Presley J F, Mayor S, McGraw T E, Dunn K W, Maxfield F R
Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
J Biol Chem. 1997 May 23;272(21):13929-36. doi: 10.1074/jbc.272.21.13929.
Treatment of Chinese hamster ovary cells with the vacuolar proton pump inhibitor bafilomycin A1 causes a 2-fold retardation in the rate of recycling of transfected human transferrin receptors back to the cell surface as measured using biochemical assays (Johnson, L. S. , Dunn, K. W., Pytowski, B., and McGraw, T. E. (1993) Mol. Biol. Cell 4, 1251-1266). We have used quantitative fluorescence microscopy to determine which step(s) in the endocytic recycling pathway are affected. We show that removal of transferrin from sorting endosomes and accumulation in the peri-centriolar endocytic recycling compartment takes place normally in bafilomycin A1-treated cells. However, the rate constant for exit of transferrin receptors from recycling endosomes (ke) is reduced from 0.063 min-1 in untreated cells to 0.034 min-1 in the presence of bafilomycin A1. This retardation appears to be dependent on the presence of internalization motifs in the cytoplasmic domain since modified receptors lacking these oligopeptide motifs do not show as large a decrease in recycling rate in the presence of bafilomycin A1. Bulk membrane recycling (measured by efflux of an internalized fluorescent lipid analog, 6-[N-[7-nitrobenzo-2-oxa-1, 3-diazol-4-yl--amino-hexoyl- sphingosylphosphorylcholine) is slowed from an exit rate constant of 0.060 min-1 without drug to 0.046 min-1 in the presence of bafilomycin A1. We conclude that bafilomycin A1 slows bulk membrane flow, but it causes additional inhibition of receptor recycling in a manner that is dependent on a peptide motif on the cytoplasmic domain.
用液泡质子泵抑制剂巴弗洛霉素A1处理中国仓鼠卵巢细胞，会使转染的人转铁蛋白受体回收到细胞表面的速率减慢两倍，这是通过生化分析测定的（约翰逊，L.S.，邓恩，K.W.，皮托夫斯基，B.，和麦格劳，T.E.（1993年）《分子生物学细胞》4，1251 - 1266）。我们使用定量荧光显微镜来确定内吞循环途径中的哪些步骤受到影响。我们发现，在巴弗洛霉素A1处理的细胞中，转铁蛋白从分拣内体的去除以及在中心粒周围内吞循环区室的积累正常发生。然而，转铁蛋白受体从循环内体排出的速率常数（ke）从未处理细胞中的0.063分钟-1降低到巴弗洛霉素A1存在时的0.034分钟-1。这种减慢似乎取决于细胞质结构域中内化基序的存在，因为缺乏这些寡肽基序的修饰受体在巴弗洛霉素A1存在时循环速率没有那么大幅的降低。大量膜循环（通过内化的荧光脂质类似物6 - [N - [7 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂萘 - 4 - 基] - 氨基己酰 - 鞘氨醇磷酸胆碱的流出测量）从无药物时的0.060分钟-1的排出速率常数减慢到巴弗洛霉素A1存在时的0.046分钟-1。我们得出结论，巴弗洛霉素A1减慢了大量膜流动，但它以一种依赖于细胞质结构域上肽基序的方式额外抑制了受体循环。
