Uchida N, Smilowitz H, Tanzer M L
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1868-72. doi: 10.1073/pnas.76.4.1868.
Procollagen and fibronectin are major products of confluent fibroblasts in culture and both are released from the cells. Procollagen is secreted by known pathways, while the mechanism of fibronectin release is controversial. We find that the secretion of both these proteins can be reduced to 20% by low concentrations (0.1-1 muM) of ionophores that have affinity for monovalent cations. In contrast, little effect upon secretion was found for similar concentrations of an ionophore that binds divalent cations. Electron microscopy showed that the inhibition of secretion is accompanied by accumulation of membranous vacuoles. We believe that the ionophores impede secretion by acting on the secretory structures rather than on the proteins themselves. Biochemical studies supported this interpretation because no changes were detected in hydroxylation or glycosylation of procollagen or glycosylation of fibronectin, nor were significant changes in cellular amino acid incorporation observed. Pulse-chase studies indicated that the rates of secretion were impaired by the ionophore without enhancing intracellular degradation. The decreased secretory rates accounted for the lower levels of procollagen and fibronectin in the culture medium; no evidence for increased catabolism of the secreted proteins was found. Secretion could be readily restored by removing the ionophore from the culture medium. The results indicate that procollagen and fibronectin may be simultaneously secreted, possibly utilizing a common pathway for secretion; the ionophores effectively interfere with cellular secretory pathways without impairing protein synthesis or protein glycosylation or altering protein catabolism.
前胶原和纤连蛋白是培养中汇合的成纤维细胞的主要产物，且二者均从细胞中释放出来。前胶原通过已知途径分泌，而纤连蛋白的释放机制存在争议。我们发现，对单价阳离子具有亲和力的低浓度（0.1 - 1μM）离子载体可将这两种蛋白质的分泌减少至20%。相比之下，类似浓度的结合二价阳离子的离子载体对分泌几乎没有影响。电子显微镜显示，分泌的抑制伴随着膜性空泡的积累。我们认为离子载体通过作用于分泌结构而非蛋白质本身来阻碍分泌。生化研究支持了这一解释，因为在前胶原的羟基化或糖基化或纤连蛋白的糖基化方面未检测到变化，在细胞氨基酸掺入方面也未观察到显著变化。脉冲追踪研究表明，离子载体损害了分泌速率，但未增强细胞内降解。分泌速率降低导致培养基中前胶原和纤连蛋白水平较低；未发现分泌蛋白分解代谢增加的证据。通过从培养基中去除离子载体，分泌可轻易恢复。结果表明，前胶原和纤连蛋白可能同时分泌，可能利用共同的分泌途径；离子载体有效地干扰细胞分泌途径，而不损害蛋白质合成、蛋白质糖基化或改变蛋白质分解代谢。
