么分选机制能够容忍转运蛋白三级结构的变化，要么胰岛素的B链和A链即使在没有C肽的情况下也能形成相对完整的三维结构。
