Proinsulin Intermolecular Interactions during Secretory Trafficking in Pancreatic β Cells
Proinsulin Intermolecular Interactions during Secretory Trafficking in Pancreatic β Cells
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DOI：
10.1074/jbc.m112.420018
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发表时间：
2013-01-18
影响因子：
4.8
通讯作者：
Arvan, Peter
中科院分区：
文献类型：
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作者：
Haataja, Leena;Snapp, Erik;Arvan, Peter
传统上，从内质网（ER）退出是限制分泌蛋白运输的速率，因为蛋白质折叠/组装发生在那里。在这项研究中，我们利用了“hPro-CpepSfGFP”，一种在胰腺β细胞中表达的携带“超级折叠”绿色荧光C肽的人胰岛素原，在胰腺β细胞中将其加工成人胰岛素和CpepSfGFP。值得注意的是，hPro-CpepSfGFP和内源性胰岛素原的稳态积累是在高尔基体区域，就好像蛋白质折叠/组装的最后阶段发生在那里。胰岛素原的高尔基区域分布是动态的，受禁食/再喂养的影响，并随着β细胞锌缺乏而增加。然而，共表达的ER-截留的突变体胰岛素原-C（A7）Y转移的野生型胰岛素原的稳态分布的ER。内源性胰岛素原与hPro-CpepSfGFP共沉淀，甚至与hProC（A7）Y-CpepSfGFP共沉淀。使用Cerulean和Venus标记的胰岛素原，我们发现WT-WT和WT-突变胰岛素原对都表现出FRET。这些数据表明，野生型胰岛素原二聚体内的ER，但积累在一个认识不足的缓慢步骤内的高尔基体区域，反映了缓慢的动力学胰岛素原六聚化，步骤中形成的新生分泌颗粒，或其他未知的分子事件。然而，在β细胞中胰岛素原亚群持续错误折叠的情况下，剩余胰岛素原转运的限速步骤转移到ER。
Classically, exit from the endoplasmic reticulum (ER) is rate-limiting for secretory protein trafficking because protein folding/assembly occurs there. In this study, we have exploited "hPro-CpepSfGFP," a human proinsulin bearing "superfolder" green fluorescent C-peptide expressed in pancreatic beta cells where it is processed to human insulin and CpepSfGFP. Remarkably, steady-state accumulation of hPro-CpepSfGFP and endogenous proinsulin is in the Golgi region, as if final stages of protein folding/assembly were occurring there. The Golgi regional distribution of proinsulin is dynamic, influenced by fasting/refeeding, and increased with beta cell zinc deficiency. However, coexpression of ER-entrapped mutant proinsulin-C(A7) Y shifts the steady-state distribution of wild-type proinsulin to the ER. Endogenous proinsulin coprecipitates with hPro-CpepSfGFP and even more so with hProC(A7) Y-CpepSfGFP. Using Cerulean and Venus-tagged proinsulins, we find that both WT-WT and WT-mutant proinsulin pairs exhibit FRET. The data demonstrate that wild-type proinsulin dimerizes within the ER but accumulates at a poorly recognized slow step within the Golgi region, reflecting either slow kinetics of proinsulin hexamerization, steps in formation of nascent secretory granules, or other unknown molecular events. However, in the presence of ongoing misfolding of a subpopulation of proinsulin in beta cells, the rate-limiting step in transport of the remaining proinsulin shifts to the ER.