Molinete M, Lilla V, Jain R, Joyce P B, Gorr S U, Ravazzola M, Halban P A
Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland.
Diabetologia. 2000 Sep;43(9):1157-64. doi: 10.1007/s001250051507.
AIMS/HYPOTHESIS: Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells.
SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity.
Secreted alkaline phosphatase showed high basal secretion (6.6% total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8% total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion.
CONCLUSION/INTERPRETATION: These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both.
目的/假设：胰岛素原向胰腺β细胞的调节性分泌途径的分选以及胰岛素在该途径的致密核心颗粒中的保留非常高效。为了监测这些事件的特异性，在INS-1细胞中研究了两种已知不携带分选或保留信息的外源性分泌蛋白在调节途径中的分泌情况。
通过转染和重组腺病毒感染分别在INS-1细胞中表达了由EGFP（具有增强荧光的突变绿色荧光蛋白）N端的信号肽和分泌性碱性磷酸酶（SEAP）组成的融合蛋白SEGFP。通过定量蛋白质免疫印迹法监测SEGFP的分泌，通过其活性监测SEAP的分泌。
分泌性碱性磷酸酶显示出较高的基础分泌水平（占总量的6.6%），但促分泌剂对其分泌的刺激作用较小（3.6倍），这与主要通过组成型途径分泌一致。相比之下，SEGFP具有与胰岛素相似的分泌模式，基础分泌水平较低（占总量的0.8%），促分泌剂可使其分泌增加16倍。通过高分辨率电子显微镜免疫细胞化学证实了SEGFP的颗粒定位。脉冲追踪实验表明，SEGFP在合成后至少24小时保留在颗粒中。分泌型SEGFP而非胞质型EGFP形成了二硫键连接的寡聚体。这可能与其调节性分泌有关。
结论/解读：这些数据表明，在INS-1细胞中，SEGFP而非SEAP意外地被当作调节性分泌蛋白处理，并与胰岛素一起储存在颗粒中。这引发了关于INS-1细胞中蛋白质分选到颗粒中的特异性和机制，或其在颗粒中的保留，或两者的问题。
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