Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line.
Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line.
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DOI：
10.1083/jcb.97.6.1762
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发表时间：
1983-12
期刊：
影响因子：
--
通讯作者：
Maxfield FR
中科院分区：
文献类型：
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作者：
Tycko B;Keith CH;Maxfield FR
内吞囊泡的酸化是包括受体再循环、病毒渗透和白喉毒素进入细胞在内的各种过程的必要步骤。然而，在形态学和生物化学上定义的内吞室中，很少有精确的pH测量。在本文中，我们发现HepG2人肝癌细胞的溶酶体内吞囊泡的内部pH值约为5.4。（我们之前报道过小鼠成纤维细胞中类似的囊泡pH值为5.0。）pH值由荧光素标记的asialo-orosomucoid （ASOR）内化后的荧光激发谱获得。为了在高自身荧光背景下进行荧光测量，我们开发了数字图像分析方法来估计单个内吞囊泡或溶酶体内的pH值。胶体金ASOR超微结构定位表明，pH测量是在配体处于缺乏酸性磷酸酶活性的管状泡状结构时进行的。用125I-ASOR进行的生化研究表明，在37℃时，酸化比降解早30分钟以上。在23℃时，配体降解几乎完全停止，但内吞噬囊泡酸化和受体循环仍在继续。这些结果表明，内吞囊泡的酸化，导致配体解离，发生在没有内吞囊泡与溶酶体融合的情况下。甲胺和莫能菌素提高了内吞囊泡的pH值，并导致了不依赖配体的受体丧失。在去除扰动后，对内吞囊泡pH值的影响迅速可逆，但对细胞表面受体的影响在甲胺作用下缓慢可逆，在莫能菌素作用下基本不可逆。这表明莫能菌素可以在一个高度敏感的步骤中阻断受体循环，而不仅仅是内吞囊泡的酸化。结合其他对内吞囊泡pH值的直接和间接估计，这些研究表明，许多细胞类型的内吞囊泡在pH值低于5.5时迅速酸化，pH值足以使受体-配体解离和一些毒素链和被包裹的病毒核衣壳渗透到细胞质中。
Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.